Cell Stress, Inflammatory Responses and Cell Death: Synopsis

Cell Signalling Biology Michael J.
Berridge
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Module 11
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Cell Stress, Inammatory Responses and Cell Death 11
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Module 11
Cell Stress, Inammatory
Responses and Cell Death
Synopsis
Cells have intrinsic signalling mechanisms that are capable of sensing various deleterious conditions,
both normal and pathological, and respond by mounting a variety of stress responses. Examples of
normal signals are the cytokines that induce inammatory responses in cells. Pathological signals
include UV and X-ray irradiation, hydrogen peroxide (H
2
O
2
), abrupt anoxia and physicochemical injury
through heat or noxious chemicals. A process of wound healing can function to repair the damage
caused by such injuries. In many cases, especially if the stress signal is not too severe, the cell can
survive and can even become tolerant to further insults. If cells are growing, such sub-lethal insults can
either cause the cell to stop growing temporarily to allow adequate time to repair the damage, or the
process of cell proliferation can be stopped more permanently and the cell will enter a state of
senescence. Another example of an evolutionarily conserved survival mechanism is autophagy, which
enables cells to cope with periods of starvation. If such stresses become too severe, however, the cell
dies, either through a process of necrosis, which is rapid and catastrophic, or through a slower and
more controlled process that is carried out by a highly regulated process of programmed cell death
known as apoptosis.
Although the morphological characteristics of necrosis and apoptosis are clearly distinct, they do
share some similarities in that they are induced by similar stimuli and often employ the same signalling
mechanism. Necrosis occurs when the cell is overwhelmed by the insult and rapidly disintegrates. The
cell volume expands rapidly, the mitochondria become swollen, and the plasma membrane suddenly
ruptures, causing release of the cellular contents into the intercellular spaces where they can elicit an
inammatory response. By contrast, apoptosis is a much more orderly affair in that proteases and
nucleases within the connes of an intact plasma membrane disassemble the cell that gradually
shrinks in size and is then engulfed by neighbouring cells, thus avoiding any inammatory reactions.
Inammatory responses
The innate immune system is our rst line of defence
against invading pathogens. Not only does it initiate a vig-
orous and rapid inammatory response to attack foreign
pathogens, but it also plays a key role in activating the
more slowly developing adaptive immune response. The
latter results in the activation of specic B and T cells to
extend the host defence system further. The initial line
of defence during the innate response is carried out by
a complex series of cellular interactions, which is known
collectively as the inammatory response. Such responses
are particularly evident during wound healing. The ma-
jor cellular players are the blood platelets, macrophages,
mast cells, neutrophils and endothelial cells (Module 11:
Figure inammation). In the following description of the
inammatory response, emphasis will be placed on the cell
signalling pathways that are used to control the participa-
tion of these different cell types, as summarized in Module
11: Figure inammation:
Green text indicates links to content within this module; blue text
indicates links to content in other modules.
Please cite as Berridge, M.J. (2012) Cell Signalling Biology;
doi:10.1042/csb0001011
1. Tissue damage. Many inammatory responses begin
with tissue damage, which can activate the complement
system to release complement factors that recruit in-
ammatory cells such as the neutrophils.
2. Endothelial cell damage. A specic form of tissue dam-
age occurs when the endothelial cells are disrupted.
The cells are induced to release inammatory medi-
ators such as thrombin and bradykinin, which are re-
sponsible for the redness, pain and swelling as the local
bloodvessels become dilatedandpermeable touidand
blood proteins. Some of these proteins are the comple-
ment factors and IgG antibodies that coat the patho-
gens, marking them out for phagocytosis. Endothelial
cells also release sphingosine 1-phosphate (S1P) that
can also have effects on vascular permeability.
3. Blood platelet aggregation and clot formation. The
thrombin produced by endothelial cell damage has a
major role to play in blood platelet aggregation and
formation of the haemostatic plug (Module 11: Figure
thrombus formation). With regard to the latter, it is
responsible for converting brinogen into brin, and
it also initiates the cascade of reactions that results in
cross-linking the brin monomers to form a brous
C
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Cell Signalling Biology Michael J. Berridge
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Module 11
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Module 11: Figure inammation
Mast cell
Antigen
S1P
Chemokines
C3a
C5a
Monocytes
Endothelial
cell
Neutrophil
Selectins
Rolling
Adhesion
Diapedesis
Macrophage
Pathogens
PAMPS
fMLP
1
Blood
platelet
Chemotaxis
Platelet
aggregation
Histamine
PDGF
TGF-
TNF-
IL-1
Thrombin
Bradykinin
Fibroblast
Tissue
damage
Complement
activation
Endothelial
permeability
Proliferation
Phagocytosis
Differentiation
2
3
4
5
6
7
8
9
Summary of the inammatory response to tissue damage and pathogens.
The innate immune system is triggered by signals derived from tissue damage and invading pathogens to activate cells such as the macrophages,
neutrophils, mast cells, blood platelets and endothelial cells that contribute to a co-ordinated series of responses to both remove the pathogens
and to repair damaged tissues. The endothelial cells are shown in three states: normal attened shape (blue), contracted to increase endothelial
permeability (light yellow) and damaged cells (bright yellow), where platelet aggregation occurs during formation of the haemostatic plug. Details of
these responses are described in the text.
meshwork that stems the ow of blood. Thrombin
contributes to the activation of the Ca
2 +
signal that
controls many of the processes of platelet aggregation
(Module 11: Figure platelet activation).
4. Endothelial permeability. Endothelial cells control the
owof substances and cells fromthe plasma into the in-
terstitial space. Under normal circumstances, this ow
is fairly restricted. However, during inammation, a
number of mediators, such as thrombin, bradykinin
and histamine, can greatly increase this permeability
by contracting the cells to open up the paracellular
pathway (Module 7: Figure regulation of paracellular
permeability).
5. Cell proliferation. During wound healing, there is a
considerable amount of cell proliferation to provide
new cells for tissue remodelling. Some of this pro-
liferation is driven by the release of platelet-derived
growthfactor (PDGF) andtransforming growthfactor-
(TGF-) (Module 11: Figure inammation). Cell
proliferation is increased in broblasts and other mes-
enchymal cells. There also is an increase in endothelial
cell proliferation as part of the process of angiogenesis
to repair the damaged blood vessels. The release of
vascular endothelial growth factor (VEGF) plays a crit-
ical role in triggering this increase in endothelial cell
proliferation (Module 9: Figure VEGF-induced prolif-
eration). Both the platelets and the endothelial cells re-
lease sphingosine 1-phosphate, which is one of the lipid
messengers formed by the sphingomyelin signalling
pathway (Module 2: Figure sphingomyelin signalling).
6. Activation of macrophages. Macrophages persist for
months or even years, positioned in a variety of organs
where they function as permanent sentinels ready to
initiate an inammatory response through two main
mechanisms. Firstly, they can respond to signals com-
ing fromthe pathogens byreleasing a number of inam-
matory mediators such as the chemokines. There also
is the possibility that macrophages may have recept-
ors capable of detecting uric acid generated from the
metabolism of nucleic acids in dying cells. Secondly,
they remove pathogens by engulng them through a
process of phagocytosis.
Pathogens initiate macrophage activation by shed-
ding pathogen-associated molecular patterns (PAMPs)
(Module 11: Figure formation and action of PAMPs).
These PAMPs act through a number of different Toll-like
receptors (TCRs) to stimulate the nuclear factor B
(NF-B) signalling pathway (Module 2: Figure Toll re-
ceptor signalling). In the case of macrophages, the PAMPs
help to regulate the transcriptional activation of a num-
ber of components that contribute to the inammat-
ory response, such as tumour necrosis factor (TNF),
interleukin-1 (IL-1) and IL-6 (Module 11: Figure macro-
phage signalling). PAMPs have a similar action in mast
cells (Module 11: Figure mast cell signalling). Pathogens
that become coated with antibodies (IgG and IgM) activ-
ate the complement system to release complement factors,
such as C3a and C5a, which function as chemoattractants
for inammatory cells such as the neutrophils. In addition,
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the coated pathogens are marked out for phagocytosis by
the macrophages (Module 4: Figure phagosome matura-
tion).
7. Activation of mast cells. Resident mast cells play an
important role in the initiation of the inammatory
response (Module 11: Figure inammation). Antigens
cross-link the IgE bound to the FcRIs to trigger
many of the mast cell signalling mechanisms that release
histamine and other inammatory mediators (Module
11: Figure mast cell signalling).
8. Neutrophil recruitment and activation. Neutrophils
have a relatively short half-life; they circulate in the
blood for a few hours before migrating into the sur-
rounding connective tissues, particularly at sites of in-
ammation, where they function for a few days only.
There appears tobe twomainpathways usedbyneutro-
phils to cross the endothelial layer of cells. The conven-
tional view is that they squeeze through the cell junc-
tions. An alternative mechanism is for them to migrate
throughthe neutrophil using a podosome toforce a way
through the cell. A process of neutrophil chemotaxis
then draws these cells to sites of inammation during
which they follow gradients of chemokines, comple-
ment factors (C3a and C5a), fMet-Leu-Phe (fMLP) and
hydrogen peroxide (H
2
O
2
) (Module 11: Figure inam-
mation). fMLP, whichis a bacterial breakdownproduct,
is a classical chemoattractant.
9. Monocyte differentiation. Monocytes follow a path-
way similar to that which occurs for the neutrophils as
they migrate through the endothelial layer to enter the
interstitial space, where they differentiate into macro-
phages.
The inammatory response is highly regulated and
depends upon pro-inammatory mechanisms that begin
early (as described above), but are gradually counterac-
ted by various anti-inammatory pathways mediated by
factors such as cytokines [interleukin-10 (IL-10)], hor-
mones and neurotransmitters [acetylcholine, vasoactive
intestinal peptide (VIP) and pituitary adenylate cyclase-
activating polypeptide (PACAP)].
Although the development of an inammatory response
is benecial, there are instances where the response gets
out of hand and begins to be deleterious, owing to an
excessive production of inammatory cytokines such as
TNF, IL-1 and IL-6 that result in oedema and tissue
injury. Indeed, acute and chronic inammation has been
associated with a number of disease states, such as sepsis,
rheumatoid arthritis, inammatory bowel disease (which
includes Crohns disease and ulcerative colitis), respirat-
ory distress syndrome, peritonitis and carditis. In the case
of the brain, many neurodegenerative diseases may result
fromactivationof Toll-like receptor 4 (TLR4) onmicroglia
cells to induce an inammatory response (Module 7: Fig-
ure microglia interactions).
Inammatory cytokines
There are a number of cytokines and related agents that
act topromote inammation(Module 1: Figure cytokines).
Two of the major cytokines are tumour necrosis factor-
(TNF) and interleukin-1 (IL-1).
Tumour necrosis factor (TNF)
Tumour necrosis factor (TNF) comes in two forms: TNF
(also known as cachectin because it mediates fever and
cachexia) and TNF (also known as lymphotoxin). For
most purposes, these are considered together as TNF,
which is a potent pro-inammatory cytokine that is re-
sponsible for many detrimental effects such as bacterial
sepsis, rheumatoid arthritis and Crohns disease. TNF acts
on the TNF receptor (TNF-R) to recruit different sig-
nalling pathways (Module 1: Figure cytokines):
TNF activates the nuclear factor B (NF-B) sig-
nalling pathway (Module 2: Figure NF-B activation).
The TNF-Rcanactivate caspase 8 toinitiate the extrinsic
pathway of apoptosis (Module 11: Figure apoptosis).
TNF activates the sphingomyelin signalling pathway
(Module 2: sphingomyelin signalling).
TNF is released from folliculostellate (FS) cells in re-
sponse to lipopolysaccharide (LPS)
The TNF receptor is inactivated through a process
of ectoderm shedding. Mutations in the cleavage site of
the TNF receptor, which prevents its down-regulation
by the ADAM enzyme responsible for its shedding, are
the cause of TNF-receptor-associated periodic febrile syn-
drome (TRAPS).
Wound healing
Detection of wounds and the subsequent healing process
depends on an orchestrated sequence of cellular events that
can be divided into four main phases:
1. Haemostasis. One of the rst processes is to stem the
ow of blood that is carried out by the blood platelets
that rapidly aggregate to forma clot (Module 11: Figure
thrombus formation)
2. Inammation. The presence of cell debris and bacteria
in the vicinity of the wound triggers an inammatory
response (Module 11: Figure inammation). An im-
portant feature of this response is the arrival of inam-
matory cells, such as the neutrophils, that are drawn
in by various attractants such as chemokines, comple-
ment factors (C3a and C5a), fMet-Leu-Phe (fMLP) and
hydrogen peroxide (H
2
O
2
).
3. Proliferation. The repair and reconstruction of wound
depends on the proliferation of various cell types.
The broblasts begin to proliferate and secrete colla-
gen. New blood vessels are formed by the process of
angiogenesis (Module 9: Figure angiogenesis).
4. Remodelling. During the nal remodelling phase, the
wound gradually adapts to its more permanent func-
tion. Those repair cells that are no longer required are
removed and the formation of collagen is rened so that
the bres are realigned along lines where tension forces
are maximal.
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Module 11: Figure platelet structure
-granules
Microfilaments
Microtubules
Pseudopodia
Tubular
invaginations
ER
Non-adhesive
circulating platelet
Adhesive
aggregating platelet
von Willebrand factor
Collagen
Thrombin
ATP
ADP
TXA 2
NO
PGI
2
Structure of non-adhesive and adhesive blood platelets.
The anucleate circulating platelet is a non-adhesive biconvex disc whose shape is maintained by a ring of microtubules. Aggregating agents induce
a dramatic structural change as the disc transform into an adhesive sphere that aggregate with each other to form a thrombus. The microtubule ring
breaks down and the peripheral actin microlaments are remodelled to form long laments located within the numerous pseudopodia. The -granules
fuse with the plasma membrane to release their contents as part of the activation process that forms a thrombus (Module 11: Figure thrombus
formation)
Blood platelets
Blood platelets that circulate freely in the plasma play a
primary role in the repair of damaged blood vessels during
wound healing by aggregating to form a clot (thrombus)
to prevent haemorrhage (Step 3 in Module 11: Figure in-
ammation). During thrombus formation, the platelets not
only provide the building blocks to construct the haemo-
static plug, but also contribute to the plasma coagulation
sequence that leads to the formation of the brin that sta-
bilizes this plug to stem the ow of blood.
Blood platelets circulate in the blood as small non-
adhesive anucleate biconvex discs about 2.5 m in length
(Module 11: Figure platelet structure). The plasma mem-
brane has numerous tubular invaginations, which often
come to lie close to segments of endoplasmic reticulum
(ER). A ring of microtubules (shown in red) lie in the
equatorial plane, and it has been suggested that they func-
tion to maintain the disc shape of the circulating platelet.
Immediately below the plasma membrane there is a layer
of actin microlaments (shown in black), which make up
about 15% of total platelet protein. The cytoplasm has
-granules that store a number of aggregating agents. In
response to a range of stimuli, the disc-shaped circulat-
ing platelet is transformed into a sphere that has numer-
ous pseudopodia containing long actin laments. The -
granules release their contents and this secretion is an im-
portant part of the platelet activation processes (Module
11: Figure thrombus formation).
