THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 276, No. 34, Issue of August 24, pp. 31738 –31744, 2001
Printed in U.S.A.
Gas6 Anti-apoptotic Signaling Requires NF-B Activation*
Received for publication, May 16, 2001, and in revised form, June 22, 2001
Published, JBC Papers in Press, June 25, 2001, DOI 10.1074/jbc.M104457200
Francesca Demarchi‡§, Roberto Verardo‡, Brian Varnum¶, Claudio Brancolini储,
and Claudio Schneider‡储**
From the ‡Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie AREA Science Park, Padriciano 99, Trieste
34012, Italy, the 储Dipartimento di Scienze e Tecnologie Biomediche, Universita’ degli Studi di Udine, piazzale Kolbe 4,
Udine 33100, Italy; and ¶Amgen Inc., Thousand Oaks, California 91320
The growth arrest-specific 6 gene product (Gas6) is a secreted
protein related to the anti-coagulant protein S. Gas6 binds,
with different affinities, to the receptors of the mammalian Axl
protein-tyrosine kinase family: Axl (also named Ark, Ufo, Tyro7), Rse (also named Sky, Brt, Tif, Dtk, Tyro3), and Mer (Eyk,
Nyk, Tyro12). Inactivation of the Gas6 gene has recently been
shown to prevent venous and arterial thrombosis in mice and
protect against fatal collagen/epinephrine-induced thromboembolism (1). This knock-out phenotype has unveiled one of the
biological functions of Gas6; however, published data of the
past several years argue for a multiplicity of functions of GAS6/
* This work was supported in part by a grant from Associazione
Italiana per la Ricerca sul Cancro and by a grant from Ministero
dell’Universita e della Ricerce Scientifica e Tecnologica (COFIN2000).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
§ Recipient of a fellowship from the International Center for Genetic
Engineering and Biotechnology.
** To whom correspondence should be addressed: Tel.: 39-040-398992; Fax: 39-040-398-990; E-mail: schneide@sci.area.trieste.it.
Axl signaling in normal and cancer cells. GAS6 has been reported to be involved in osteoclastic bone reabsorption (2) and
in pluripotential hematopoietic stem cells growth support (3).
A large body of evidences support a role of Gas6/Axl signaling
in cell growth and protection from apoptosis in normal and
cancer cells. Simultaneous inactivation of Axl, Sky, and Mer in
the mouse causes progressive death of differentiating germ
cells and infertility (4). Axl is overexpressed in metastatic colon
carcinoma (5, 6) and aggressive melanoma (7). Gas6 is mitogenic for fibroblasts (8, 9), Schwann cells (10), and contactinhibited mouse mammary gland cells (11), and it can enhance
the mitogenic activity of thrombin in injured vascular smooth
muscle cells (12). In addition, Gas6 can also act as a survival
factor. It protects mouse fibroblasts and human umbilical vein
endothelial cells from apoptosis in response to serum withdrawal and TNF1 cytotoxicity (8, 9, 13).
Gas6-mediated survival involves the phosphatidylinositol
3-OH kinase (PI3K) and the AKT/protein kinase B (PKB) pathways in serum-starved NIH 3T3 mouse fibroblasts (14, 15).
Activation of these pathways has been reported to induce nuclear translocation of the transcription factor nuclear factor
kappa B (NF-B) in response to platelet-derived growth factor
(PDGF) (16), tumor necrosis factor (TNF) (17), and interferon
␣/ (18). Moreover, Mer-dependent protection from apoptosis
induced by interleukin-3 withdrawal in hematopoietic cells
involves PI3K induction and NF-B transcriptional activation
(19). Altogether these pieces of evidences suggest that NF-B
could play a role in Gas6/Axl signaling.
NF-B is present in the cytoplasm of the majority of cell
types as homodimer or heterodimer of a family of structurally
related proteins (20). Five members have been identified in
mammalian cells: RelA (p65), cRel, RelB, NF-B1 (p50/p105),
and NF-B2 (p52/p100). They are present in an inactive form
associated with inhibitory proteins that mask their nuclear
localization signal. Inhibitors belong either to the IB family
(IB␣, , ⑀, and Bcl3) or are the precursors of NF-B1 and
NF-B2: p105 and p100, respectively. A wide variety of stimuli
are known to activate NF-B, including cytokines, growth factors, bacterial and viral products, oxidative stress, UV irradiation, and some pharmaceutical drugs and chemicals. Upon
cell stimulation IB is rapidly phosphorylated at two conserved
serine residues near its amino terminus (21). Similarly, p105 is
phosphorylated in its carboxyl-terminal region (22). Phosphorylation of the inhibitors triggers their proteolytic degradation
1
The abbreviations used are: TNF, tumor necrosis factor; PI3K,
phosphatidylinositol 3-OH kinase; PKB, protein kinase B; NF-B, nuclear factor kappa B; PDGF, platelet-derived growth factor; GSK, glycogen synthase kinase; FGF, fibroblast growth factor; FCS, fetal calf
serum; EGF, epidermal growth factor; CMV, cytomegalovirus; IP, immunoprecipitation; PAGE, polyacrylamide gel electrophoresis; TK, thymidine kinase; LUC, luciferase.