When a blood vessel is damaged, the endothelial cells
that line the vessel wall no longer function as a barrier
and blood begins to ooze through the extracellular matrix
(ECM), and it is this contact between the blood and the
ECM that triggers the platelet activation sequence (Steps
ag in Module 11: Figure thrombus formation):
a. One of the rst interactions to occur is the adhesion of
platelets to ECM components such as von Willebrand
factor (vWF) that sits on the surface of the collagen
bres. Since the interaction between WF and the gly-
coprotein (GP) 1bGPIXGPV receptor complex on
the surface of the platelet is not particularly strong and
is easily made and broken, the platelet rolls over the
surface of the ECM.
b. As a result of this rolling over the ECM surface, the
glycoprotein receptor (GPIV) on the platelet surface
makes more meaningful contacts with glycogen to initi-
ate the process of platelet activation and secretion. Dur-
ing this activation process, the platelets undergo a dra-
matic change in shape. The non-adhesive disc is trans-
formed into a spherical shape with numerous pseudo-
podia and becomes highly adhesive, enabling the activ-
ated cells to aggregate.
c. In addition to the change in shape, the activated plate-
lets begin to secrete the contents of their -granules
to release molecules such as ATP and ADP that play
such an important role in the subsequent aggregation
response. Presumably, the available collagen surfaces
are rapidly encrusted with activated platelets, and ad-
ditional aggregating stimuli are necessary to activate
incoming platelets to build up the clot.
d. The release of ADP and the formation of other activ-
ators such as the eicosanoid thromboxane A
2
(TXA
2
)
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Module 11: Figure thrombus formation
Endothelial
cell
Rolling
Blood
platelet
Platelet
activation and
secretion
Clot
retraction
Aggregation
Platelet
inhibition
Adhesion
b
a
d
e
f
g
c
Collagen
NO
Fibrin
Haemostatic
plug
PGI
2
vWF
Red blood cells and plasma escaping
from ruptured blood vessel
ADP
Thrombin
TXA
2
__
__
+
+
+
Extracellular matrix
(Collagen, fibrinogen etc)
Platelet activation and thrombus formation.
When the endothelial cells are damaged, blood leaks out of blood vessel and the platelets are activated by components of the extracellular matrix
such as von Willebrand factor (vWF) and collagen. The process of platelet activation follows a sequence of events (ag) as described in the text.
and thrombin create a local environment that provides
a positive-feedback loop whereby the initial platelets
activated on the collagen surface are able to activate
platelets as they ow into the region of the developing
clot.
e. The incoming platelets activated by the soluble medi-
ator (ADP, thrombin and TXA
2
) are highly adhesive
and rapidly stick to the developing clot. The devel-
opment of the platelet aggregate is facilitated by both
integrin signalling and by the ephrin (Eph) receptor sig-
nalling system. Fibrin bres begin to appear around the
aggregated platelets
f. Once the clot reaches a certain size, a clot retraction
process occurs whereby a concerted contraction of the
platelets somehow pulls the aggregated cells and brin
closer together to form a water-tight seal (the haemo-
static plug).
g. To ensure that the positive-feedback processes respons-
ible for rapid platelet activation and clot formation do
not get out of hand, the surrounding endothelial cells
orchestrate a negative-feedback process by releasing
prostaglandin I
2
(PGI
2
) and nitric oxide (NO) to switch
off further platelet activation.
This platelet activation sequence is controlled by a num-
ber of signalling pathways as illustrated in Module 11: Fig-
ure platelet activation:
1. When platelets come into contact with the extracellu-
lar matrix (ECM) at the sites of vascular injury, one of
the rst interactions to occur is for the GP1bGPIX
GPV complex on the platelet surface to interact with
vWF that sits on the surface of collagen. The GP1b
GPIXGPV complex consists of four different GPs.
There are two GPIb subunits, two GPIb subunits,
two GPIX subunits and a single GPV subunit. A di-
sulphide bond links together the GPIb and GPIb
subunits. The GPIb subunit seems to be particularly
important because it has the binding sites for vWF and
its cytoplasmic tail is connected to the cytoskeleton.
The interaction between vWF and GP1bGPIXGPV
is relatively weak and this enables the platelet to roll
over the surface of collagen (see Step a in Module
11: Figure thrombus formation) and this presumably
sets the stage for the next interaction. Bernard-Soulier
syndrome has been linked to mutations in some
of the components of the GP1bGPIXGPV
complex.
2. One of the key steps in platelet activation sequence is
the interaction between collagen and the collagen re-
ceptor GPVI (Module 11: Figure platelet activation).
GPVI has two immunoglobulin-like domains, a trans-
membrane helix and a short cytoplasmic domain. The
receptor function of GPVI depends upon a close associ-
ation with the Fc receptor (FcR) chains, which have
the immunoreceptor tyrosine-based activation motifs
(ITAMs) that provide the docking sites to assemble
the transducing mechanism. Signal transduction be-
gins when the non-receptor protein tyrosine kinases
Fyn and Lyn, which are bound to GPVI through their
Src homology 3 (SH3) domains, phosphorylate the IT-
AMs on the FcR chains to provide binding sites for
phospholipase C2 (PLC2). The resulting activation
of the inositol 1,4,5-trisphosphate (InsP
3
)/Ca
2 +
sig-
nalling cassette (Module 2: Figure InsP
3
and DAG
formation) appears to be a major control mechanism
for platelet activation (see below).
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3. Activation of InsP
3
and the release of Ca
2 +
are also
used by a number of the other aggregating agents such
as ADP, thrombin and TXA
2
which all act through
G protein-coupled receptors (GPCRs) that activate
phospholipase C (PLC).
4. Platelets express the P2X1 isoform of the ionotropic
P2X receptors (Module 3: Figure P2X receptor struc-
ture), that provide an inux of external Ca
2 +
that con-
tributes to platelet activation (Module 11: Figure plate-
let activation). Inaddition, theyexpress the Orai 1 chan-
nel that responds to store depletion by an increase in
Ca
2 +
entry(Module 3: Figure SOCsignalling compon-
ents). The membrane depolarization induced by these
Ca
2 +
channels is counteracted by the presence of Kv1.3
channels that hyperpolarize the membrane to maintain
the driving force for Ca
2 +
entry.
5. One of the signalling functions of Ca
2 +
is to stimu-
late the release of the -granules that release aggregat-
ing agents such as ADP, which thus provide positive-
feedback processes by stimulating further Ca
2 +
release
through Step 4 as described above.
6. Another important positive amplication step is the
formation of TXA
2
, which is produced by the Ca
2 +
-
dependent activation of phospholipase A
2
(PLA
2
). The
resulting arachidonic acid (AA) is converted into TXA
2
(Module 1: Figure eicosanoids), which feeds back to
activate the TP receptor. The AA is also converted
into PGI
2
, which builds upper towards then end of the
aggregation process to activate an inhibitory pathway
(Step 12 in Module 11: Figure platelet activation).
7. An increase in Ca
2 +
may contribute to phospholipid
scrambling that result in phosphatidylserine (PS) trans-
fer from the inner to outer plasma membrane leaf-
let, which then contributes to formation of the tenase
and prothrombinase complexes responsible for gener-
ating thrombin. Scott syndrome may result frommuta-
tions in ABCA1, which is one of the ATP-binding cas-
sette (ABC) transporters responsible for the transfer of
PS.
8. Platelets express two integrin receptor types,
2
/
1
and
11b
/
3
, which can link to collagen and brinogen
respectively (Module 1: Figure integrin heterodimeric
complexes). The former interaction plays an import-
ant role in the initial attachment to collagen, whereas
the
11b
/
3
interaction with brinogen functions later
to bind the aggregating platelets together (Module 11:
Figure platelet activation). The collagen receptor GPVI
plays an important role in activating the integrin recept-
ors earlyinthe activationprocess throughthe inside-out
mechanism whereby intracellular signals coming from
other receptors induce a conformational change in the
integrin receptors that greatly enhance their afnity for
external ligands (Module 1: Figure integrinreceptor). In
the case of platelets, the
2
/
1
receptor binds collagen
whereas the
11b
/
3
receptor binds brinogen.
9. The high-afnity
2
/
1
receptor is activated by colla-
gen (Module 11: Figure platelet activation), which not
only increase the platelet adhesiveness to the ECM,
but also activates integrin signalling mechanisms that
remodel the actin cytoskeleton that contributes to
formation of the pseudopodia of the activated plate-
let (Module 11: Figure platelet structure
10. The activated
11b
/
3
integrin binds to brinogen to
help establish cellcell interactions between the aggreg-
ating platelets (Module 11: Figure platelet activation).
In addition, the activated integrin receptors can as-
semble a large signalling complex capable of both re-
modelling the actin cytoskeleton and inducing a num-
ber of signalling pathways (Module 6: Figure integrin
signalling). The development of actin bres stabilized
by non-muscle myosin II (NMII) extend across the
cell and function in clot retraction (Step f in Module 11:
Figure thrombus formation). Glanzmanns thrombas-
thenia is caused by mutations in the
3
integrin subunit.
11. The aggregating platelets communicate with each other
through the bidirectional ephrin (Eph) receptor sig-
nalling system (Module 1: Eph receptor signalling).
This ephrin signalling system can generate a number
of signals that appear to contribute to platelet activa-
tion. One of its actions may be to phosphorylate the
3
integrin subunit to enhance the activity of the
11b
/
3
integrin receptor (Module 11: Figure platelet activa-
tion).
12. The cyclic AMP signalling pathway has an import-
ant function in platelet aggregation by activating a
negative-feedback process to ensure that the positive-
feedback processes responsible for rapid platelet ac-
tivation and clot formation do not get out of hand
(Step g in Module 11: Figure thrombus formation).
The inhibitor action of cyclic AMP is not clear, but
it may act by inhibiting the Ca
2 +
signal (Module 11:
Figure platelet activation). Another function of cyclic
AMP is to activate the phosphorylation of vasodilator-
stimulated phosphoprotein (VASP), which is a mem-
ber of the Ena/vasodilator-stimulated phosphoprotein
(VASP) family (Module 4: Figure Ena/VASP family)
resulting in a decrease in the actin-dependent processes
associated with clotting. One of the potent activators
of cyclic AMP formation is PGI
2
acting on its IP re-
ceptor (Module 1: Figure eicosanoids). ADP acting on
the P2Y
12
receptor is coupled to the G protein Gi
2,
which acts to inhibit adenylyl cyclase (Module 2: Table
heterotrimeric Gproteins). ADPthus has two effects: it
stimulates Ca
2 +
signalling through the P2Y
1
receptor
(Step 3), while simultaneously lowering the level of the
inhibitory cyclic AMP signal by acting on the P2Y
12
receptor.
Mast cells
Mast cells are widely distributed throughout the body,
where they are positioned close to blood vessels and epi-
thelial cell layers. They are particularly evident in locations
that are exposed to the outside world, such as the lung,
gastrointestinal tract and skin. Since they take up perman-
ent residence in these locations, they are always on hand to
respond to infections that most commonly occur during
wounding. Basophils have a similar function to mast cells,
except that they circulate in the blood and are recruited
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Module 11: Figure platelet activation
Ca
2+
Ca
K
2+
+
InsP
3
+
+
+
+
- -
+
+
+
InsP R
3
5
1
4
3
2
7
8
11
12
9
10
6
PLC
A
B
C
A
1
P
L
C
G
S
G
I
AC
ADP
FcR
vWF
ADP
ADP
ATP
AA
NMII
Actin
Cyclic
AMP Fibrinogen
TXA
2
TXA
2
PGI
2
Thrombin
Thrombin
Prothrombin
Prothrombinase
Collagen
Collagen Low
affinity
PS
PLA
2
P S A V
Gp1b
Gp1b
G
P
V
GPIX
2 1
11b 3
4 A h p E
1 B - n i r h p E
P2Y
12
IP
GPVI
PAR1
P2Y1
Kv1.3
P2X
1
TP
S
S
Orai 1
n o i t e r c e S
TXA
2
Platelet activation signalling pathways.
Platelets are sensitive to a large number of stimuli that act through a wide range of receptors. The numbers refer to the receptors and signalling
pathways responsible for controlling the processes of thrombus formation, which is summarized in Module 11: Figure thrombus formation.
to sites of infection. Once they arrive and settle down,
basophils begin to function much like mast cells.
Mast cells play a central role in the innate immune re-
sponse and are particularly important in helping to recruit
other cells, such as the basophils, neutrophils and lympho-
cytes (Module 11: Figure inammation). In the case of skin
infections, mast cells contribute to the mediators of the in-
ammatory soup that act on sensory neurons to cause
pain (Module 10: Figure inammatory soup). This role
in inammatory responses depends on the capability of
the mast cells to release an enormous range of mediators
(Module 11: Figure mast cell signalling). There are three
main mast cell release mechanisms.
Mast cells have been implicated in a number of aller-
gic diseases, such as asthma, and they also contribute to
chronic inammatory conditions such as atherosclerosis,
vasculitis and rheumatoid arthritis.
Mast cell release mechanisms
Mast cells release mediators through three main mechan-
isms (Module 11: Figure mast cell signalling):
Mast cell granule release
A characteristic feature of mast cells is the large num-
ber of secretory granules that ll the cytoplasm. These
large vesicles contain many different mediators, including
biogenic amines (histamine and 5-hydroxytryptamine),
growth factors and enzymes. The earliest response to stim-
ulation of mast cells is the fusion of these vesicles with the
plasma membrane. Since the granules are so large, not all
the granules have access to the membrane, so they can fuse
with each other to produce proles that resemble a bunch
of grapes.
Mast cell release of lipid-derived mediators
Mast cells have the capacity to rapidly synthes-
ize prostaglandins and leukotrienes from the
arachidonic acid (AA) released following activation
of phospholipase A
2
(PLA
2
). The pathways for form-
ing these eicosanoids are shown in Module 1: Figure
eicosanoids.
Mast cell synthesis and release of inammatory
cytokines and immunoregulators
In addition to the rapid release of granules and formation
of lipid-derived mediators, mast cells also have a slower,
but more prolonged, release of a bewildering array of cy-
tokines and immunoregulators, as shown in Module 11:
Figure mast cell signalling. This slower release mechanism
depends upon activating the genes responsible for coding
all of these mediators.
These multiple secretory processes are controlled by a
number of different mast cell signalling mechanisms.
Mast cell signalling mechanisms
Mast cells have a large number of signalling mechanisms to
control the different mast cell release mechanisms (Module
11: Figure mast cell signalling). A number of different
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8
stimuli engage signalling pathways that can either activ-
ate or inhibit secretion.
Mast cell FcRI signalling pathway
The FcRI is responsible for activating signalling path-
ways that control all three secretory processes (Module
11: Figure mast cell signalling). It acts through the phos-
phoinositide signalling pathway to produce inositol 1,4,5-
trisphosphate (InsP
3
), which mobilizes the Ca
2 +
that con-
tributes to the release of secretory granules, which is
very dependent on the monomeric G proteins Rac and
Cdc42 (see below). This release of internal Ca
2 +
is aug-
mented by various Ca
2 +
entry channels that belong to
the transient receptor potential (TRP) ion channel fam-
ily, such as canonical TRP 1 (TRPC1), vanilloid TRP 2
(TRPV2) and melastatin-related TRP 2 (TRPM2). The
increase in Ca
2 +
also contributes to the activation of
phospholipase A
2
(PLA
2
), which release the precursor
arachidonic acid that is converted into the prostagland-
ins and leukotrienes (Module 1: Figure eicosanoids).
The FcRI also relays information out through differ-
ent mitogen-activated protein kinase (MAPK) signalling
pathways to regulate all three secretory processes. It uses
the extracellular-signal-regulated kinase (ERK) pathway
to contribute to the activation of both PLA
2
and the tran-
scriptional events responsible for the synthesis of inam-
matory cytokines and immunoregulators. It also activates
transcription through the Janus kinase (JNK) pathway.
The link between the receptor and JNK seems to depend
on the monomeric G proteins Cdc42 and Rac. The lat-
ter also play a critical role in controlling granule release,
which is unusual because it is very dependent on GTP. Rac
and Cdc42 seem to function by preparing the granules for
release, whereas Ca
2 +
functions more as a modulator of
exocytosis.
The way in which the FcRI is coupled to these different
signalling pathways is shown in Steps 17 in Module 11:
Figure FcRI mast cell signalling:
1. The FcRI consists of four subunits: an subunit, a
subunit and two subunits that are connected together
through a disulphide bridge. The subunit has a very
high afnity for IgE, which is permanently bound to
the FcRI complex. Signalling begins when a bivalent
antigen binds to two IgE molecules to bring together
two receptor complexes that then interact to initiate the
process of signal transduction.
2. These FcRI subunits lack enzyme activity, and thus
have to recruit various transducing elements, such
as the non-receptor tyrosine kinases Fyn, Lyn and
Syk. Lyn plays an early role by phosphorylating
immunoreceptor tyrosine-based activation motifs (IT-
AMs) (the red bars on the and subunits). These
ITAMs are the same as those used during signal trans-
duction by the T cell receptor (TCR) (Module 9: Fig-
ure TCR signalling). Another similarity to T cell sig-
nalling is that the FcRI also makes use of the scaffold-
ing proteins LAT (linker of activated T cells) and Src
homology 2 domain-containing leukocyte protein of
76 kDa (SLP-76). The phosphorylated ITAMs on the
FcRI then recruit Syk, which is also activated by Lyn
phosphorylation (Module 11: Figure FcRI mast cell
signalling).
3. Activated Syk has a number of actions. It phos-
phorylates the scaffolding proteins LAT and SLP-76
to provide binding sites for a number of signalling
components. It also phosphorylates phospholipase C
(PLC1) and Brutons tyrosine kinase (Btk), which in-
teract with each other during the activation of PLC1.
4. The activated PLC1 hydrolyses PtdIns4,5P
2
to form
both InsP
3
and diacylglycerol (DAG).
5. Phosphorylation of LAT recruits growth factor recept-
or-bound protein 2 (Grb2), which sets up a nucleation
centre to assemble the signalling components that res-
ult in the formation of phosphorylated ERK1/2, which
activates both PLA
2
and gene transcription.
6. PhosphorylatedLATrecruits SLP-76 and Grb2-related
adaptor protein (GADS) to set up a signalling complex
that activates monomeric G proteins such as Vav and
Rac that stimulate JNK, and they also function to stim-
ulate granule release.
7. The FcRI can also stimulate the PtdIns 3-kinase (PI
3-K) signalling pathway. The Fyn that is recruited to
the activated receptor phosphorylates Gab2 and Btk,
which contribute to the activation of PI 3-K, which
then phosphorylates PtdIns4,5P
2
to formthe lipid mes-
senger PtdIns3,4,5P
3
.
Mast cell GPCR signalling pathways
Mast cells express a number of G protein-
coupled receptors (GPCRs) that function to either
stimulate or inhibit mast cell secretory functions. The
stimulatory actions of the GPCRs are shown in Module
11: Figure mast cell signalling. For example, stimuli such
as PGE
2
, leukotriene C
4
(LTC
4
) and the complement
factors C3a and C5a act through these GPCRs, which are
coupled to the G
q
phospholipase C (PLC) signalling
pathway that generates inositol 1,4,5-trisphosphate
(InsP
3
) and diacylglycerol (DAG). The InsP
3
mobilizes
Ca
2 +
, which contributes to granule release and also
functions to stimulate phospholipase A
2
(PLA
2
). On the
other hand, noradrenaline (norepinephrine) stimulates
2
-adrenoceptors that act through G
s
and adenylyl
cyclase (AC) to generate cyclic AMP, which inhibits some
of these secretory pathways (Steps 5 and 6 in Module 11:
Figure mast cell inhibitory signalling). The mode of action
of cyclic AMP is not clear, but there are indications that
it may act to inhibit certain aspects of the Ca
2 +
signalling
system.
Mast cell Toll receptor signalling pathway
Mast cells are sensitive to many of the fragments
coming from various pathogens that are known
as pathogen-associated molecular patterns (PAMPs)
(Module 11: Figure formation and action of PAMPs).
These PAMPs act through the Toll receptor signalling
pathway that engages the nuclear factor B (NF-B)
signalling pathway to stimulate gene transcription
(Module 2: Figure Toll receptor signalling). Mast cells are
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Module 11: Figure mast cell signalling
PLC
P
L
C
2
IP
3
Ca
2+
+
+ +
AA
JNK
PAMPS
GPCRs
IgE
MAST
CELL
Antigen
PGE
LTC
4
2 C3a, C5a
TRPC1, TRPV2
TRPM2
TLR
Rac
R
a
c
Fc RI
R
a
f
Preformed granules
Lipid-derived mediators
Histamine
5-hydroxytryptamine (5-HT)
TNF-
VEGF
FGF2
Tryptase
Chymase
Carboxypeptidase
2 2
Prostaglandins (PGE ;PGD )
4 4
Leukotrienes (LTC ;LTB )
ERK1/2
NF- B
PLA
Inflammatory cytokines and
immunoregulators
TNF TGF VEGF
IL-6; IL-9; IL-10; IL-12
IL-13; IL-15; IL-16; IL-18
CCL2, CCL3; CCL4; CCL5
CCL11; CCL20
CXCL1; CXCL2; CXCL8;
CXCL9; CXCL10; CXCL11
IL-1 IL-3; IL-4; IL
+
+
+
Cdc42
The function and mechanisms of mast cell signalling.
Mast cells release an enormous number of components that fall into three separate groups that are controlled by different signalling mechanisms.
The preformed granules are released quickly through a typical Ca
2 +
-dependent exocytotic mechanism. The lipid-dependent mediators that are
produced from the arachidonic acid (AA) following stimulation of phospholipase A
2
(PLA
2
). Finally, a whole range of inammatory cytokines and
immunoregulators are produced and released as a result of increases in gene transcription. The signalling mechanism that controls these release
processes is described in the text. This gure represents only those signalling pathways that activate release processes. The signalling pathways that
inhibit release are described in Module 11: Figure mast cell inhibitory signalling.
somewhat unusual in that they do not express the CD14
co-receptor, but they appear to use a soluble CD14 present
in plasma to facilitate the transfer of PAMPs on to the
Toll-like receptors (TLRs). The PAMPs such as lipopoly-
saccharide (LPS) can activate transcription independently
of the other cellular processes, such as the release of gran-
ules or the lipid-derived messengers.
Mast cell FcRIII signalling pathway
The FcRIII receptor on mast cells exerts an inhibitory
action on FcRI through Steps 14 in Module 11: Figure
mast cell inhibitory signalling:
1. FcRIII can bind IgE and is thus drawn into the re-
ceptor complex containing FcRI, where it begins to
exert its inhibitory action.
2. The Lyn attached to FcRI phosphorylates immunore-
ceptor tyrosine-based inhibitory motifs (ITIMs; purple
region on FcRIII). These phosphorylated residues
then provide binding sites for various negative regu-
lators.
3. The Src homology 2 (SH2) domain-containing inos-
itol 5-phosphatase (SHIP), which is one of the inositol
polyphosphate 5-phosphatases, dephosphorylates the
lipid second messenger PtdIns3,4,5P
3
.
4. The Src homology 2 (SH2) domain-containing protein
tyrosine phosphatase-1 (SHP-1) acts by reversing the
phosphorylations responsible for activating Syk, which
relays information to a number of signalling pathways
(Module 11: Figure FcRI mast cell signalling).
Macrophages
There are two distinct macrophage types: M1 macrophages
sometimes known as killers and the M2 healer macro-
phages.
The M1 macrophages function to increase inammat-
ory responses and can kill pathogens. They respond to
pathogen-associated molecular patterns (PAMPs) to initi-
ate inammatory responses by releasing inammatory cy-
tokines, chemokines and immunoregulators (Module 11:
Figure inammation). The M1 macrophages have high
levels of iNOS to generate nitric oxide (NO) to com-
bat bacterial and viral infections and to destroy tumour
cells.
M2 macrophages (healers) ingest dead and damaged
host cells and micro-organisms such as bacteria and
protozoa through a process of phagocytosis. They also
play a role in enhancing collagen synthesis by releasing
transforming growth factor (TGF-).
Pathogen-associated molecular patterns
(PAMPs)
The pathogen-associated molecular patterns (PAMPs)
are specic components of invading pathogens that
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Module 11: Figure FcRI mast cell signalling
Rac
Grb2
Sos
Ras
MEK
MEK
Raf
L
A
T
Vav
Fyn
Gab2
Btk
Btk
PI 3-K
DAG
PLA activation Granule release Gene transcription 2
Ca
2+
IgE
Fc RI
PKC
Lyn Syk
InsP
3
2 2
3
PtdIns4,5P PtdIns4,5P
PtdIns3,4,5P
PLC 1
ERK1/2 JNK PKB
P P P
P
P
P
P
P
P
P
P
P
P
P
P
P P P
GADS
6 7 - P L S
1
2
3
4
5
6
7
Antigen
The mast cell FcRI complex relays information to a number of signalling pathways.
The FcRI complex is composed of , and subunits that lack enzymatic activity. Signal transduction is carried out by non-receptor tyrosine
kinases (Fyn, Lyn and Syk) that phosphorylate different elements that then recruit the signalling components of a number of pathways to control
phospholipase A
2
(PLA
2
) activation, granule release and gene transcription, as described in the text.
Module 11: Figure mast cell inhibitory signalling
Cyclic AMP
Noradrenaline
Antigen
IgE
IgE
Fc RI Fc RIII
- Adrenoceptor
Lyn
3
2
PtdIns4,5P
PtdIns 3,4,5P
P
P
P
1
2
5
6
SHIP SHP-1
L
A
T
Ca
2+
2
Syk
InsP
InsP R
3
3
2
PtdIns4,5P
PLC 1
P
P
P
P
P
3
4
+
E R
G
S
AC
P
P
P
Inhibition of mast cell release by FcRIII and
2
-adrenoceptors.
Release of inammatory mediators by mast cells is inhibited by FcRIII and
2
-adrenoceptors. The former bind IgE and are drawn into the FcRI
complex, where they are phosphorylated by Lyn and this enables them to bind the phosphatases Src homology 2 (SH2) domain-containing inositol 5-
phosphatase (SHIP) and SH2 domain-containing protein tyrosine phosphatase-1 (SHP-1) that can dephosphorylate the lipid messenger PtdIns3,4,5P
3
and phosphorylated proteins respectively. The
2
-adrenoceptors produce cyclic AMP that appears to inhibit the Ca
2 +
signalling pathway.
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Module 11: Figure formation and action of PAMPs
Bacteria
Eubacteria Yeast
Mycobacteria
Lipoprotein
Peptidyl-
glycan
PAMPs
TLRs TLR2
TLR4 TLR5
(Lipid A, and
Lipoteichoic
acid)
Flagellin
CpG DNA
Virus
dsRNA
ssRNA
Endosome
LPS
Lipoarabino-
mannan
PATHOGENS
Zymosans
INFLAMMATION
Toll receptor signalling pathways
MAP kinase signalling pathways
Signalling pathways
Inflammatory response
TLR3
TLR7/8
TLR9
The role of pathogen-associated molecular patterns (PAMPs) in triggering the inammatory response.
Unique components derived from different pathogens make up the pathogen-associated molecular patterns (PAMPs) that activate the Toll-like
receptors (TLRs) located on macrophages and some other cell types. These TLRs are located either on the plasma membrane or on the endosomal
membranes. The TLRs, such as TLR4, act through the Toll receptor signalling pathway (Module 2: Figure Toll receptor signalling). The TLRs on
endosomal membranes function in virus recognition (Module 2: Figure virus recognition). CpG DNA, cytidine-phosphate-guanosine DNA; dsRNA,
double-stranded RNA; ssRNA, single-stranded RNA; MAP kinase, mitogen-activated protein kinase.
are used to stimulate resident macrophages to initiate
an inammatory response (Module 11: Figure formation
and action of PAMPs). Many of the PAMPs are derived
from the surface coat of the pathogens, whereas others
are unique nucleic acid sequences. For example, viruses do
not contain many PAMPs because both the viral coat pro-
tein and lipids are derived from the host. However, they
do provide double-stranded RNA (dsRNA) and single-
stranded RNA (ssRNA) fragments that can be detected
by the virus recognition and antiviral response system
(Module 2: Figure virus recognition).
One of the most active ingredients of the PAMPs is
lipopolysaccharide (LPS), which is composed of lipid A
derived from the external membrane of Gram-negative
bacteria, and lipoteichoic acid from Gram-positive bac-
teria. At the sight of infection, there will therefore
be a complex pattern of pathogen-derived molecules,
which is then interpreted by a corresponding pattern of
TLRs (Module 11: Figure formation and action
of PAMPs). These TLRs then recruit various downstream
signalling pathways, such as the Toll receptor signalling
pathway (Module 2: Figure Toll receptor signalling) or
the mitogen-activated protein kinase (MAPK) signalling
pathway, to generate an inammatory response to match
the kind of pathogens that are invading the organism.
Modulation of inammatory responses is carried out by
a number of inammatory regulators that exert both pro-
and anti-inammatory responses.
Modulation of inammatory responses
The Toll receptor signalling pathway (Module 2: Fig-
ure Toll receptor signalling) responds to PAMPs by
activating both nuclear factor B (NF-B) signalling
and the p38 pathway to induce the transcription of
a large number of inammatory mediators (Module
11: Figure macrophage signalling). Some of these gene
products, such as cyclooxygenase 2 (COX-2), act to
increase the formation of eicosanoids (Module 1: Fig-
ure eicosanoids). The COX-2 increases the conver-
sion of arachidonic acid (AA) into prostaglandin E
2
(PGE
2
), which sets up a negative-feedback loop because
PGE
2
acts through cyclic AMP to inhibit transcription
(see below).
The ability of the Toll receptor signalling path-
way to elicit an inammatory response can be
modulated by a number of regulatory mechan-
isms, which are either pro- or anti-inammatory
(Module 11: Figure macrophage signalling). Regulation of
the macrophage response is highly dynamic in that there
are processes that act to speed up the onset of the response,
which are then counteracted by an anti-inammatory re-
sponse that comes into play to ensure that the response
does not get out of hand. Some of these positive- and
negative-feedback responses are endogenous (i.e. they oc-
cur within the macrophage), whereas others are imposed
from the outside. Understanding this dynamic balance
is of considerable interest with regard to pathology in
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Module 11
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Module 11: Figure macrophage signalling
iNOS
Ca
2+
+
+
+
+
+
+
Cyclic
AMP
COX-2
PKA
CREB
IKK
MEKK1
MEK3/6
p38
PDE4B
mRNA
CaMKII
P
P
P
P
P
P
P
P
P
P
TBP
Pol II
TATA
CBP
IL-1
IL-1
NO
NO TNF
TNF
Inflammatory
mediators
I B
NF- B
NF- B
Toll
signalling
pathway
IP
3
Arachidonic
acid
cPLA2
lysoPL
PAF
PAF
PGE
2
PGE
2
PGE
2
TLRs
AC
Gs
VIP
PACAP NA
AMP
P2X7 nAChR
ATP
ACh
Gq
PLC
SOC
PL
PAMPs
Oestradiol
B
C
R
E
iNOS mRNA
COX-2
PDE4B
IL-1 mRNA
TNF mRNA
Modulation of inammatory responses.
Pathogen-associated molecular patterns (PAMPs) acting through Toll-like receptors (TLRs) use the Toll receptor signalling pathway to activate the
transcriptional processes that result in the release of inammatory mediators such as nitric oxide (NO), tumour necrosis factor (TNF) and interleukin
1 (IL-1). These inammatory signalling pathways can be modulated by both the cyclic AMP and Ca
2 +
signalling pathways, as described in the
text. PAF, platelet-activating factor; (lyso)-PL, (lyso)-phospholipid.
that they reveal novel strategies for the discovery of anti-
inammatory drugs.