31738
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The growth arrest-specific 6 gene product Gas6 is a
growth and survival factor related to protein S. Gas6 is
the ligand of Axl receptor tyrosine kinase; upon binding
to its receptor Gas6 activates the phosphatidylinositol
3-OH kinase (PI3K) and its downstream targets S6K and
Akt. Gas6 anti-apoptotic signaling was previously
shown to require functional PI3K and Akt and to involve
Bad phosphorylation in serum-starved NIH 3T3 cells.
Here we demonstrate that Gas6 induces a rapid and
transient increase in nuclear NF-B binding activity
coupled to transcription activation from NF-B-responsive promoters and increase in Bcl-xL protein level. Gas6
survival function is impaired in cells lacking p65/RelA
and in NIH 3T3 cells transfected with a dominant negative IB, indicating that NF-B activation plays a central role in promoting survival in this system. Moreover,
NF-B activation can be blocked by a dominant negative
Akt and by wortmannin, an inhibitor of PI3K, thus suggesting that NF-B activation is a downstream event
with respect to PI3K and Akt, as already described for
other growth factors. In addition, we show that glycogen
synthase kinase 3, which is phosphorylated in response
to Gas6, can physically associate with NFKB1/p105 in
living cells and can phosphorylate it in vitro. Furthermore, Gas6 treatment is coupled to a decrease in p105
protein level. Altogether these data suggest the involvement of NF-B and glycogen synthase kinase 3 in Gas6
anti-apoptotic signaling and unveil a possible link between these survival pathways.
Involvement of NF-B and GSK3 in Gas6 Signaling
EXPERIMENTAL PROCEDURES
Cell Culture and Reagents—293 cells, NIH 3T3 cells, p50⫺/⫺ and
p65⫺/⫺ mouse fibroblasts were grown in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal calf serum (FCS), penicillin (100
units/ml), and streptomycin (100 g/ml). Medium was replaced with
0.5% FCS containing serum for 48 h to induce growth arrest by serum
starvation.
Wortmannin, sodium fluoride, and sodium orthovanadate were from
Sigma Chemical Co., St. Louis, MO. Basic fibroblast growth factor
(bFGF) was kindly supplied by Dr. C. Grassi (Farmitalia, Milano).
Epidermal growth factor (EGF) was kindly provided by A. Ullrich.
Recombinant Gas6 was supplied by Amgen Inc.
Transfection and Luciferase Reporter Assay—NIH 3T3 cells at 60 –
80% confluency were transiently transfected using LipofectAMINE
Plus reagent (Life Technologies, Inc.) according to the manufacturer’s
instructions. IB⌬N in pCMV4 expression vector was kindly provided
by Dr. Shao-Cong Sun and is described in a previous study (37), pCMV6
plasmids containing HA-AKT K⫹ and AKT K⫺ (K179M) were kindly
provided by Dr. A. Bellacosa. Basic TKluc containing the herpes simplex virus thymidine kinase (TK) promoter in front of luciferase and
mPRDII TKluc, with two NF-kB binding sites in front of the TK promoter, were a kind gift of Dr. G. Manfioletti and have been described
previously (38). bcl-x promoter and bcl-x promoter triple mutant linked
to luciferase expression vector were a kind gift of Dr. Perez Polo and are
described in a previous study (29). PRL-null Renilla luciferase expression vector was from Promega (Madison, WI). Briefly, 1–2 g of plasmid
DNA were diluted in Opti-MEM (Life Technologies, Inc.) and mixed
with LipofectAMINE and Plus reagent. Complexes were allowed to
form for 15 min prior to addition to the cells. Meanwhile the culture
medium was replaced with Opti-MEM. The medium was replaced with
0.5% FCS containing medium 5 h after transfection to induce growth
arrest. 48 h later the medium was replaced with serum-free medium
with or without Gas6, and the cells were lysed 5 h later in Passive Lysis
Buffer (Promega). Extracts were collected and cleared by centrifugation
at high speed. The substrates were obtained using the Dual Luciferase
Reporter assay system (Promega), and relative light units (firefly/Renilla) were measured using a luminometer (Turner Design).