The cyclic AMP signalling pathway is particularly im-
portant in carrying out some of the anti-inammatory re-
sponses. The macrophage responds to a number of agon-
ists, such as noradrenaline, vasoactive intestinal peptide
(VIP)/pituitary adenylate cyclase-activating polypeptide
(PACAP) and PGE
2
, which are coupled to the form-
ation of cyclic AMP. The latter acts through protein
kinase A(PKA) to not only inhibit mitogen-activated pro-
tein kinase (MAPK)/extracellular-signal-regulated kinase
(ERK) kinase kinase 1 (MEKK1), thereby reducing the
phosphorylation of the basal transcription factor TATA-
box-binding protein (TBP), but it also phosphorylates and
activates cyclic AMP response element-binding protein
(CREB), which enters the nucleus to compete with NF-B
for a binding site on CREB-binding protein (CBP). In this
way, cyclic AMP inhibits the formation of inammatory
mediators.
The inhibitory action of cyclic AMP is terminated by
phosphodiesterase PDE4B, which is one of the genes
activated by the TLRs, which thus sets up an internal
positive-feedback loop. By increasing the expression level
of PDE4B, the macrophage will reduce the inhibitory ef-
fect of cyclic AMP, thereby enhancing the inammatory
response (Module 11: Figure macrophage signalling).
An increase in the level of Ca
2 +
brought about by a
number of receptor mechanisms has both pro- and anti-
inammatory effects. These different actions of Ca
2 +
may
depend on the way the Ca
2 +
signal is presented both in
time and space. These Ca
2 +
signals are generated either
by activation of receptor-operated channels (ROCs) such
as the nicotinic acetylcholine receptors (nAChRs) or the
purinergic P2X7 receptor. In addition, Ca
2 +
is released
frominternal stores by inositol 1,4,5-trisphosphate (InsP
3
)
generated by Gprotein-coupled receptors (GPCRs) activ-
ated by ATP or oestradiol. One of its anti-inammatory
responses depends upon inhibition of the synthesis of in-
ammatory mediators. On the other hand, Ca
2 +
can also
be pro-inammatory by promoting gene transcription by
stimulating the phosphorylation of inhibitor of NF-B
(IB) kinase (IKK) and NF-B, which enhances the re-
lease of inammatory mediators such as interleukin 1
(IL-1).
Neutrophils
Neutrophils are one of the major cell types contribut-
ing to innate immunity, which is the rst line of defence
against invading pathogens. During an inammatory re-
sponse, the neutrophils move out of the blood and mi-
grate towards the site of infection (see Step 8 in Module
11: Figure inammation). Neutrophils are guided towards
these inammatory sites by responding to gradients of
chemokines, complement factors (C3a and C5a) or fMet-
Leu-Phe (fMLP). They sense these gradients while still in
the blood vessel, and start their journey through a typical
response that begins with them attaching to the surface of
the activated endothelial cells. The neutrophils then begin
to roll along the surface by interacting with molecules of
P-selectin, which is packaged within WeibelPalade bod-
ies and released on to the endothelial cell surface following
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Module 11: Figure SHIP1 and PIP
3
polarity
Src homology 2 (SH2) domain-containing inositol 5-phosphatase 1 (SHIP1) controls neutrophil polarity and motility.
A. Wild-type (WT) and Src homology 2 (SH2) domain-containing inositol 5-phosphatase 1 (SHIP1)
/
neutrophils were placed in an EZ-chamber and
their positions were monitored at 30 s intervals as they responded to a gradient of fMet-Leu-Phe (fMLP). The WT cells moved in a relatively straight line
up the gradient, whereas the SHIP1
/
cells moved more slowly. B. When viewed at higher magnication, the WT cells were rounded, but assumed
an elongated shape with a pseudopod at one end when placed in a fMLP gradient. By contrast, the SHIP1
/
neutrophils remained rounded with
pseudopodia appearing all round the cell. If recorded over a longer period, these cells did develop a partial polarity and there was some motion up
the gradient (see panel C). Reproduced by permission from Macmillan Publishers Ltd: Nishio, M., Watanabe, K., Sasaki, J., Taya, C., Takasuga, S.,
Iizuka, R., Balla, T., Yamazaki, M., Watanabe, H., Itoh, R., Kuroda, S., Horie, Y., Forster, I., Mak, T.W., Yonekawa, H., Penninger, J.M., Kanaho, Y., Suzuki,
A. and Sasaki, T. (2007) Control of cell polarity and motility by the PtdIns(3,4,5)P
3
phosphatase SHIP1. Nat. Cell Biol. 9:3644. Copyright (2007);
http://www.nature.com/ncb; see Nishio et al. 2007.
activation by histamine (i.e. during Step 4). As the attach-
ment to the selectins strengthens, the increase in adhesion
causes the neutrophils to atten out in preparation for the
processes of diapedesis, during which they squeeze their
way through the gaps that have appeared between the en-
dothelial cells. When the neutrophils have passed through
the endothelium, a process of neutrophil chemotaxis draws
them towards the sites of inammation.
Neutrophil chemotaxis
Resting neutrophils are spherical, but once they detect a
chemoattractant, they rapidly develop a polarity charac-
terized by an elongated shape with a pseudopod at the
front and a bulbous uropod at the rear (Module 11: Figure
neutrophil chemotaxis). These two regions have a charac-
teristic organization of actin laments. At the front, there
is an actin network that helps to push out the pseudo-
pod, whereas the uropod has an actin/myosin II network
that functions to contract and pull up the rear end as the
cell moves forward. There also is a microtubule organ-
izing centre (MTOC) that aligns the microtubules in the
direction of movement and helps to stabilize cell polarity.
The microtubules may also be important to directing the
ow of vesicles that enter the cell by endocytosis at the
rear of the cell and are released at the front end. Another
important aspect of motility, which concerns adhesion to
the matrix, is apparent when cells are viewed from the
side. In order for cells to move over a matrix, they have to
strike a balance between motility and adhesion. They re-
quire a certain level of adhesiveness to provide the traction
to move forward. This delicate adhesion/motility balance
seems to depend on the integrins that attach to the surface
at the front and detach at the back. The ow of vesicles
mentioned earlier may provide a mechanism for moving
integrin receptors from the back to the front.
The process of chemotaxis enables neutrophils to seek
out the sites of inammation by moving up a chemoat-
tract gradient. This directed movement is clearly evident
when the position of neutrophils are recorded at 30 s inter-
vals as they move up a chemoattractant gradient in an EZ-
Taxiscanchamber (see panel AinModule 11: Figure SHIP1
and PIP
3
polarity). A number of external stimuli func-
tion as chemoattractants (Module 11: Figure neutrophil
chemotaxis). One of the main attractants is the tripeptide
fMet-Leu-Phe (fMLP), which can attract neutrophils even
when applied in a very shallowgradient such that the front
of the cell experiences a concentration that is just 12%
higher than that experienced at the back end. Since the re-
ceptors for fMLP are distributed equally over the surface,
the cell has to detect this small difference in concentration
to develop a polarity that enables them to move towards
the source of the gradient. The chemoattractant gradient
will result in more receptors being occupied at the front
compared with the back, and this small difference in occu-
pancy is somehow translated into a polarized cell capable
of migrating in a directed way towards the source of the
gradient. In effect, the difference in receptor occupancy is
transduced into an internal compass that is then used to
direct the motile machinery to move in one direction. Just
how this compass is set up is still somewhat of a mystery.
In addition to following a pre-existing chemoattractant
gradient such as fMLP, neutrophils also create a localized
gradient of ATPand its breakdown product adenosine that
are concentrated at the front of the cell (yellow halo in
Module 11: Figure neutrophil chemotaxis). Hemichannels
in the region of the pseudopod release ATP, some of which
is converted into adenosine. ATP and adenosine then feed
back in an autocrine manner to activate receptors at the
front and thus contribute to chemotaxis by amplication
of early polarity signalling.
The signalling pathways activated during the onset of
chemotaxis have been divided into the following sequence
of events (Module 11: Figure neutrophil chemotactic sig-
nalling):
C
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Module 11
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14
Module 11: Figure neutrophil chemotaxis
Integrin
detachment
Integrin
attachment
MTOC
MTOC
Ca flicker
2+
High
Ca
2+
High
Ca
2+
Low
Ca
2+
Low
Ca
2+
fMLP gradient
fMLP
receptor
Myosin II
P2Y2
Adenosine
Hemichannels
Uropod
BACK
FRONT
Actin
Microtubules
Microtubule
Uropod
contraction
Actin
assembly
Top-view
Side-view
ATP
ATP
ATP
ATP
A3
Pseudopod
Pseudopod
Structural organization of neutrophil motility and chemotaxis.
The top view shows a neutrophil moving up a gradient of a chemoattractant such as fMet-Leu-Phe (fMLP). The exploratory pseudopod at the front has
an extensive actin meshwork. The actin and myosin II located at the rear enables the uropod to contract to propel the cell forwards. The microtubule-
organizing centre (MTOC) aligns the microtubules in the direction of movement and helps to stabilize cell polarity. The side view illustrates how polarity
is established by fMLP acting on receptors to promote actin assembly and pseudopod formation at the front and contraction of the uropod at the rear.
During movement, integrins attach at the front and detach at the rear. The yellow halo at the front represents a local concentration of ATP released from
the cell through hemichannels. The ATP and its hydrolytic product adenosine feed back in an autocrine manner to activate P2Y2 and A
3
purinergic
receptors that function to amplify the chemotactic response. Information on the standing Ca
2 +
gradient (see the red shading showing the high level
at the back and the low level at the front) and the Ca
2 +
ickers was taken from Wei et al. (2009).
Early gradient-sensing mechanisms (setting the com-
pass)
Amplication of early polarity signalling
Actin assembly, pseudopod formation and uropod con-
traction
Early gradient-sensing mechanisms (setting the
compass)
One of the most impressive aspects of chemotaxis is the
way the various signalling components are polarized in
order to direct different motile processes such as actin
assembly at the front and actin/myosin II assembly and
contraction at the back of the cell. In Module 11: Fig-
ure neutrophil chemotactic signalling, the major signalling
components have been positioned as near as possible to
their functional locations within the cell. An expression of
the polarity of signalling components is the location of the
Rho family (Cdc42, Rac and Rho) of monomeric G pro-
teins. Cdc42 and Rac are active at the front, whereas Rho
functions at the back. Just how this differential activation
of G proteins is set up may hold the key to understanding
the gradient sensing mechanism that establishes the com-
pass. One hypothesis considers that a G protein signalling
and chemotactic orientation mechanismprovides the com-
pass. The proposed polarized function of these G proteins
is illustrated within the yellowarrowin Module 11: Figure
neutrophil chemotactic signalling). Another suggestion is
that a relationship between Ca
2 +
signalling microdomains
and chemotactic orientation is the basis for the compass.
Migrating neutrophils have unusual Ca
2 +
signals. In ad-
dition to a standing gradient of Ca
2 +
, which is high at the
rear and low at the front (see the red shading of the cells in
Module 11: Figure neutrophil chemotaxis), there also are
localized Ca
2 +
ickers that are restricted to the front of
the cell. The latter may play a role in cell orientation.
These two mechanisms, G protein or Ca
2 +
signalling,
are not mutually exclusive and they may interact with each
other to provide a robust orientation system to guide cells
as they migrate along a chemotactic gradient.
G protein signalling and chemotactic orientation
mechanism
The fMet-Leu-Phe (fMLP) receptor, which sets up the
compass, is a typical G protein-coupled receptor (GPCR)
that acts through heterotrimeric G proteins (Module 2:
Figure heterotrimeric G protein signalling). Another ex-
pression of the signalling polarity that develops during
chemotaxis is the fact that fMLP receptors at the front
are coupled to G
i
, whereas those at the back operate
through G
12/13
(Module 11: Figure neutrophil chemotactic
signalling). Just why fMLP receptors at the front couple
to G
i
, whereas those at the back select G
12/13
is a mystery
and is clearly something that must be closely linked to set-
ting up the compass because so many other events follow
from the differential activation of the different G proteins
at either end of the cell. Signalling events at the front will
be considered rst before turning to what happens at the
back end.
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15
Module 11: Figure neutrophil chemotactic signalling
PI3,4P
2
Cofilin
Myosin II
M
y
o
s
in
II
+
Integrin
detachment
Uropod
contraction
12/13
I
P
PIP
3
PIP
3
PIP
3
PIP
3
PI4,5P
2
fMLP
PI 3-K
S
H
IP
1
WAVE
Arp2/3
Arp2/3
+
+
+
+
Actin
assembly
Compass ?
Cdc42
GTP
WASP
Cdc42
Rac
GDP
GEFs
+
+
+
+
+
+
-
Rac
fMLP
FRONT BACK
ROK
Rho
Rho
Rho
GTP
GDP
+
p115
RhoGEF
p190
RhoGAP
Matrix
Smurf1
Integrin
attachment
PAK
-
Neutrophil chemotactic signalling mechanisms.
Chemotaxis of neutrophils in response to a gradient of a chemoattractant such as fMet-Leu-Phe (fMLP) that elicits different signalling mechanism at
the front and back. The large yellow arrow encloses those signalling components that may function as the compass responsible for setting up the
polarity that drives directed cell movement. At the front, fMLP acts through guanine nucleotide exchange factors (GEFs) to activate both Cdc42 and
Rac. The activated Cdc42 and Rac initiates an amplication loop by stimulating phosphoinositide 3-kinase (PI 3-K) that establishes a high level of the
lipid second messenger PtdIns3,4,5P
3
(PIP
3
) in the front of the cell. The localized PIP
3
together with Cdc42 and Rac, then act together to assemble
actin in the pseudopod. At the rear of the cell, the fMLP receptor activates Rho that then stimulates the Rho kinase (ROK) to induce contraction of the
uropod.
When fMLP binds to its receptor, it dissociates the G
protein into its two components G
i
and G. The func-
tion of the former is unclear, but the G
subunit seems to
play a critical role in chemotaxis where it has two actions:
it can activate the guanine nucleotide exchange factors
(GEFs) that switchonthe monomeric Gproteins andit can
also activate PtdIns 3-kinase (Module 2: Figure heterotri-
meric G protein signalling). The Cdc42 signalling mech-
anism (Module 2: Figure Cdc42 signalling) and the Rac
signalling mechanism (Module 2: Figure Rac signalling)
are particularly active at the front of the cell. For example,
this G-dependent activation of Cdc42 GEFs such as -
Pix (Module 2: Table monomeric G protein toolkit) con-
vert inactive Cdc42.GDP into active Cdc42.GTP, may be
an essential part of the compass. A similar mechanism is
responsible for producing active Rac.GTP, which drives
actin assembly, as described later.
Different signalling mechanisms operate at the back of
the cell to activate the monomeric G protein Rho (Module
11: Figure neutrophil chemotactic signalling). The fMLP
receptors dissociate the heterotrimeric G
12/13
into the two
components G
12/13
and G. The G
12/13
then activ-
ates the p115-RhoGEF (Module 2: Table monomeric G
protein toolkit) to convert inactive Rho.GDP into active
Rho.GTP. The fact that Rhoactivityis connedtothe back
may depend not only on its activation through G
12/13
at
the back, but also on the fact that it is activity seems to
be switched off by Cdc42 and Rac at the front. A number
of mechanisms have been proposed for this inhibition of
Rho. For example, Rac may inhibit Rho by stimulating
the p190-RhoGAP that inactivates Rho.GTP by promot-
ing the hydrolysis of GTP to form the inactive Rho.GDP.