Western Blot—Western blot analysis was performed by preparing
whole cell extracts in 2⫻ SDS sample buffer containing 1 g/ml aprotinin, 1 g/ml pepstatin, and 1 g/ml leupeptin on ice. 20 mM -glycerophosphate, 10 mM sodium orthovanadate was added to the extracts to
be analyzed with anti-phospho-GSK3 antibodies. Western blotting antibodies were purchased from the following companies: IB (New England BioLabs, Inc., Beverly, MA), NF-B p50/105 (Santa Cruz Biotechnology, Inc.), Bcl-x (Transduction Laboratories, Lexington, KY), GSK3
(BIOSOURCE International Inc.), Phospho GSK3 (Cell Signaling), and
actin (Sigma). After incubation with primary antibodies, blots were
incubated with horseradish peroxidase secondary antibodies (Sigma)
and visualized using ECL chemiluminescence reagents (Amersham
Pharmacia Biotech, Piscataway, NJ).
Gel Retardation Assays—Nuclear extracts and oligonucleotide probe
were prepared as described previously (39). 5 g of nuclear extracts was
incubated with [␥-32P]ATP (Amersham Pharmacia Biotech UK)-labeled
NF-B-specific oligonucleotide and analyzed in native 5% gel as described (39). Super-shift assays were performed by preincubating nuclear extracts with specific antibodies for 15 min. NF-B p50, p65, and
cRel antibodies were from Santa Cruz Biotechnology, Inc.
Immunoprecipitation and in Vitro Kinase Assay—293 cells grown on
10-cm plates were lysed in a buffer containing 20 mM Tris, pH 8, 100 mM
KCl, 1 mM EDTA, 0.5% EDTA, 1 mM phenylmethylsulfonyl fluoride, 1
g/ml aprotinin, 1 g/ml pepstatin,1 g/ml leupeptin, 20 mM -glycerophosphate, and 10 mM sodium orthovanadate on ice. Cells were disrupted by repeated aspiration through a 26-gauge needle, and cellular
debris were removed by centrifugation. The lysates were incubated with
4 g of p50/105 antibody or GSK3 antibody (Santa Cruz Biotechnology)
overnight at 4 °C, and subsequently protein A-Sepharose beads (Amersham Pharmacia Biotech) were added and incubated for 2 h. At this
point the IP products to be used for kinase assay were centrifuged and
washed twice with kinase buffer (20 mM Tris, pH 7.5, 10 mM MgCl2, 5
mM dithiothreitol, 20 M ATP, 20 mM -glycerophosphate, and 10 mM
sodium orthovanadate) and resuspended in the same buffer. Kinase
assay was performed by adding to the immunoprecipitation product: 5
Ci of [␥-32P]ATP and 0.5 l of recombinant GSK3 (Cell Signaling)
and incubating 15 min at 37 °C. Reactions were terminated by adding
SDS-PAGE loading buffer and analyzed on a 12.5% SDS-PAGE after
boiling for 2 min. The IP reactions to be analyzed by SDS-PAGE were
centrifuged and washed three times with lysis buffer. Afterward, the
beads were resuspended in SDS loading buffer, boiled, and separated on
a 12.5% SDS-PAGE.
RESULTS
Gas6 Treatment Is Coupled to a Decrease in IB␣ Protein
Level and to an Increase in Nuclear NF-B Binding Activity in
NIH 3T3 Mouse Fibroblasts—Gas6 anti-apoptotic activity was
previously shown to absolutely require the phosphatidylinositol 3-OH kinase (PI3K) (14) and its substrate AKT/PKB (15) in
serum-starved NIH 3T3 mouse fibroblasts. AKT has been
shown to drive transcription activation of NF-B both indirectly through IKK and subsequent IB phosphorylation (16,
17) or directly by p65/RelA subunit phosphorylation (42, 43).
As a first approach to verify whether Gas6 could lead to
NF-B induction we analyzed the effect of Gas6 treatment on
the protein level of the NF-B inhibitor IB-␣. NIH 3T3 were
grown for 48 h in 0.5% containing medium to achieve quiescence. Thereafter the medium was replaced with serum-free
containing medium in the presence or absence of Gas6 and
lysates prepared at different time points (5, 15, 30, 60 min).
Some plates were replaced with 20% FCS containing medium
as control for IB␣ down-regulation. The blot shown in Fig. 1A
demonstrates that both Gas6 and 20% FCS treatment are
coupled to a transient and rapid decrease in IB␣. Actin was
used as loading control.
To analyze the effect of Gas6 on nuclear NF-B binding
activity, gel retardation assays were performed with a 32Plabeled oligonucleotide containing a NF-B consensus as already described (39). NIH 3T3 cells were serum-starved (48 h
in 0.5% FCS) and then shifted to 20% FCS or to serum-free
medium with or without Gas6, and nuclear extracts were prepared 60 min later. Extracts were prepared also from cells left
in 0.5% FCS as negative control. Fig. 1B shows the retarded
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via the ubiquitin-proteasome pathway. The released NF-B
dimer can then translocate to the nucleus, where it directly
binds to its cognate DNA sequence to activate gene
transcription.