On the other hand, the degradation of Rho in the front of
the cell may be facilitated by Cdc42. The Cdc42 located
in the front of the cell interacts with the E3 ubiquitin li-
gase Smad ubiquitin-regulatory factor 1 (Smurf1), which
then promotes ubiquitination and degradation of Rho in
the front of the cell thus leaving high levels of Rho at the
back. Smurf1 is a component of the Smad signalling toolkit
(Module 2: Table Smad signalling toolkit).
Although the polarization of early signalling events
leading to Cdc42 and Rac activation at the front and Rho
at the back appear to be enough to initiate the gradient
sensing mechanism, they are not sufcient to drive the
large-scale morphological changes necessary to drive cell
motility. The next step appears to be an amplication of
early polarity signalling.
Ca
2 +
signalling microdomains and chemotactic
orientation
Neutrophils may use Ca
2 +
signalling as an orientation
mechanism. There are two components to neutrophil
Ca
2 +
signalling. First, migrating neutrophils have a stand-
ing gradient of Ca
2 +
with low concentrations at the lead-
ing end and higher concentrations at the rear end (Module
11: Figure neutrophil chemotaxis). The latter may play a
C
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Module 11
r
r
16
role in driving the cytoskeletal events that occur at the back
to trigger uropod contraction to drive the cell forwards in
the direction of the gradient (Module 11: Figure neutrophil
chemotactic signalling). Secondly, there are brief localized
pulses of Ca
2 +
called ickers that are another example of
an elementary Ca
2 +
event. These localized Ca
2 +
ickers
depend on Ca
2 +
entering the cell through TRPM7 chan-
nels, which are thought to be stretch-activated channels
that open when the membrane is deformed as occurs at
the leading edge where the membrane is being thrown
out to form the pseudopods. The TRPM7 channels gate a
small amount of Ca
2 +
that is then amplied by activating
the type 2 Ins 1,4,5P
3
receptors (Ins1,4,5P
3
R2s) respons-
ible for generating these ickers. When the direction of
the chemotactic gradient is altered, the ickers are concen-
trated on the side of the cell that turns towards the gradient
suggesting that these local pulses of Ca
2 +
may play a role
in cell orientation.
Neutrophils express P2Y2 receptors, which are activ-
ated by ATP that is released from the front of the cell
(Module 11: Figure neutrophil chemotaxis), are coupled
to G
q/11
that normally stimulates phospholipase C (PLC)
to form inositol 1,4,5-trisphosphate (InsP
3
) and diacylgly-
cerol (DAG). This formation of InsP
3
may thus be import-
ant in maintaining this Ca
2 +
-mobilizing messenger at a
level that will keep the Ins1,4,5P
3
R2s sufciently sensitive
to respond to the small pulses of trigger Ca
2 +
introduced
by the TRPM7 channels. This possibility is consistent with
the observation that the deletion of P2Y2 receptors causes
a marked loss in gradient sensing (see the right-hand panel
in Module 11: Figure purinergic chemotaxis).
Amplication of early polarity signalling
Amplication of the early gradient-sensing signals seems
to be an important feature of neutrophil chemotaxis. At
least two amplication steps have been identied. One of
these steps depends on the rapid and polarized formation
of the lipid second messenger PtdIns3,4,5P
3
at the leading
edge. The other is the release of ATP, which is hydrolysed
to adenosine, and these two purinergic stimuli feedback
in an autocrine manner to enhance the early chemotactic
signals (Module 11: Figure neutrophil chemotaxis).
One of the earliest indicators of polarity in neutro-
phils, which are responding to a chemoattractant gradi-
ent, is the rapid formation of the lipid second messen-
ger PtdIns3,4,5P
3
(PIP
3
in Module 11: Figure neutrophil
chemotactic signalling) at the front of the cell. While G
is able to induce some activation of the PtdIns 3-kinase,
the real surge in the formation of PIP
3
seems to depend
upon various positive-feedback loops. For example, there
are indications that Rac.GTP can activate PtdIns 3-kinase
and this will lead to further formation of PIP
3
, which in
turn will lead to more Rac.GTP formation. Also there are
indications that once actin starts to be formed at the lead-
ing edge, it can recruit PtdIns 3-kinase to induce more
PIP
3
formation, resulting in a large accumulation of this
signalling lipid in the membrane at the front of the cell.
Although these various feedback mechanisms need to be
dened more precisely, there are indications that the early
appearance of PIP
3
localized to the leading edge is a critical
feature of chemotaxis.
This localized build up of PIP
3
at the front of the cell
is very dependent on the activity of one of the type II
inositol polyphosphate 5-phosphatases called Src homo-
logy 2 (SH2) domain-containing inositol phosphatase 1
(SHIP1), whichhydrolyses PIP
3
. As the PIP
3
formedat the
front diffuses within the plane of the membrane towards
the rear of the cell, it is hydrolysed, and this helps to main-
tain the sharp anterior/posterior gradient of this signalling
lipid. If SHIP1 is knocked out in mice, PIP
3
increases
throughout the membrane, and there is less evidence of
cellular polarity(Module 11: Figure SHIP1 andPIP
3
polar-
ity). The SHIP1
/
neutrophils were not able to polarize
correctly; they retained their spherical shape and appeared
to put out pseudopodia in all directions. When their po-
sition and shapes were recorded over a period of 800 s,
it is clear that the wild-type (WT) cells have an elongated
shape and moved in a direct line, whereas the SHIP
/
cells are much more rounded and move very little (see
panel C in Module 11: Figure SHIP1 and PIP
3
polarity).
Since the SHIP
/
cells can still display some polarity, it
seems that the polarized distribution of PIP
3
may not be
so important in setting up the compass, but it is critical
for the mechanism that translates this polarity signal into
directional movement through actin assembly, pseudopod
formation and uropod contraction.
Another major amplication step depends upon the re-
lease of ATP from hemichannels that are activated by the
membrane deformation of the pseudopods at the front of
the cell (Module 11: Figure neutrophil chemotaxis). Some
of the ATP is hydrolysed by ectoenzymes such as CD39
and CD73 to form adenosine. In this way, the migrat-
ing cell sets up its own chemotactic gradient that contrib-
utes to both the gradient sensing and motility components
of chemotaxis. Both ATP and adenosine feed back in an
autocrine manner to stimulate G protein-coupled recept-
ors (GPCRs). ATP and adenosine appear to have slightly
different effects on the chemotactic response. ATP, which
acts through P2Y2 receptors, contributes to gradient sens-
ing whereas adenosine that act through A
3
receptors en-
hance cell motility (Module 1: Table G protein-coupled
receptors).
The P2Y2 receptors, which are activated by ATP, are
coupled to G
q/11
that normally stimulates phospholipase
C (PLC) to form inositol 1,4,5-trisphosphate (InsP
3
) and
diacylglycerol (DAG) (Module 2: Figure InsP
3
and DAG
formation). It is not clear yet whether these two second
messengers function in neutrophil chemotaxis. Certainly,
the formation of InsP
3
would contribute to the Ca
2 +
sig-
nalling microdomains and chemotactic orientation mech-
anism by enhancing the likelihood of generating the Ca
2 +
ickers (Module 11: Figure neutrophil chemotaxis). The
dissociation of G
q/11
into G
q/11
and G could also be
signicant because the G subunit could feed into the
putative compass to augment the subunits introduced
by the fMet-Leu-Phe (fMLP) receptor (Module 11: Figure
neutrophil chemotactic signalling). Each of these possib-
ilities would be in line with the observation that the de-
letion of P2Y2 receptors causes a marked loss in gradient
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17
Module 11: Figure purinergic chemotaxis
Disruption of chemotaxis by deletion of P2Y2 and A
3
purinergic receptors.
The rst panel shows how wild-type (WT) mouse neutrophils migrate in a directed way towards a source of fMet-Leu-Phe (fMLP). The middle panel
shows that neutrophils lacking the adenosine A
3
receptor (A3
/
) retain a sense of direction, but their motility is severely impaired. On the other
hand, deletion of the P2Y2 receptor (P2Y2
/
) does not affect motility, but there is a loss of gradient sensing. Reproduced from Chen, Y., Corriden,
R., Inoue, Y., Yip, L., Hashiguchi, N., Zinkernagel, A., Nizet, V. Insel, P.A., and Junger, W.G. (2006) ATP release guides neutrophil chemotaxis via P2Y2
and A3 receptors. Science 314:17921795; with permission from AAAS; http://www.sciencemag.org; see Chen et al. 2006.
sensing (see right hand panel in Module 11: Figure pur-
inergic chemotaxis).
Adenosine has a different action to ATP in that it seems
to contribute to cell motility. Deletion of the adenosine
A
3
receptors does not effect gradient sensing, but it does
markedly reduce motility (see the middle panel in Module
11: Figure purinergic chemotaxis). The A
3
receptors are
not uniformly distributed over the surface, as are the P2Y2
receptors, but are concentrated at the anterior end. Just
how this receptor polarity is established is unclear. There
also is little information on the mode of action of these A
3
receptors, which are known to be coupled to G
i
, i.e. the
same G protein used by fMLP. It would be interesting to
learn more about the action of these P2Y2 and A
3
recept-
ors, as this could provide more insights into the signalling
mechanisms that control chemotaxis.
Actin assembly, pseudopod formation
and uropod contraction
The polarization of signalling elements into the front and
back of the cell, as described above, provides the localized
instructions to protrude a pseudopod at the front and to
contract the uropod at the rear (Module 11: Figure neutro-
phil chemotactic signalling). The activation of Cdc42 and
Rac at the front of the cell orchestrate the assembly of an
actin network that contributes to protruding the pseudo-
pod. The ways in which the Cdc42 signalling mechan-
isms function to assemble actin are described in Module
2: Figure Cdc42 signalling. Similarly, the ways in which
the Rac signalling mechanisms function to assemble actin
are described in Module 2: Figure Rac signalling. These
two G proteins have subtly different actions on the pro-
cess of actin remodelling (Module 4: Figure actin remodel-
ling). Cdc42.GTP stimulates Wiskott-Aldrich syndrome
protein (WASP) and actin-related protein 2/3 complex
(Arp2/3 complex) to form long actin laments, whereas
Rac.GTP favours the formation of branches (Module 11:
Figure neutrophil chemotactic signalling). BothCdc42 and
Rac activate p21-activated protein kinase (PAK), which
is highly concentrated in the front of the cell where it
has a number of functions. It acts through LIM kinase
1 (LIM-K1) to phosphorylate colin, thus preventing it
from cutting actin so that assembly can occur. PAK also
phosphorylates myosin light chain kinase (MLCK), res-
ulting in a decrease in the activity of the myosin II motor
at the front of the cell. During movement, integrins attach
at the front and detach at the rear. Actin laments together
with myosin form the cables that attach to the integrin
receptors and it is important that this contractile system
remains quiescent at the front of the cell. Activation of
Cdc42 and Rac thus ensures that actin assembly is rapid
at the front of the cell to facilitate the protrusion of the
pseudopod and to provide stable attachments.
Different cytoskeletal events occur at the back to trig-
ger contraction of the uropod to drive the cell forwards
in the direction of the gradient. This local contraction is
controlled by Rho, which is activated at the back during
the operation of the early gradient-sensing mechanisms
(setting the compass). There are a number of Rho sig-
nalling mechanisms (Module 2: Figure Rho signalling). In
the case of neutrophil chemotaxis, the main function of
Rho.GTP is to activate contraction by stimulating Rho
kinase (ROK) (Module 11: Figure neutrophil chemotactic
signalling). ROK induces a signalling cascade that results
C
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Module 11
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r
18
in the activation of non-muscle myosin IIA (NMIIA) to
trigger contraction of the uropod.
Insummary, the localizedactionof Cdc42 andRac at the
front assembles the actin network that forms the pseudo-
pod. While at the back of the cell, Rho acts to stimulate
the myosin IIactin network to contract the uropod to
propel the cell forward. A challenge for the future is to
determine the exact nature of the signalling mechanisms
that constitutes the compass that orchestrates these direc-
ted motile mechanisms that are responsible for neutrophil
chemotaxis.
Senescence
Senescence is a process whereby cells lose the ability to
proliferate. They can survive for a prolonged period in a
state of suspended animation with regard to cell division.
There are two main states of senescence that are arrived at
by separate mechanisms (Module 11: Figure senescence).
There is replicative senescence, which depends upon the
loss of telomeres, and stress-induced senescence, which is
produced by various cell stresses. One of these stresses is
caused by the activation of oncogenes that are potent activ-
ators of stress-induced senescence. Senescence-associated
-galactosidase (SA--gal) is a biochemical marker used
to detect senescence.
Replicative senescence
Normal mammalian cells have a nite capacity to divide,
as was rst recognized when cells were placed in culture.
For example, when human cells from a young individual
are placed in culture, they will divide about 50 times before
stopping. Cells from older individuals will divide less.
The number of divisions appears to be determined by
the telomeres located at the ends of the chromosomes.
These telomeres, which are essential for DNA synthesis,
are made up of approximately 20 repetitive TTAGGG
tracts followed bya single strandoverhang onthe 3
G-rich
strand. Since these telomeres shorten during each round
of cell division, the number of repeats decline to a point
where DNA synthesis is no longer possible and the cell
enters a state of replicative senescence (Module 11: Figure
senescence).
Such replicative senescence is avoided by stem cells or
bycancer cells bythe expressionof the enzyme telomerase,
which is able to maintain the telomeres by replicating the
telomere tracts. One of the prerequisites for a cancer to
develop is therefore the expression of telomerase, thus en-
abling the cancer cell to divide repeatedly without running
the risk of replicative senescence.
Stress-induced senescence
As the name implies, stress-induced senescence is activated
by various cell stresses such as DNA damage or through
the appearance of oncogenes that begin to activate prolif-
eration. The latter is particularly relevant to the onset of
cancer, because such stress-induced senescence is an im-
portant mechanism to suppress the early stages of cancer.
For example, this form of senescence has now been de-
scribed in a number of precancerous tissues such as nevi
(skin moles) and early-stage prostate abnormalities.
Stress-induced senescence is activated by components
of the proliferative pathways that normally act to stimu-
late proliferation. This apparent paradox of the same sig-
nalling mechanisms being able to activate both senescence
and proliferation can probably be resolved by the fact
that these two outcomes may have different thresholds.
Normal levels of signalling can activate the proliferative
pathways that lead to cyclin D activation (Module 9: Fig-
ure proliferation signalling network), whereas abnormally
high signals are required to activate the pathways that de-
ect the cell towards stress-induced senescence (Module
11: Figure senescence).
There appear to be two main signalling pathways that
are activated by excessive proliferative signalling to switch
on stress-induced senescence. One pathway depends upon
the tumour suppressor p16
INK4a
that is activated by the
E2F/retinoblastoma protein (Rb) complex. The onset of
senescence may be facilitated by Suv39h1, which binds to
Rb and methylates histones to enhance formation of het-
erochromatin that will silence DNA. The other pathway
depends upon the tumour promoter alternative reading
frame (ARF) that activates p53. The latter plays a key role
in preventing cancer, since it can drive cells towards either
senescence or apoptosis (Module 9: Figure proliferative
signalling network).
Immortalization is the term given to a process whereby
cultured cells escape senescence and acquire the ability to
growin culture indenitely. The frequency of spontaneous
immortalization is species-specic, being very efcient in
rodent cells, but occurs rarely in human and avian cells.
There appear to be a limited number of genes that change
during immortalization. An alteration in p53 is often as-
sociated with immortalization.