NF-B is a regulator of inflammation and immune response
as well as of cellular proliferation and apoptosis (23). Although
NF-B has been shown to be pro-apoptotic in certain experimental settings, a number of data argues for a role of NF-B as
regulator of survival. RelA (p65) knock-out in mice is embryonically lethal as a result of extensive liver apoptosis. In addition, cells derived from these mice show enhanced sensitivity to
TNF-induced apoptosis (24). NF-B is required for protection
from apoptosis occurring after growth factors withdrawal in
various systems, including fibroblasts (16), hematopoietic cells
(19), hepatoma cells (25), Chinese hamster ovary (CHO) (26),
and PC12 cells, (27). A number of anti-apoptotic proteins encoded by NF-B-inducible genes have recently been identified,
namely Bcl-xL (28, 29), A1 (30, 31), and TNF receptor-associated proteins 1 and 2 (32).
Besides NF-B induction, activation of Akt/PKB has been
reported to determine partial inactivation of glycogen synthase
kinase 3 (GSK3) in response to insulin (33), Gas6 (11), and
FGF1 (34). GSK3 is a serine threonine protein kinase regulating cell-fate specification and tumorigenesis (35). Recently,
GSK3 function has been shown to be required for NF-Bmediated anti-apoptotic response to TNF-␣ (36). Disruption of
the mouse GSK3 gene determines severe liver degeneration
during mid-gestation, as observed in mice lacking genes involved in the activation of NF-B (36).
In the present manuscript we have investigated the role of
NF-B in modulating Gas6 survival signaling and started to
address the question of possible cross-talk between NF-B and
GSK3 signaling.
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Involvement of NF-B and GSK3 in Gas6 Signaling
bands. Shifting the cells to serum-free induces a slight increase
in binding activity (lane 2) as compared with growth-arrested
control cells (lane 1). Gas6 (lane 4) induces a clear increase in
the complex binding to the B-specific oligonucleotide, just as
20% serum (lane 3), used as positive control, because it was
previously shown to induce NF-B (40). The specificity of the
retarded complex was assessed by competition experiments
with cold specific and unspecific oligonucleotides as already
described (39) (data not shown).
NF-B Is Required for Gas6-mediated Protection from Apoptosis—Because we have observed that Gas6 treatment is coupled to the induction of nuclear NF-B binding activity, we
investigated the effect of other growth factors in our experimental setting. Serum-starved cells were shifted to serum-free
medium, and FGF, EGF, or Gas6 were added to the cells.
Control cells were shifted to serum-free medium without
growth factors. Treated cells were either used to prepare nuclear extracts 30 min later, or to measure viability 20 h later.
The graph reported in Fig. 2A indicates the increase in cell
viability with respect to the control cells grown in serum-free
containing medium. Both Gas6 and FGF, but not EGF, efficiently protect serum-starved NIH 3T3 fibroblasts from apoptosis, as already reported (8). Interestingly, protection from
apoptosis correlates with a clear increase of NF-B binding
activity, as shown in Fig. 2B. The B binding activity induced
by FGF and Gas6 are comparable, whereas it is significantly
lower in extracts from EGF-treated cells.
To test the importance of NF-B induction in Gas6-mediated
protection from apoptosis, we challenged growth-arrested
mouse fibroblasts lacking either p50 (41) or p65 (24) subunits of
NF-B with serum-free medium in the presence or absence
Gas6. As reported in the graph of Fig. 2C, p50⫺/⫺ cells can be
rescued by Gas6, as for wild type NIH 3T3 cells, whereas
p65⫺/⫺ cells cannot. This result demonstrates that Gas6-mediated protection from apoptosis induced upon serum with-
FIG. 2. Induction of NF-B binding activity correlates with
protection from apoptosis. A, serum-starved cells (48 h in 0.5%
serum containing medium) were incubated with the FGF, EGF, and
Gas6 in serum-free medium for 20 h; then the cells were trypsinized and
counted. The graph indicates the increase in viability with respect to
the control cells incubated in serum-free medium without growth factors. B, serum-starved cells were incubated with FGF, EGF, and Gas6
in serum-free medium for 1 h, and nuclear extracts were prepared and
analyzed in gel retardation assays with a NF-B-specific oligonucleotide. C, effect of Gas6 on cell viability of p50⫺/⫺ and p65⫺/⫺ murine
fibroblasts. NIH 3T3 cells were used as control. The graph indicates the
increase in cell viability of Gas6-treated cells with respect to control
cells incubated in serum-free medium. D, effect of a dominant negative
IB (IB ⌬N) on Gas6-mediated protection from apoptosis. NIH 3T3
were transfected with dominant negative IB or with an empty vector
as transfection control. 6 h later, the cells were serum-starved for 48 h
to achieve growth arrest. Thereafter, the cells were incubated in serumfree medium in the presence or absence of Gas6 or in 0.5% containing
medium for additional 20 h. Afterward, the cells were trypsinized and
counted. The graph shows the average of the number of recovered cells
in three independent experiments.
drawal requires a functional cRel/p65, whereas p50 is
dispensable.
To further investigate the relevance of NF-B induction for
Gas6 survival properties, we analyzed the effect of a dominant
negative IB␣ (IB⌬N), which cannot be phosphorylated and is
therefore resistant to degradation. NIH 3T3 cells were transfected with an expression vector for IB⌬N or with an empty
vector as control using LipofectAMINE. Six hours after transfection the cells were shifted to a medium containing 0.5%
serum to induce growth arrest. After 48 h the cells were rinsed
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FIG. 1. Gas6 effect on IB protein and NF-B binding activity
in serum-starved NIH 3T3 fibroblasts. A, the Western blot demonstrates that both serum and Gas6 treatment are coupled to a rapid and
transient decrease in IB protein level. Actin is used as loading control.
B, gel retardation assays were performed using a 32P-labeled oligonucleotide containing a consensus sequence for NF-B. Nuclear extracts
were from serum-starved NIH 3T3 cells (lane 1), starved cells incubated
for 1 h in serum-free medium, as negative control (lane 2), 20% serum
containing medium as positive control (lane 3), or serum-free medium
containing Gas6 (lane 4).
Involvement of NF-B and GSK3 in Gas6 Signaling
in phosphate-buffered saline and incubated in serum-free plus
or minus Gas6 or again with 0.5% serum containing medium
for additional 20 h. At this point the cells were trypsinized and
counted. The graph of Fig. 2D reports the average of the results
obtained in three independent experiments and clearly demonstrates that blocking NF-B activation by a dominant negative
IB impairs Gas6 survival activity.
NF-B Binding Activity Induction by Gas6 Is Rapid and
Transient and Contains p50 and p65 Subunits—To analyze the
kinetics of NF-B binding activity induction, we prepared nuclear extracts at different time points after shifting serumstarved NIH 3T3 cells to serum-free medium containing Gas6
as indicated in Fig. 3. The extracts were used in a gel retardation assay with a probe specific for NF-B. The results of this
time course experiment, shown in Fig. 3A, indicate that the
induction starts already at 15 min, peaks at 30 min, and subsequently declines.
To identify the polypeptides present in the shifted complex
we preincubated the nuclear extracts either with p50-, p65-, or
cRel-specific antibodies before performing the gel retardation
assay. As shown in Fig. 3B, both p50 and p65 antibodies can
partially super-shift the retarded complex, whereas cRel-specific antibody cannot.
These data indicate that Gas6-induced complex contains
both p50 and p65 subunits of NF-B but not cRel.
Gas6 Induces NF-B-dependent Transcription Activation—
The effect of Gas6 on NF-B-dependent transcription was studied by analyzing its effect both on an artificial promoter containing two NF-B binding sites and on the bcl-x promoter.
This promoter has recently been shown to contain three NF-B
binding sites and to be responsive to NF-B induction (29, 28,
31). NIH 3T3 cells were transfected with luciferase expression
plasmids driven by the herpes simplex virus thymidine kinase
FIG. 4. Effect of Gas6 on transcription activity on promoters
containing NF-B binding sites. A, NIH 3T3 mouse fibroblasts were
transfected with a luciferase expression vector driven by a Tk promoter
or a Tk promoter with two NF-B binding sites. A Renilla expression
vector was co-transfected as normalization control. 6 h after transfection growth arrest was achieved by replacing the medium with 0.5%
serum containing medium for 48 h. Thereafter the medium was replaced with serum-free medium or serum-free medium containing Gas6
and luciferase activity (firefly/Renilla) was measured 5 h later. B, effect
of Gas6 on transcription activity of the bcl-x promoter and the bcl-x
promoter in which the three NF-B binding sites have been mutated.
Transfection and luciferase assay were performed as described for A. C,
time course of Gas6 effect on Bcl-xL protein. Actin is used as the loading
control. The blot shows an increase in Bcl- xL protein level coupled to
Gas6 treatment.
(TK) promoter or the same promoter containing two NF-B
binding sites (38). A Renilla expression vector was included in
each transfection experiment for normalization. 6 h after transfection the cells were rinsed and the medium was replaced with
0.5% FCS containing medium to induce growth arrest. 48 h
later the medium was changed with serum-free medium with
or without Gas6 for an additional 5 h. Afterward, relative
luciferase activity (firefly/Renilla) was measured with a
luminometer.
Fig. 4A shows the increase of relative luciferase (LUC) activity occurring for each transfected expression vector using
extracts from Gas6-treated cells as compared with control cells.