Autophagy
Autophagy, whichmeans self-eating, has a number of func-
tions in both normal cellular physiology and in various
pathological processes, especially those related to nutrient
starvation and hypoxia. Its normal day-to-day role is to re-
move both damaged organelles and proteins. Autophagy
is also activated by various stressful stimuli and most at-
tention will focus on how this process is activated by a
decline in energy metabolism due to a loss of nutrients or
hypoxia.
Cell survival depends critically on a continuous sup-
ply of energy. There is a metabolic energy network that
manages the energy status of the cell (Module 7: Figure
metabolic network). During periods of starvation, this en-
ergy network can be maintained for a considerable period
by using energy stored in reserves such as fat and gly-
cogen. However, when these easily accessible reserves
are depleted, survival can be prolonged by a process of
autophagy whereby the cell begins to catabolize its cyto-
plasmic components (Module 11: Figure autophagy). The
autophagic process depends on an orderly sequence of
events that begins with the process of induction whereby
a small region of the endoplasmic reticulum (ER) and
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Module 11
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Module 11: Figure senescence
Cancer
Inactivation of
tumour suppressors
Normal
cell
ARF MYC
Ras E2F/Rb p16
p53
Replicative
senescence
Stress-induced
senescence
Oncogene
activation
Telomere
loss
Telomerase
expression
Mechanisms of cell senescence.
Normal cells can transforminto a non-proliferating senescent state through two main mechanisms. Loss of telomeres results in replicative senescence.
A variety of cellular stresses, including the activation of oncogenes, results in stress-induced senescence. The latter is induced by two main
pathways. Oncogenes such as Ras and Myc can activate tumour suppressors, such as p16 and alternative reading frame (ARF), that act through
E2F/retinoblastoma protein (Rb) and p53 respectively to divert cells into stress-induced senescence. These senescence pathways are avoided or
switched off during the development of cancer cells. The expression of telomerase avoids the replicative senescence pathway, whereas the inactivation
of the tumour suppressors such as Rb, p16 or p53 prevents the emerging cell from being diverted into stress-induced senescence.
perhaps also the mitochondria begin to form a cup-like
bud, which is dened by a local accumulation of the phos-
pholipid PtdIns3P. After this cup detaches itself from the
ER/mitochondria, a process of vesicle nucleation enlarges
this cup to form a membrane envelope that engulfs large
volumes of the cytoplasm containing organelles such as
ribosomes and mitochondria to form an autophagic va-
cuole. This autophagic vacuole then fuses with lysosomes
to form an autophagosome, where all the macromolec-
ular components are broken down into metabolites that
can be fed to the mitochondria to provide ATP for sur-
vival. Much of the control of autophagy is carried out by
a large family of proteins coded for by autophagy-related
genes (Atg). With regard to cell signalling, therefore, the
problem is to understand how the signalling pathways
that have been implicated in the control of autophagy
have an impact on the function of this ensemble of Atg
proteins.
Autophagy is controlled by a number of signalling path-
ways (Module 11: Figure autophagy). The major control
is carried out by the target of rapamycin (TOR), which
is regulated both by growth factors and by the energy
state of the cell (Module 9: Figure target of rapamycin
signalling). Growth factors acting through class I PtdIns
3-kinase (PI 3-K) stimulate the PtdIns 3-kinase signalling
pathway (Module 2: Figure PtdIns 3-kinase signalling) to
maintain the ability of TOR to inhibit autophagy. As part
of this mechanism, the PtdIns3,4,5P
3
-dependent activa-
tion of PKB inhibits the TSC1/TSC2 complex that serves
to remove the inhibition of Rheb that is responsible for
the activation of TOR (Module 11: Figure autophagy).
Under conditions where energy becomes limiting, the
inhibitory effect of TOR declines and autophagy begins.
Many of the factors that induce autophagy act by reducing
TOR activity through various mechanisms. An important
signal for autophagy is a decrease in the cells energy state
and this is transmitted through the AMP signalling path-
way (Module 2: Figure AMPK control of metabolism).
One reection of energy state is the level of essential amino
acids that enter the cell through specic carriers. Glutam-
ine enters through a high-afnity transporter SLC1A5 and
its build up within the cell is then used by the heterodi-
meric bidirectional transporter SLC7A5/SLC3A2 to ex-
change this glutamine for essential amino acids (Module
11: Figure autophagy). The presence of essential amino
acids acts through the GTPase Rag family, which are a
members of the Ras family of small monomeric G pro-
teins (Module 2: Table monomeric G protein toolkit). A
RagB/RagD heterodimer interacts with TOR and directs
it to the surface of the late endosomal/lysosomal compart-
ment where TOR activity is maintained to inhibit auto-
phagy. The amino acids also act to inhibit the activity of
AMPK, but when essential amino acids are limiting this
inhibition is reversed and AMPK can activate TCS2 to
inhibit Rheb and TOR to induce hypertrophy. A similar
AMPK-dependent mechanism occurs when the level of
ATP falls since the resulting increase in AMP stimulates
AMPK resulting in the inhibition of TOR through the
same sequence of events just described for the decline in
essential amino acids.
The next question to consider is how TOR acts to reg-
ulate the induction of autophagy by acting on members of
C
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Module 11
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20
Module 11: Figure autophagy
TOR
Rheb
TSC1
PKB
PI3K
AMPK
ATP
SLC7A5 SLC1A5
SLC3A2-
L-Glutamine
Essential
amino acids
L-Glutamine
TSC2
Growth
factors
_ _
_ _
_ _
_ _
_ _
PIP
3
PIP
PIP
2
2
+
+
+
+
+
+
+
+
+
+
+
+
+
2+
Ca
InsP
3
PLC AC
Ras
Cyclic
AMP
PtdIns3P
B
e
c
l
i
n

1
InsP R
3
4 3 S P V h
CaMKK
P
R
a
b
5
Rap2B
EPAC
ULK1-2
Atg13
FIP200
RagD
Autophagic
vacuole
Lysosome
Degradation
Induction
Elongation
Vesicle
nucleation
RagB
Control of autophagy.
Autophagy is the large-scale degradation of cytoplasmic components. During an induction process, a membrane protrusion buds off from the ER
and begins to enlarge (vesicle nucleation) to form a cup-shaped vesicle that then elongates and breaks away from the ER to form an isolation
membrane that engulfs mitochondria and ribosomes to form an autophagic vacuole, which then fuses with a lysosome where degradation occurs.
These membrane events are controlled by a variety of signalling pathways many of which are channelled through the target of rapamycin (TOR) to
activate the induction processes. Another pathway depends on various signalling processes that are directed towards the class III PI 3-kinase hVPS34
to generate PtdIns3P that acts to control the process of vesicle nucleation. For further details of the way these signalling pathways control autophagy
see Module 11: Figure autophagy signalling mechanisms.
the Atg family. In the case of the early induction step, the
action of TOR is to control three components of this Atg
family: ULK1, Atg13 and FAK family interacting protein
of 200 kDa (FIP200) (Module 11: Figure autophagy sig-
nalling mechanisms). Under nutrient-rich conditions, act-
ive TOR phosphorylates and inactivates ULK1 and Atg13
to inhibit autophagy. When nutrients decline and TOR
is inactive, these inhibitory phosphates are removed and
the ULK1/Atg13/FIP200 complex is able to initiate auto-
phagy by activating the initial induction step when mem-
brane is removed from the ER to initiate the formation of
the autophagic vacuole. This early membrane trafcking
step is also facilitated by Atg14Lthat acts together with the
integral membrane protein p150 to stimulate the class III
PI 3-Kinase hVPS34 to produce the PtdIns3P that denes
the ER membrane that is being budded off to form the
autophagic vacuole (Module 2: Figure localized inositol
lipid signalling). This hVPS34/p150 complex is then car-
ried over to function in the next phase of vesicle nucleation
and elongation.
Vesicle nucleation is driven by a large macromolecu-
lar complex that depends on the formation of PtdIns3P
by hVPS34. This lipid is a messenger that operates in the
PtdIns3P signalling cassette that functions in the control
of a number of vesicle trafcking processes. During this
vesicle nucleation process, hVPS34 can be activated by
two mechanisms. First, Rab5 interacts with and stimu-
lates hVPS34. Secondly, another member of the Atg family
called Beclin-1 is an important component of a large reg-
ulatory complex that also controls the activity of hVPS34
(Module 11: Figure autophagy). A number of proteins
bind to Beclin-1 to regulate its activity both positively
and negatively. The positive regulators include activat-
ing molecule in Beclin-1 regulator 1 (ambra-1), Rubicon
and UV radiation resistance-associated gene protein (UV-
RAG) (Module 11: Figure autophagy signalling mechan-
isms). Beclin-1 inhibition depends on its interaction with
the InsP
3
receptor (InsP
3
R) through a mechanism that is
modulated by a number of other proteins such as Bcl-2 and
by nutrient-deprivation autophagy factor-1 (NAF-1). The
ability of Beclin-1 to inhibit hVPS34 depends on its inter-
action with Bcl-2, NAF-1 and the InsP
3
R. Phosphoryla-
tion of Bcl-2 by JNK1 causes Beclin-1 to dissociate from
Bcl-2 resulting in the activation of hVPS34 and the form-
ation of the PtdIns3P necessary to activate vesicle nucle-
ation. One of the functions of PtdIns3P is to recruit the
early autophagic protein WPP-domain interacting protein
1 (WIP-1), whichis a serine/threonine proteinphosphatase
that is also known as protein phosphatase 1D magnesium-
dependent (PPM1D). The function of PtdIns3P in vesicle
nucleation in autophagy is very reminiscent of endosome
vesicle fusion to early endosomes that occurs during en-
docytosis (Module 4: Figure endosome vesicle fusion).
The myotubularin MTMR14, which is also known as
Jumpy, inactivates the messenger function of PtdIns3P by
hydrolysing it back to PtdIns (Module 11: Figure auto-
phagy signalling mechanisms). Inactivation of MTMR14
enhances the onset of either basal or starvation-induced
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Module 11
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Module 11: Figure autophagy signalling mechanisms
TOR
ULK1-2
Atg13
FIP200
Autophagic
vacuole
Lysosome
Degradation
A
m
b
r
a
-
1
R
u
b
ic
o
n
_ _
Elongation
2+
Ca
InsP R
3
h
V
P
S
3
4
h
V
P
S
3
4
PtdIns3P
PE
PtdIns
p150
PtdIns
WIP-1
LC3
LC3-I
LC3-II
Atg12
Atg12
Atg12
Atg12
Atg4
Atg5
Atg5
Atg5
Atg16
Atg16
Atg16
Atg10
Atg3
Atg10
Atg7
Atg7
Beclin 1 N
A
F
-1
Bcl-2
M
T
M
R
1
4
JNK1
U
V
R
A
G
+
P
Rab5
Atg12 Atg5
Atg14L
p150
AMPK
+
Induction
Vesicle
nucleation
Autophagy signalling mechanisms.
A family of autophagy-related gene (Atg) products carry out the signalling mechanisms that activate autophagy. When the target of rapamycin (TOR) is
switched off, the ULK1/Atg13/FIP200 complex is activated to stimulate the induction of autophagy. Vesicle nucleation is activated by a macromolecular
complex that controls the hVPS34 responsible for forming PtdIns3P. Elongation depends on two ubiquitin-like pathways carried out by a series of Atg
proteins that result in the conjugation of the Atg protein LC3-I to phosphatidylethanolamine (PE) to form LC3-II. See the text for further details.
autophagy and thus highlights the signicance of PtdIns3P
invesicle nucleation. Amissense mutationinMTMR14 has
been identied in centronuclear myopathy (CNM).
Inadditiontoplaying a role inmodulating the activityof
hVPS34, the InsP
3
Ralso functions to release internal Ca
2 +
that may also play a role in controlling autophagy (Module
11: Figure autophagy). There are a number of mechanisms
for activating the phospholipase C isoforms to elevate the
level of InsP
3
(Module 2: Figure PLC structure and func-
tion). For example, formation of InsP
3
by phospholipase
C (PLC) has been implicated in the control of auto-
phagy. This PLC can be activated through Ras (Module 2:
Figure Ras signalling) or through the EPAC/Rab2b com-
ponents of the cyclic AMP signalling pathway (Module 2:
Figure cyclic AMP signalling). The InsP
3
-induced release
of Ca
2 +
has been implicated in a number of autophagy
control mechanisms. However, the proposed actions of
Ca
2 +
are somewhat contradictory in that Ca
2 +
has been
reported to be both stimulatory and inhibitory.
Such positive and negative actions of Ca
2 +
are not un-
common and often reect a dual role depending on the
location and concentration of Ca
2 +
. Such a dual mechan-
ism might explain its contradictory actions in autophagy
with normal lowlevels of Ca
2 +
having an inhibitory effect
andhigher levels associatedwithstress maystimulate auto-
phagy. For example, there are reports that a constitutive
release of Ca
2 +
from the ER may operate to inhibit auto-
phagy. The nature of this constitutive Ca
2 +
release by
the InsP
3
R is unclear, but it may depend on a continuous
formation of InsP
3
. This constitutive release of Ca
2 +
then
operates throughthe ER-mitochondrial shuttle (Module 5:
Figure ER/mitochondrial shuttle) to maintain mitochon-
drial energymetabolismandthe increase inATPformation
will reduce AMPK activation to prevent autophagy. The
intimate communication between the ER and mitochon-
dria means that this Ca
2 +
signalling is highly localized and
may not be detected by some of the other cellular Ca
2 +
-
sensitive processes. The other reports of Ca
2 +
being able
to activate autophagy may be explained through a more
global action of Ca
2 +
acting on another set of targets.
One of these stimulatory actions of Ca
2 +
is to act through
CaMKK to stimulate AMPK (Module 2: Figure AMPK
control of metabolism), which will result in inhibition of
TOR and the induction of autophagy. Another action of
Ca
2 +
is to stimulate hVPS34 to increase the formation of
the PtdIns3P that plays a role in driving the early mem-
brane trafcking events.
Following vesicle nucleation, the developing membrane
cup begins to elongate and this process seems to be reg-
ulated by two ubiquitin-like pathways carried out by a
series of Atg proteins (Module 11: Figure autophagy sig-
nalling mechanisms). The conjugation of Atg5 to Atg12 is
carried out by Atg7 (E1-like enzyme) and Atg10 (E2-like
enzyme). The Atg5 conjugate then interacts with Atg16
to form a large complex containing Atg5/Atg12/Atg16
tetramers. The other pathway begins with microtubule-
associated protein 1A/1B-light chain 3 (LC3) that is hy-
drolysed at its C-terminus by the protease Atg4B to
form LC3-I. The latter then undergoes a series of con-
jugation reactions carried out by Atg7 and Atg3 result-
ing in the LC3-I being attached to phosphatidylethan-
olamine (PE) to form LC3-II. The Atg5/Atg12/Atg16
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22
complex can also participate in the conversion of LC3-
II into LC3-III, which plays a role in the elongation
step.
Apoptosis
Apoptosis is particularly evident during development,
when unwanted cells are pruned during organogenesis.
For example, ngers and toes begin to form when cells are
selectively removed between developing digits. The devel-
oping nervous system also produces a large excess of both
neurons and oligodendrites, many of which are destroyed
as the neural circuitry is formed. Massive apoptosis oc-
curs in the developing immune system when autoreactive
T- and B cells are destroyed. The completion of devel-
opment does not mark the end of apoptosis, because it
continues to play a vital role in the adult organism during
the normal turnover of cells. For example, the human body
spawns several million newcells every second. If cell num-
bers are to remain constant, several million cells must die
every second. This massive turnover is necessary to replace
aged and damaged cells. For example, the cells of the hair
bulb die by apoptosis during the catagen phase of each
hair follicle cycle (Module 8: Figure hair follicle cycle).
Any distortion of this precise homoeostasis between cell
birth and cell death can have very severe consequences.