These results indicate that Gas6 can specifically increase the
transcription rate from a promoter containing binding sites for
NF-B. Similar results were obtained by transfecting a LUC
expression vector driven by the bcl-x promoter or a bcl-x promoter in which the three putative NF-B binding sites have
been mutated (29). As shown in Fig. 4B, Gas6 can positively
affect the bcl-x promoter but not the same promoter lacking
NF-B binding sites. Altogether these data argue that Gas6
can activate NF-B-dependent transcription.
To verify the biological relevance of the increase in bcl-x
promoter activity in response to Gas6, we analyzed its effect on
Bcl- xL protein level in a time-course experiment. Cell lysates
from serum-starved NIH 3T3 cells incubated for different times
with Gas6 were analyzed in a Western blot decorated with an
antibody specific for Bcl- xL; actin was used as loading control.
As shown in Fig. 4C, Gas6 treatment is coupled to an increase
in Bcl- xL protein level, suggesting that one of the mechanisms
by which Gas6 can protect from apoptosis is by up-regulating
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FIG. 3. NF-B activation is transient and contains p50 and p65
polypeptides. A, a time-course gel retardation assay was performed
with a 32P-labeled NF-B-specific oligonucleotide and nuclear extracts
from serum-starved NIH 3T3 fibroblasts incubated for increasing times
in serum-free medium containing Gas6 as indicated in the figure. B,
super-shift analysis of the Gas6-induced complexes. Nuclear extracts
from Gas6-treated cells were preincubated with antibodies against the
p50 (lane 2), p65 (lane 3), or cRel (lane 4) and subsequently used for gel
retardation assay. The control assay, preincubated without antibodies,
is shown in lane 1.
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Involvement of NF-B and GSK3 in Gas6 Signaling
the anti-apoptotic protein Bcl-x. To further assess the relevance of NF-B activation by Gas6 in Bcl-x up-regulation, the
same type of analysis was performed in serum-starved p65⫺/⫺
and p50⫺/⫺ cells. Fig. 5A shows that bcl-xL promoter activity
appears to be susceptible to Gas6 induction in p50⫺/⫺ cells,
whereas it is unaffected in p65⫺/⫺ cells. The blot of Fig. 5B
shows that Gas6 induces Bcl-xL protein increase in p50⫺/⫺
cells, but not in p65⫺/⫺ cells, suggesting that a functional p65
is required for the noticed effect of Gas6.
NF-B Activation by Gas6 Involves the PI3K and AKT Pathways—Previous studies have demonstrated the absolute requirement both of PI3K and AKT for Gas6 survival property
(14, 15). To verify whether NF-B activation was a downstream
event in these pathways we analyzed the effect of a dominant
negative Akt on bcl-x promoter activation in response to Gas6
in serum-starved NIH 3T3. As shown in Fig. 6A, the increase in
the bcl-x promoter activity upon Gas6 addition can be blocked
by co-transfecting an expression plasmid encoding for a dominant negative kinase dead Akt.
Moreover, wortmannin, a potent inhibitor of PI3K, efficiently
prevents NF-B binding activity, as induced by Gas6, and is
analyzed in the gel retardation assay of Fig. 6B. Altogether
these data suggest that the induction of NF-B-mediated transcription is linked to PI3K and AKT activation in the used
experimental settings.
Involvement of GSK3 in Gas6 Anti-apoptotic Signaling—
Gas6 was recently reported to induce GSK3 phosphorylation in
mouse C57MG (11). We found that Gas6 had the same effect on
GSK3 in serum-starved NIH 3T3 as shown in Fig. 7A.
GSK3 substrates contain either clustered serines spaced at
four-amino acid intervals (-catenin), or a conserved proline
residue to the carboxyl side of a targeted serine/threonine (cJun, cyclin-D1, myelin basic protein, Tau) (35). Because human
p105 has three serines with this spacing at positions 899, 903,
and 907 and a proline to the carboxyl side of serine 907 we
investigated whether p105 could be an in vitro substrate for
GSK3. We therefore immunoprecipitated p105 from 293 cells
lysates and used it in an in vitro kinase assay with recombinant
GSK3; a mock immunoprecipitation product was used as negative control. A radioactive band of 105-kDa apparent molecular mass specifically appears after incubation of the p50/p105
immunoprecipitation product with GSK3, as shown in Fig. 7B.
As a first approach to investigate whether this in vitro phosphorylation could have any biological significance, we investigated whether the endogenous p105 and GSK3 do interact in
living cells. To this end we probed a p50/p105 immunoprecipitation product with a GSK3-specific antibody. A total lysate
and a GSK3 IP were used as control. As shown in Fig. 7C,
GSK3 can be specifically immunoprecipitated with p50/p105.
To address the question whether Gas6 signaling affects the
association between p105 and GSK3, we analyzed the p105
protein level in cells treated with Gas6 and in control, untreated cells. As shown in Fig. 7D, the level of p105 significantly decreases in response to Gas6. This result indicates that
the association between p105 and GSK3 may be regulated
indirectly by Gas6, through a reduction in p105 protein level.
DISCUSSION
The data presented in this manuscript demonstrate the requirement of transcription factor NF-B activation in Gas6/Axl
signaling and protection from apoptosis. We have used NIH
3T3 mouse fibroblasts as a model system to investigate Gas6/
Axl function in the control of apoptosis, as already characterized (8, 14, 15). Previous studies have shown that Axl receptor
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FIG. 5. Effect of Gas6 on bcl-x promoter activity and Bcl-xL
protein in p50ⴚ/ⴚ and in p65ⴚ/ⴚ mouse fibroblasts. A, cells were
transfected with a luciferase expression vector driven by the bcl-x
promoter and a Renilla expression vector for normalization, as described in the legend of Fig. 4. The graph shows the relative luciferase
activity (firefly/renilla) of lysates from p50⫺/⫺ and p65⫺/⫺ cells incubated in serum-free medium in the presence or absence of Gas6. B, the
blot shows an increase in Bcl- xL protein level coupled to Gas6 treatment in p50⫺/⫺ mouse fibroblasts. Bcl- xL protein level appears unaffected in p65⫺/⫺ cells.
FIG. 6. Akt and PI3K are involved in NF-B activation by Gas6.
A, effect of a dominant negative, kinase dead Akt kinase on the transcription activity of the bcl-x promoter. Bclx-LUC was co-transfected
either with an expression vector for wild type Akt kinase or for kinase
dead, dominant negative Akt. Transfection was performed as described
in the legend of Fig. 4. The graph indicates the relative increase in LUC
activity (firefly/Renilla) of GAS6-treated cells with respect to control
cells incubated in serum-free. B, effect of PI3K inhibitor, wortmannin
on the induction of NF-kB binding activity by Gas6. Gel retardation
assays were performed using a 32P-labeled NF-B-specific oligonucleotide, and nuclear extracts from cells were preincubated 1 h with wortmannin prior to Gas6 addition or from control cells stimulated with
Gas6.
Involvement of NF-B and GSK3 in Gas6 Signaling
activation by Gas6 switches the PI3K/AKT pathways on and
that both of them are absolutely required for protection from
apoptosis in this system (14, 15). In addition, Gas6 treatment is
coupled to phosphorylation and inactivation of the pro-apoptotic protein Bad (15). Here we show that NF-B induction
and transcription activation require functional PI3K and Akt
kinase. Therefore, on the basis of published data and the results reported here we can suggest that the Gas6 survival
pathway involves consecutive activation of Axl, PI3K, and Akt,
ultimately leading to an increase in nuclear NF-B binding
activity and subsequent induction of NF-B-responsive antiapoptotic genes like bcl-xL.
Protection from apoptosis through the PI3K and subsequent
NF-B activation has been shown to occur in response to platelet-derived growth factor (PDGF), tumor necrosis factor (TNF),
and interferon ␣/ (16 –18). In the case of PDGF and TNF the
Akt kinase can directly phosphorylate and activate the IKK␣
kinase with subsequent IB phosphorylation and degradation.
On the other hand, other reports argue for a direct action of Akt
on the p65 subunit of NF-B leading to transcription activation
(42). In the present study we observed a transient decrease in
IB␣, suggesting that also in our experimental settings the
classical pathway of NF-B activation could be switched on.
Namely: activation of a IB kinase by Akt, phosphorylation and
ubiquitination-dependent degradation of IB␣, and subsequent
nuclear translocation of the active transcription factor. However, we cannot exclude a direct effect of Akt on p65; indeed,
this polypeptide plays a crucial role in our system. We have
shown that p65 is one of the major components of the induced
complex by means of super-shift analysis and that p65⫺/⫺ cells
are not responsive to the survival effect of Gas6.
One of the means by which NF-B displays its pro-survival
function is through the up-regulation of genes encoding for
anti-apoptotic proteins (23). These proteins include Bcl-xL,
TRAF1, TRAF2, c-IAP1, c-IAP2, numerous cytokines and
growth factors, as well as adhesion molecules (23). Bcl-xL protein, like Bcl 2, is known to dimerize with Bax and Bad proteins, and it has been shown that the balance between the
expression of these apoptosis-protecting and apoptosis-inducing proteins is critical for cell survival or death (44). Previous
studies have shown that one of the effects of Gas6/Axl antiapoptotic signaling is Bad phosphorylation (15). Here we investigated the effect of Gas6 on Bcl-xL, because the promoter of its
gene has recently been shown to contain three binding sites for
NF-B and to be responsive to NF-B activation (28, 29). We
have shown that induction of NF-B binding activities is coupled both to bcl-x promoter activation and increase in Bcl-xL
protein levels. These findings further support the relevance of
the role played by Gas6 in modulating cell survival by targeting
different members of the Bcl 2 family. This effect occurs as an
early response through phosphorylation of Bad via Akt, followed by increased Bcl-xL expression level through NF-B induction. In our study we have analyzed the effect of Gas6mediated NF-B activation on Bcl-xL; it is likely that also other
NF-B-responsive genes might be involved.