Autoimmune diseases and cancer are associated with a de-
crease in apoptosis. Conversely, an increase in apoptosis
occurs during neurodegenerative and neuromuscular dis-
eases, ischaemia/reperfusion damage during a stroke or
heart failure, and with various infectious diseases such
as AIDS and those caused by toxin-producing micro-
organisms.
The orderly programme of cell death is controlled by an
apoptotic signalling network. There are two major path-
ways for initiating apoptosis: an extrinsic pathway that
responds to external signals such as the Fas ligand (FasL)
and tumour necrosis factor (TNF), and an intrinsic
pathway where a process of Ca
2 +
-induced apoptosis is
induced following an alteration in the normal operation
of the endoplasmic reticulum (ER)/mitochondrial Ca
2 +
shuttle. These two pathways activate the caspase cascade
responsible for carrying out the orderly cell death pro-
gramme. The Bcl-2 superfamily, which has both pro- and
anti-apoptotic members, plays a major role in regulating
apoptosis, with much of its activity focused on the intrinsic
pathway where Ca
2 +
-induced apoptosis is an important
element. The Bcl-2 superfamily control of Ca
2 +
signalling
plays an important role in regulating apoptosis, and fea-
tures signicantly in the relationship between apoptosis
and cancer.
Control of apoptosis is regulated by both hormonal
and transcriptional processes. The hormonal modulation
of apoptosis is carried out by a number of the cell signalling
pathways activated by growth factors and hormones that
act either directly through the covalent modication of
pre-existing components of the apoptotic system (e.g. the
Bcl-2 superfamily) or by activating gene transcription to
alter the balance between the pro- and anti-apoptotic regu-
latory components. This balance is also regulated by tran-
scriptional processes activated during p53-induced apop-
tosis, which is one of the primary mechanisms used by this
tumour suppressor to destroy developing cancer cells.
Apoptotic signalling network
A complex apoptotic signalling network controls the fate-
ful decision of whether a cell survives or dies. The sig-
nalling system is made up of a number of discrete pro-
cesses that are linked together into an integrated network
(Module 11: Figure apoptosis):
1. Cytokines such as the Fas ligand (FasL) and tumour
necrosis factor (TNF) act on death receptors that
engage the extrinsic pathway, which has two ma-
jor outputs (Module 11: Figure TNF apoptotic sig-
nalling).
2. It activates caspase 8, which is one of the initiator
caspases of the caspase cascade.
3. It can also recruit the intrinsic pathway by activating
Bid, which is one of the pro-apoptotic members of the
Bcl-2 superfamily. This conversionof BidintotBidcan
also be carried out by cathepsin D that is activated by
ceramide during sphingomelin signalling (Module 2:
Figure sphingomyelin signalling).
4. The initiator caspases (e.g. caspases 8, 9 and10) activate
the executioner caspases (e.g. caspases 3, 6 and 7) that
are responsible for driving the processes of apoptosis.
5. The intrinsic pathway depends upon an inter-
action between the endoplasmic reticulum (ER)
and the mitochondria. These two organelles are
tied together through an endoplasmic reticulum
(ER)/mitochondrial Ca
2 +
shuttle, which seems to
play a major part in determining how Ca
2 +
functions
together with the Bcl-2 superfamily to regulate the
initiation of apoptosis. Both organelles can provide
output signals that can activate apoptosis. A process
of endoplasmic reticulum (ER) stress signalling gen-
erates a number of output signals, one of which is
the activation of caspase 12 (Module 2: Figure ER
stress signalling), which feeds into the caspase cascade.
However, most of the output signals fromthe intrinsic
pathway come from the mitochondria, which release
a variety of apoptotic factors, such as cytochrome c
and a second mitochondrial derived activator of cas-
pase (SMAC) that feed into the caspase cascade, and
apoptosis-inducing factor (AIF) and endonuclease G
(EndoG), which feed into the caspase-independent
pathway.
6. The Bcl-2 superfamily contains both pro- and anti-
apoptotic factors that play a major role in modulating
the intrinsic pathway.
7. Anumber of cell signalling pathways canmodulate the
apoptotic signalling network. This hormonal modu-
lation of apoptosis is carried out by a variety of clas-
sical cell signalling pathways or through various stress
stimuli that act by either modulating the performance
of pre-existing Bcl-2 family members or altering the
expression of pro- and anti-apoptotic factors.
8. Some of the signalling pathways modulate apoptosis
by adjusting the activity of the Bcl-2 superfamily [e.g.
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Module 11: Figure apoptosis
APOPTOSIS
Growth factors
Hormones
Cell signalling
pathways
PI 3-kinase
MAPK
ROS
NO
Apoptotic
signalsome
Extrinsic
pathway
Initiator
caspases
Executioner
caspases
Caspase
cascade
Caspase-
independent
cascade
Intrinsic pathway
2+
Ca
1
4
6
7
10
8
9
FAS-L
UV
p53
Ceramide
tBid
Bid
Neurotoxins
Casp 12, Cyto c, Smac
AIF, Endo G
Genotoxic
stress
Ischaemia
Stroke
TNF
ER-mitochondrial
calcium shuttle
Bcl-2
Bcl-xL
Bcl-w
Mcl-1
A1
Bfl-1
BHRF-1
Bax
Bak
Bad
Bid
Bim
Noxa
Puma
Anti-
apoptotic
Pro-
apoptotic
Apoptotic
factors
Bcl-2
Superfamily
3
2
5a
5b
ER
The apoptotic signalling network.
The onset of apoptosis is controlled by a number of interconnecting processes. Particular attention is focused on how these different processes
interact with each other as outlined in the text.
protein kinase B (PKB) phosphorylation of Bad; see
Module 11: Figure Bcl-2 family functions].
9. Some of the pathways can regulate the transcription
of components of the apoptotic signalsome. A par-
ticularly important aspect of this remodelling process
is that it can alter the balance between the pro- and
anti-apoptotic factors, thereby altering the sensitivity
of cells to apoptosis.
10. Genotoxic stress resulting in the activation of the tran-
scription factor p53, which functions as a tumour sup-
pressor by increasing the expression of a large number
of apoptotic factors (Module 4: Figure p53 function).
Extrinsic pathway
The extrinsic pathway is activated by external signals such
as the Fas ligand (FasL) and tumour necrosis factor
(TNF), which are typical cytokines (Module 1: Figure
cytokines). The FasL and TNF act through death rece-
ptors.
The DR6 receptor, which is another member of the su-
perfamily of tumour necrosis factor receptors (TNF-Rs),
functions to control apoptosis in neurons (see step 11 in
Module 12: Figure amyloid cascade hypothesis).
Death receptors
The best-characterized death receptors belong to the
tumour necrosis factor (TNF) receptor family that
also mediate inammatory responses (Module 1: Figure
cytokines). Members of this receptor family, such as the
TNF receptor (TNF-R) and Fas, have cysteine-rich extra-
cellular domains, while their cytosolic regions contain a
death domain (DD). When these death receptors engage
their ligands, they form trimers and are then able to activ-
ate various signalling pathways. One of these is the nuclear
factor B (NF-B) signalling pathway (Module 2: Figure
NF-B activation). A separate pathway is used to activate
the extrinsic pathway, as outlined in Steps 16 in Module
11: Figure TNF apoptotic signalling:
1. Apoptotic signals such as TNF and FasL bind to the
trimeric TNF receptor family (TNF or Fas) to form
a platform that then binds a variety of adaptor proteins
capable of relaying information to different signalling
pathways.
2. Activation of death receptors results in the recruitment
of adaptor proteins such as tumour-necrosis-factor-
receptor-associated death domain (TRADD) or Fas-
associated death domain (FADD) that bind to the death
domain (DD) on the trimeric TNF receptor.
3. The next event is the recruitment of pro-caspase 8 to
form the death-inducing signalling complex (DISC).
4. The pro-caspase 8 is cleaved to release caspase 8, which
can have two functions depending on the cell type. In
some cells, such as lymphocytes, there is a direct ac-
tivation of the caspases downstream of caspase 8 (Step
5). In other cells, such as hepatocytes and pancreatic
-cells, there is an additional caspase cascade amplic-
ation pathway that depends on the formation of tBid
(Step 6). The X-chromosome-linked inhibitor of apop-
tosis protein (XIAP) seems to play a prominent role in
determining which of these pathways is activated.
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Module 11: Figure TNF apoptotic signalling
APOPTOSIS
Intrinsic
pathway
Initiator
caspase
Executioner
caspases
1
6
7
8
4
5
TNF R
or
Fas
TNF
or
FasL
TRADD
or
FADD
Sphingomyelin
signalling
NF- B
signalling
Bid
cFLIP
Death-inducing
signalling
complex (DISC)
tBid
Pro-caspase 8
Caspase 8
3
2
D
D
D
D
D
D
D
E
D
D
E
D
D
E
D
D
E
D
D
E
D
D
E
D
D
E
D
D
D
Tumour necrosis factor (TNF) activation of the extrinsic apoptotic pathway.
Apoptotic ligands such as tumour necrosis factor (TNF) or Fas ligand (FasL) bind to members of the trimeric TNF receptor family (TNFR or
Fas) to initiate the extrinsic signalling cascade, as described in the text.
5. Apoptosis can be inhibited by cellular FLICE-inhib-
itory protein (cFLIP), which acts by binding to the
death-inducing signalling complex (DISC).
6. Caspase 8 then functions as an initiator caspase to feed
in to the executioner caspases. Through these rapid
proteinprotein interactions, the death receptors can
activate the caspase cascade within seconds, causing the
cell to die within hours.
7. Caspase 8 can also convert Bid into tBid, which feeds
into the intrinsic pathway. In addition, the ceramide
formed during sphingomyelin signalling can activate
cathepsin D that carries out a similar conversion of Bid
into tBid.
8. In addition to this direct fast-track signalling pathway
to the caspase cascade, these death receptors can also ac-
tivate other signalling systems, such as the NF-B sig-
nalling pathway (Module 2: Figure NF-B activation)
and the sphingomyelin signalling pathway (Module 2:
Cellular FLICE-like inhibitory protein (cFLIP)
Cellular FLICE-inhibitory protein (cFLIP), which is also
known as the Casp8 and FADD-like apoptosis regulator
and is encoded by the CFLAR gene, inhibits apoptosis by
binding to the death-inducing signalling complex (DISC)
that consists of a member of the TNF receptor family
(TNFR or Fas), an adaptor such as FADD or TRADD
and pro-caspase 8 (Module 11: Figure TNF apoptotic
signalling). Such a mechanism may act to prevent the ap-
optosis of B-cells in the germinal centre during the process
of B-cell differentiation in the lymph node (Module 8: Fig-
ure B cell maturation signalling). Resistance of breast can-
cer tumour cells to the TNF-related apoptosis-inducing
ligand (TRAIL) may depend on the expression of cFLIP.
The expression of cFLIP seems to be regulated by the
NF-B signalling pathway.
Intrinsic pathway
The mitochondria, whichare anessential component of the
intrinsic apoptotic pathway, harbour an array of apoptotic
factors. Release of these stored apoptotic factors from the
mitochondria are triggered by inputs fromeither signalling
pathways (e.g. ceramide fromthe sphingomyelinsignalling
pathway) or from various stresses (UV, DNA damage,
chemotherapeutic agents and neurotoxins) (Module 11:
Figure apoptosis). This intrinsic pathway is also respons-
ible for inducing apoptosis following cardiac ischaemia
or stroke when cells are overwhelmed by Ca
2 +
. Indeed,
Ca
2 +
-induced apoptosis is a signicant component of the
intrinsic pathway. The latter is also regulated by the Bcl-2
superfamily of apoptotic regulators.
In response to these various stimuli, apoptotic factors
are released into the cytoplasm, where they feed into the
caspase cascade to induce apoptosis (Step 5 in Module
11: Figure apoptosis). Two of the major factors released
from the mitochondria are cytochrome c and apoptosis-
inducing factor (AIF). The cytochrome c combines with
apoptosis-activating factor 1 (Apaf-1) in the cytoplasm to
form a large scaffolding complex called the apoptosome,
which draws in and activates pro-caspase 9, which then
C
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Module 11
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25
activates pro-caspase 3 (Module 11: Figure Bcl-2 family
functions).
Ca
2 +
-induced apoptosis
Intracellular Ca
2 +
is an important regulator of both cell
proliferation and apoptosis. This example of Ca
2 +
hav-
ing opposite effects in cells can be explained by the
way that this signal is presented in both time and space.
During normal signalling, Ca
2 +
is usually presented as
brief transients, where information is encoded in the
temporal properties of the transients (Module 6: Figure
encoding oscillatory information). Following each tran-
sient, there is a corresponding ux through the mito-
chondria through the operation of the endoplasmic retic-
ulum (ER)/mitochondrial Ca
2 +
shuttle (Module 5: Figure
ER/mitochondrial shuttle). Normally, most of the Ca
2 +
storedinthe cell resides inthe ER, except during signalling,
when some of the Ca
2 +
released from the ER takes up
temporary residence in the mitochondria. However, if this
equilibrium position is changed such that there is a large
transfer of Ca
2 +
from the ER to the mitochondria, a pro-
cess of Ca
2 +
-induced apoptosis is triggered through activ-
ation of the intrinsic pathway. A number of stress signals
can repartition this stored Ca
2 +
such that it moves from
the ERlumen into the mitochondrion. For example, inhib-
ition of the sarco/endo-plasmic reticulum Ca
2 +
-ATPase
(SERCA) pump by thapsigargin leads to a decrease in lu-
minal Ca
2 +
concentration and activation of endoplasmic
reticulum (ER) stress signalling. In addition, the shift of
Ca
2 +
from the ER into the mitochondrion leads to an el-
evation of Ca
2 +
in the mitochondrial matrix, which results
in activation of the mitochondrial permeability transition
pore (MTP). Another consequence of mitochondrial Ca
2 +
loading is to stimulate the generation of reactive oxygen
species (ROS), which have also been linked to formation
of the MTP (Module 5: Figure mitochondrial Ca
2 +
sig-
nalling).
The Bcl-2 superfamily control of Ca
2 +
signalling may
depend upon their ability to regulate the leak of Ca
2 +
from the ER, which will thus reduce the probability of
MTP opening because less Ca
2 +
will be transferred to the
mitochondria.
Bcl-2 superfamily
The Bcl-2 superfamily of apoptotic regulatory factors is
a primary regulator of the intrinsic pathway. This highly
dynamic internal regulatorysystemhas bothpro- andanti-
apoptotic family members (Module 11: Figure apoptosis).
Some of this dynamism depends upon the Bcl-2 super-
family domain structure and function, which is domin-
ated by the Bcl-2 homology (BH) domains that enable
family members to interact with each other (Module 11:
Figure Bcl-2 superfamily domain structure). Such Bcl-2
superfamily interactions enable the anti-apoptotic factors
to curb the activity of the pro-apoptotic factors, and the
balance between these two opposing factors is critical
in determining whether a cell lives or dies. This critical
balance can be altered through the hormonal modula-
tion of apoptosis (Step 7 in Module 11: Figure apop-
tosis), which operates either in the short term through
post-translational modications such as phosphoryla-
tion/dephosphorylation reactions, or in the longer term
by the transcriptional control of the Bcl-2 superfamily.
The Bcl-2 superfamily control of Ca
2 +
signalling is an-
other way in which these apoptotic regulators may control
apoptosis.
In addition to these regulatory functions, the Bcl-2 su-
perfamily may also have a structural role in that they may
contribute to the formation of channels in the outer mi-
tochondrial membrane (OMM) responsible for the release
of the apoptogenic factors.