What is the physiological significance of Axl/Gas6 anti-apoptotic signaling? In the testis, one of the body districts were
both Gas6 and Axl are expressed, Gas6/Axl signaling has been
shown to exert anti-apoptotic functions. Mice lacking all the
three receptors are indeed sterile because of progressive death
of differentiating germ cells (4). Interestingly, bcl-xL is highly
expressed in human spermatogonia (45) and its overexpression
in mouse male germinal cells make them more resistant to
apoptotic-inducing conditions (46).
Another system where Axl is highly expressed both during
development and in the adult is the central nervous system (47,
48). In this system NF-B could also play a survival function
(49, 50). In addition, gene targeting has revealed that bcl-xL is
required for neuronal survival during neuronal development
and for post-natal CNS neurons (51). A cross-talk between
GSK3 and NF-B molecular pathways has been recently suggested because cells lacking GSK3 are defective in NF-B
signaling (36). Moreover, both GSK3 and RelA gene disruption result in embryonic lethality caused by severe liver degeneration (24, 36).
We have observed that Gas6 treatment is coupled to GSK3
phosphorylation in serum-starved NIH 3T3, as already described for the murine C57MG cells (11). This coupling could
underlie an involvement of GSK3 in Gas6 anti-apoptotic signaling, as suggested for FGF. The herein reported physical
association of endogenous p105 and GSK3 in living cells and
the in vitro phosphorylation of p105 by GSK3 could be one of
the molecular basis of a cross-talk between NF-B and GSK3.
We speculate that GSK3 could be involved in regulating p105
protein stability. When GSK3 is phosphorylated and inactivated upon treatment with Gas6 or other growth factors, p105
could become a target of inducible kinases like ikB kinase,
which target it to degradation, thus leading to NF-B activation. Indeed we have observed that Gas6 treatment is coupled
to a decrease in p105 protein level, as already reported for other
NF-B inducers. Further studies are required for a full understanding of the role played by GSK3 in Gas6-Axl signaling and
NF-B activation.
Besides its reported physiological roles in the hematopoietic
and reproductive systems, Gas6/Axl signaling has also been
suggested to play a role in some disorders, including glomerulonephritis (52), osteoclastic bone re-absorption (2), rheumatoid
arthritis (13), and certain metastatic tumors (7). A molecular
Downloaded from http://www.jbc.org/ by guest on June 15, 2020
FIG. 7. Gas6-GSK3-NF-B: a possible cross-talk. A, the blot
shown in the upper panel was decorated with anti-phospho GSK3 and
shows that GSK3 is phosphorylated in Gas6-treated cells. The blot
shown in the lower panel was decorated with anti-GSK3 for control. B,
GSK3 can phosphorylate p105 in vitro. Commercial GSK3 was used
for a kinase assay with a p50/105 immunoprecipitation product (lane 2);
a mock IP product was used as negative control (lane 1). C, an antibody
specific for p50/p105 can co-immunoprecipitate GSK3. 293 cell lysates
were immunoprecipitated with p50/p105 goat-specific antibody (lane 4)
or goat GSK3-specific antibody (lane 2). A total lysate (lane 1) and a
mock IP (lane 3) were used as controls. The gel was blotted and decorated with a monoclonal GSK3 antibody. D, p105 protein level decreases in response to Gas6 treatment. NIH 3T3 cells were serumstarved and then shifted to a serum-free medium in the presence or
absence of Gas6. The cell lysates were used for Western blot experiments. The blot was decorated with a p50/105-specific antibody. Actin
was used as the loading control.
31743
31744
Involvement of NF-B and GSK3 in Gas6 Signaling
dissection of the pathways involved therefore represents an
important step in the development of new tools for therapeutic
intervention.
Acknowledgments—We thank Dr. Alexander Hoffmann from the laboratory of Prof. David Baltimore for providing p50⫺/⫺ and p65⫺/⫺
cells, and Dr. Guidalberto Manfioletti, Dr. Perez-Polo Jr., Dr. A Bellacosa,
Dr. Shao-Cong Sun for kindly providing DNA constructs and mutants.
We thank Stefania Marzinotto for preliminary microinjection experiments and Dr. Cosetta Bertoli for help in tissue culture.
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Gas6 Anti-apoptotic Signaling Requires NF-κB Activation
Francesca Demarchi, Roberto Verardo, Brian Varnum, Claudio Brancolini and Claudio
Schneider
J. Biol. Chem. 2001, 276:31738-31744.
doi: 10.1074/jbc.M104457200 originally published online June 25, 2001
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