Bcl-2 superfamily domain structure and function
The Bcl-2 superfamily has a domain structure that is dom-
inated by the Bcl-2 homology (BH) domain (Module 11:
Figure Bcl-2 superfamily domain structure). Many of the
family members also have a hydrophobic C-terminal re-
gion resembling a transmembrane (TM) domain, which
may assist in the attachment of these factors to the mem-
branes of the mitochondria and endoplasmic reticulum
where they exert their actions.
The superfamily is divided into the anti-apoptotic and
pro-apoptotic families. The anti-apoptotic factors, such as
Bcl-2 and Bcl-X
L
, have four BH domains and most have a
TM domain except for A1. The pro-apoptotic factors are
divided into the multidomain (BH) and BH3-only famil-
ies. Examples of the latter are Bad, Bid, Bim, Noxa and
Puma, which act as sensors to pick up information from
various inputs that are then relayed to the multidomain
(BH) factors such as Bak and Bax, which function as ef-
fectors responsible for the permeabilization of the outer
mitochondrial membrane (Module 11: Figure Bcl-2 family
functions). The importance of Bak and Bax, which have
been referred to as a gateway to the intrinsic pathway, is
evident fromthe fact that their removal renders cells resist-
ant to a variety of death signals, such as a Ca
2 +
overload,
ceramide and oxidative stress. It is not therefore surpris-
ing to nd that many of the other Bcl-2 family mem-
bers act in one way or another to modulate the activity
of these two pro-apoptotic factors. An important feature
of the modulatory function within the Bcl-2 superfamily
is the way they interact with each other through direct
proteinprotein interactions. The -helical BH3 domain
of one protein inserts into a specic binding site formed
by the BH1, BH2 and BH3 domains of another Bcl-2 fam-
ily member.
Bcl-2
B cell leukaemia-2 (Bcl-2) is a prominent anti-apoptotic
factor. Two mechanisms have been proposed to explain
this inhibitory action of Bcl-2. One mechanism depends
upon the ability of Bcl-2 to bind to pro-apoptotic factors
such as Bax, thereby preventing them from forming chan-
nels in the outer mitochondrial membrane (Module 11:
Figure Bcl-2 family functions). The second mechanism,
which is somewhat more controversial, depends upon
growing evidence of a Bcl-2 superfamily control of Ca
2 +
signalling.
The transcription factor microphthalamia-associated
transcription factor (MITF) regulates the expression of
C
r
Module 11
r
r
26
Module 11: Figure Bcl-2 superfamily domain structure
BH1
BH1
BH1
BH1
BH2
Bcl-2, Bcl-xL, Mcl-1
BH2
Bcl-w
A1
TM
Bcl-xS
BH2
BH2 Bak, Bax, Bok
Bim, Blk, Hrk, Puma
BH3
BH3
BH3
BH3
BH3
BH3
BH3 Bid, Bad
BH4
ANTIAPOPTOTIC
PROAPOPTOTIC
Multidomain (BH)
BH3-only
BH4
BH4
BH4
BH4
Domain structure of the Bcl-2 superfamily.
The function of the different domains is described in the text. Many members of the superfamily have a transmembrane (TM) domain that enables
them to insert into membranes.
Bcl-2 and this contributes to the survival of melanocytes
during melanogenesis (Step 5 in Module 7: Figure melano-
genesis).
One of the functions of Bcl-2 is to contribute to
inositol 1,4,5-trisphosphate receptor (InsP
3
R) modulation
(Module 3: Figure InsP
3
R regulation) and this may play
a role in a number of cellular processes and some neural
diseases:
Bcl-2 interacts with Beclin-1 to regulate the process of
autophagy (Module 11: Figure autophagy).
Bcl-2 gene single nucleotide polymorphisms (SNPs)
have been associated with a risk of developing manic
depressive illness.
There may be a protective role for Bcl-2 in Alzheimers
disease (AD). The calcium hypothesis of Alzheimers
disease (AD) suggests that memory loss may be caused
by an elevation of the resting level of Ca
2 +
and some
of this may result from an increase in InsP
3
-dependent
Ca
2 +
release (Module 12: Figure amyloids and Ca
2 +
signalling). The ability of Bcl-2 to reduce the symptoms
of Alzheimers disease (AD) may be explained by its
ability to reduce the release of Ca
2 +
by inhibiting InsP
3
receptors.
The expression of Bcl-2 is repressed by miR-15 and
miR-16. These two miRs are often deleted or down-
regulated in many cases of chronic lymphocytic leukaemia
(CLL).
Bcl-X
L
The Bcl-X
L
anti-apoptotic factor functions much like Bcl-
2 to inhibit apoptosis by curbing the activity of Bak
(Module 11: Figure Bcl-2 family functions).
C
r
Module 11
r
r
27
BH3-only family
Bad
Bad promotes apoptosis by binding to Bcl-X
L
, which is a
potent inhibitor of cell death. Following phosphorylation
by protein kinase B (PKB) or protein kinase A (PKA),
Bad binds instead to 14-3-3 protein, thereby releasing the
Bcl-X
L
that then inhibits apoptosis by binding to the pro-
apoptotic factor Bak(Module 11: Figure Bcl-2 familyfunc-
tions).
Bid
BH3-interacting domain death agonist (Bid) is con-
stitutively expressed in the cytoplasm, where it has a spe-
cic role inlinking together the extrinsic andintrinsic path-
ways (Step 3 in Module 11: Figure apoptosis). Caspase 8,
which is activated by the extrinsic pathway (Module 11:
Figure TNF apoptotic signalling), cleaves Bid to a C-
terminallytruncatedfragment (tBid), whichtranslocates to
the mitochondrion where it triggers the activation of Bak
and Bax by inducing the oligomerization and permeabiliz-
ation of the mitochondrial membrane (Module 11: Figure
Bcl-2 family functions). This conversion of Bid into tBid
can also be carried out by cathepsin D that is activated
by ceramide during sphingomyelin signalling (Module 2:
Bim
The Bcl-2 interacting mediator of cell death (Bim) acts
as a pro-apoptotic factor by binding to and neutralizing
anti-apoptotic factors such as Bcl-2 and Bcl-X
L
.
Noxa
The expression of Noxa is enhanced by the pro-apoptotic
transcription factor p53 (Module 9: Figure proliferation
signalling network).
Puma
Like Noxa, the expression of p53 up-regulated modulator
of apoptosis (Puma) is increasedbythe pro-apoptotic tran-
scription factor p53, and thus contributes to p53-induced
apoptosis (Module 9: Figure proliferation signalling net-
work).
Multidomain (BH) family
Bak
Bak is normally resident in the outer mitochondrial mem-
brane (OMM), where it exerts its pro-apoptotic function
by oligomerizing to form channels through which apop-
togenic factors such as cytochrome c are released. The
activity of Bak depends upon the balance of other pro-
and anti-apoptotic factors. Bak activity can be neutralized
by binding to Bcl-X
L
. If the Bak/Bcl-X
L
dimer is disrup-
ted by the removal of Bcl-X
L
by Bad, the monomeric Bak
can oligomerize through a process that can be facilitated
by tBid (Module 11: Figure Bcl-2 family functions).
Bax
(Bax) is a pro-apoptotic factor that is highly homolog-
ous with Bak and acts in much the same way in that it
oligomerizes to form channels in the mitochondrial mem-
brane. As its name implies, it can form dimers with the
anti-apoptotic protein Bcl-2, and this inhibits its activity
(Module 11: Figure Bcl-2 family functions).
Bcl-2 superfamily control of Ca
2+
signalling
It has been known for some time that members of the Bcl-2
superfamilynot onlybindtothe mitochondria, but are also
found on the endoplasmic reticulum(ER), where they may
act to modulate Ca
2 +
signalling by adjusting the operation
of the endoplasmic reticulum (ER)/mitochondrial Ca
2 +
shuttle (Module 5: Figure ER/mitochondrial shuttle).
There is general agreement that Bcl-2 seems to act by re-
ducing the amount of Ca
2 +
being transferred from the ER
to the mitochondria, but there is some controversy as to
the exact mechanism. The opposing views are centred on
the question of whether or not Bcl-2 acts to reduce the
level of Ca
2 +
in the ER lumen. Some consider that Bcl-2
promotes the passive leak of Ca
2 +
through the inositol
1,4,5-trisphosphate (InsP
3
) receptor, which then reduces
the amount of Ca
2 +
stored in the lumen. The alternative
viewis that Bcl-2 acts byaltering the sensitivityof the InsP
3
receptor, thereby reducing the amount of Ca
2 +
being re-
leasedduring stimulation. Despite this uncertaintyas tothe
exact mechanism, there does seem to be some agreement
that Bcl-2 plays an important role in regulating apoptosis
by interacting with the InsP
3
receptor to reduce the load
of Ca
2 +
that is imposed upon the mitochondrion by the
ER during cell signalling. The pro-apoptotic factors Bak
and Bax can promote Ca
2 +
-induced apoptosis by binding
Bcl-2, thereby removing the ability of this anti-apoptotic
factor to reduce Ca
2 +
signalling.
Bcl-2 has been shown to interact with the InsP
3
receptor
to control the phosphorylation of Ser-1755, which seems
tobe critical for altering the release of Ca
2 +
. The Bcl-2 may
act either by promoting the activity of the protein kinase
A (PKA)/A-kinase-anchoring protein (AKAP) complex
that carries out the phosphorylation or by removing the
calcineurin (CaN) that dephosphorylates Ser-1755.
Bcl-2 gene single nucleotide polymorphisms have been
associated with a risk of developing bipolar disorder.
Caspase cascade
Signals coming either from the death receptors or from the
mitochondria activate the caspase cascade (Module 11: Fig-
ure apoptosis). A family of caspases, which exist in the cell
as dormant proenzymes, respond to the various induction
signals by initiating a proteolytic cascade that produces
active heterodimers responsible for the nal degradation
phase. The sequence begins with the initiator caspases (e.g.
caspases 8, 9 and 10). Caspases 8 and 10 contain a tandem
motif of death effector domains (DEDs), whereas caspase
9 has a caspase-recruitment domain (CARD) (Module 11:
Figure TNF apoptotic signalling). These initiator cas-
pases are responsible for activating the executioner cas-
pases (e.g. caspases 3, 6 and 7). The latter then cleave
specic polypeptides [e.g. poly(ADP-ribose) polymerase,
DNA-dependent protein kinase, lamins and actin]. Cas-
pase 3 cleaves the inhibitor of caspase-activated DNAase
C
r
Module 11
r
r
28
Module 11: Figure Bcl-2 family functions
Apaf-1
Procaspase 9
Procaspase 3
Caspase 3
Cyt c
XIAP
SMAC/
DIABLO
Apoptosome
Cyp
D
Mitochondrial
matrix
H
+
MTP
VDAC
PBR
Bax
Bax
tBid
Bax
Bcl-2
Bcl-2
Bcl-2
Bim
Bim
Bad
P
Bad
Bcl-xL
Bcl-xL
Bak
Bak
ANT
OMM
IMM
1
4
-
3
-
3
PKB
_
_
Intrinsic apoptotic events at the mitochondrion responsible for the release of cytochrome c and the formation of the apoptosome.
A critical event of the intrinsic pathway is the release of cytochrome c (Cyt c) through mechanisms that are still being investigated. One hypothesis
considers that formation of the mitochondrial permeability transition pore (MTP) provides an avenue for cytochrome c to pass across the outer
mitochondrial membrane (OMM). Another hypothesis is that Cyt c passes across channels formed by the polymerization of pro-apoptotic factors such
as Bak and Bax, which are members of the Bcl-2 superfamily. Whether or not Bax and Bak can polymerize depends on a complex web of interactions
with other members of the Bcl-2 superfamily. Bax is kept quiescent by being bound to the anti-apoptotic factor Bcl-2, but the inhibitory action can be
negated by the pro-apoptotic factor Bim. Likewise, Bak is inhibited by Bcl-X
L
, but this inhibition can be reversed by Bad. The ability of Bad to remove
Bcl-X
L
is regulated by the PtdIns 3-kinase signalling pathway, which acts through protein kinase B (PKB) that phosphorylates Bad, which is then taken
out of action by being bound to 14-3-3 protein.
(iCAD) to release CAD that begins to hydrolyse DNA.
Through this co-ordinated sequence of protein and DNA
hydrolytic events, the cell begins its regulated disassembly.
Inhibitor of apoptosis family of proteins (IAPs)
There are ve members of the inhibitor of apoptosis
(IAP) family of proteins, which function to inhibit
specic caspases. A prominent member of the family
is X-chromosome-linked inhibitor of apoptosis protein
(XIAP), which is a potent inhibitor of caspases 3, 7 and
9, can prevent cell death induced by TNF-, Fas and UV
light. This inhibitory activity of XIAP is, in turn, inhibited
by the second mitochondrial-derived activator of caspases
(SMAC), which is also known as the direct IAP-binding
protein with low pI (DIABLO). SMAC/DIABLO is re-
leased from the mitochondria to enhance apoptosis by
reducing the activity of XIAP (Module 11: Figure Bcl-2
family functions).
A t(11;18)(q21;q21) chromosomal translocation of the
MALT1 gene to the genomic locus of the inhibitor of ap-
optosis gene cIAP to give an API2-MLT fusion gene has
been linked to MALT lymphomas.
Another member of the IAP family is the baculoviral
inhibitor of apoptosis repeat-containing 5 (BIRC5, also
known as survivin) that is controlled by the hippo sig-
nalling pathway (Module 2: Figure hippo signalling path-
way).
Caspase-independent pathway
Some of the factors such as apoptosis-inducing factor
(AIF) and endonuclease G (EndoG) released by the mi-
tochondria during the operation of the intrinsic pathway
can activate apoptosis independently of caspases (Step 5a
in Module 11: Figure apoptosis). These two factors trans-
locate into the nucleus, where they function to degrade
DNA.
Hormonal modulation of apoptosis
Apoptosis is tightly regulated by a number of signalling
mechanisms that operate to either adjust the activity of
pre-existing components or to alter the expression levels
of these components (Steps 8 and 9 in Module 11: Fig-
ure apoptosis). Both mechanisms can be controlled by the
PtdIns 3-kinase signalling pathway, which plays a major
role as a negative regulator of apoptosis. One of its func-
tions depends onthe phosphorylationof the pro-apoptotic
factor Bad on Ser-136, which increases its interaction with
14-3-3 protein and thus prevents Bad from inactivating
C
r
Module 11
r
r
29
Bcl-X
L
(Module 11: Figure Bcl-2 family functions). PtdIns
3-kinase signalling can also modulate apoptosis by altering
the expression of the pro-apoptotic factor Bim (Module
4: Figure FOXO control mechanisms). Survival factors
use this signalling pathway to remove Forkhead box O
(FOXO) from the nucleus, thereby reducing the expres-
sion of Bim. When survival factors are removed and sig-
nalling through the PtdIns 3-kinase pathway is decreased,
FOXO returns to the nucleus to begin the transcription
of Bim, which will help to bias the repertoire of apoptotic
factors towards apoptosis.
The operation of the anti-apoptotic factor Bcl-2 is also
inuenced by phosphorylation. For example, the phos-
phorylated form of Bcl-2 is less effective in binding to
Bim and Bax. The dephosphorylation of Ser-70 on Bcl-2
by calcineurin (CaN) seems to be necessary for its anti-
apoptotic activity. Phosphorylation may also inuence the
Bcl-2 superfamily control of Ca
2 +
signalling, because Bcl-
2 is better able to modulate endoplasmic reticulum (ER)
Ca
2 +
signalling when it is dephosphorylated.
Pathogens such as Shigella exneri and Salmonella en-
terica serotype Typhimurium, which cause bacillary dys-
entery and food poisoning respectively, manipulate this
hormonal adaptation of apoptosis to prolong the survival
of their host cells.
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