Gynecol Surg (2010) 7:189–193

DOI 10.1007/s10397-009-0546-7

ORIGINAL ARTICLE

HMG-CoA reductase inhibitor lovastatin upregulates

plasminogen activator production through RhoA-signaling
in peritoneal cell line Met5A
Noriko Suzuki & Atsushi Imai

Received: 21 October 2009 / Accepted: 3 December 2009 / Published online: 22 December 2009
# Springer-Verlag 2009

Abstract This study was conducted to determine if Introduction

hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor
statin, known to protect postoperative adhesion in animal Since the advent of surgery, peritoneal adhesions have been
model, affect the expressing tissue-type plasminogen activator a significant and often inevitable postoperative complica-
(tPA) in peritoneal cells in culture. Human peritoneal Met5A tion [1, 2]. It is a common consequence of serosal repair,
cells were used to examine the effects of hydrophobic statin occurring in 93–100% of patients following laparotomy,
lovastatin on the level of tPA. PA concentrations were and may lead to serious complications such as intestinal
measured by real-time polymerase chain reaction and obstruction, pelvic pain, and infertility. Peritoneal adhesions
enzyme-linked immunosorbent assay. Active RhoA form are defined as fibrous bands of tissue that join together
was also examined. Lovastatin caused concentration- organs that are normally separated. The plasma fibrinolytic
dependent tPA expression associated with fall of RhoA active system is primarily responsible for the degradation of
level in Met5A cells. These lovastatin-induced changes were fibrin. Tissue-type plasminogen activator (tPA) is consid-
significantly overcome by the addition of geranylgeranyl ered to play an important role in the onset of the extrinsic
pyrophosphate (intermediate of HMG-CoA pathway). A fibrinolytic route [3, 4]. It has been proposed that the
RhoA protein inhibitor C3 transferase mimicked the effects persistence of fibrin, due to impaired tPA activity, results in
of lovastatin on the Met5A cells. These results suggest that the formation of adhesions between damaged serosal
lovastatin may be an effective stimulator of local peritoneal surfaces.
fibrinolytic activity, as it upregulates tPA expression in Hydroxymethylglutaryl-CoA (HMG-CoA) reductase is
peritoneal Met5A cells through the reduction of RhoA the rate-limiting enzyme in the conversion of HMG-CoA to
geranylgeranylation. The extra-cholesterol lowering action mevalonate, an intermediate in the de novo synthesis of
of statin provides a new rationale to prevent peritoneal cholesterol [5–7]. Apart from the conversion of mevalonate
adhesion in postoperative patient. to cholesterol via a number of enzymatic steps, several lipid
isoprenoid intermediate such as geranylgeranyl pyrophos-
Keywords Statin . Tissue-type plasminogen activator . phate (GGPP) and farnesyl pyrophosphate are enzymatically
RhoA . Peritoneal cells . Postoperative adhesion generated from mevalonate. Small guanosine triphosphate
(GTP)-binding proteins (G-protein), including Rho, that
N. Suzuki : A. Imai play pivotal roles in normal and oncogenic signaling,
Department of Obstetrics and Gynecology, undergo posttranslational modification, termed isopreny-
Gifu University School of Medicine,
lation, allow the attachment of G proteins to internal cell
Gifu, Japan
membranes by means of a lipid anchor [5]. HMG-CoA
A. Imai (*) reductase inhibitors, commonly referred to as statins
Atsushi Imai at Institute of Endocrine-Related Cancer, including lovastatin, are in wide use for the treatment of
Matsunami General Hospital,
hypercholesterolemia.
Kasamatsu,
Gifu 501-6062, Japan Recent studies have elucidated additional effects of statin
e-mail: aimai@matsunami-hsp.or.jp beyond their impact on serum cholesterol levels [5–9].
190 Gynecol Surg (2010) 7:189–193

Aarons et al. reports that statins decrease postoperative AssayMax tPA ELISA kit. The absorbance (optical density)
adhesion by increasing peritoneal fibrinolytic activity in rat was measured at 450 nm.
model [3]. This beneficial effect beyond lowering cholesterol
might be due in part to the ability of statins to inhibit the tPA mRNA
synthesis of GGPP that interferes with G-protein-mediated
RhoA activation [10, 11] using peritoneal cells derived from Total RNA was isolated with CellAmp™ Direct RNA Prep
the omentum. In this study, we investigated whether kit for One Step reverse transcription (RT)-polymerase
lovastatin increases fibrinolytic potential by the induction chain reaction (PCR; Takara Bio.Co., Ohtsu, Japan)
of tPA through inhibiting the RhoA-dependent pathway in according to the manufacturer's protocol. The designed
human peritoneal Met5A cells. oligonucleotide sequences were verified to amplify unique
sequence, tPA: (F) 5′-AGCAGGCCCTGTACTTCTC and
(R) 5′-TCTGCAGTAGTTGTGGTTCC, ß-actin (as a
Materials and methods housekeeping gene): (F) 5′-AGAAAATCTGGCACCA
CACC and (R) 5′-AGAGGCGTACAGGGATAGCA,
Materials respectively. Real-time PCR analysis was performed using a
One Step SYBR PrimeScript RT-PCR Kit (Takara Bio. Co.)
Lovastatin and GGPP were obtained from Calbiochem and analyzed with a Takara Smart cycler system using a two-
(Darmstat, Germany) and Sigma (St. Louis, MO, USA), step program consisting of 5 s at 95°C and 30 s at 60°C for 40
respectively. C3 transferase was a product of Cytoskelton cycles. ß-Actin was included as an endogenous normalization
(Denver, CO, USA). Human tPA enzyme-linked immuno- control.
sorbent assay (ELISA) kit was purchased from AssayPro
(St. Charles, MO, USA). All other chemicals were of Measurement of active Rho
reagent grade. This study did not require approval of our
institutional review board because it did not imply active The activation state of Rho-GTP was measured using
involvement of patients. G-LISA RhoA Activation Assay Biochem Kit from
Cytoskelton. The absorbance was measured at 490 nm.
Cell culture
Statistics
Human mesothelial (peritoneal) Met5A cells were obtained
from ATCC (Manassas, VA, USA). The Met5A cells were The variables followed normal distribution, and statistical
cultured in a 5% CO2 humidified atmosphere in Medi- analysis was performed with the Student's t test, and a P
um199 with 10% fetal bovine serum, 75 mM L-glutamine, value of less than 0.05 was considered significant.
1.25 g/l sodium bicarbonate, 3.3 nM epidermal growth
factor, 400 nM hydrocortisone, 870 nM insulin, 20mM
HEPES, 100 IU penicillin, and streptomycin. Lovastatin Results
was converted from its inactive form to active form
following the manufacturer's instructions by dissolving Lovastatin showed the effects of dose-dependent increase in
52 mg of the compound in 1.04 ml of ethanol and then the production of tPA (Fig. 1). To determine whether the
adding 813µl of 1 N NaOH. The resulting solution was lovastatin effects were mediated by the isoprenylation, we
neutralized with 1 N HCl to a pH of 7.2 and brought up to a incubated Met5A cells with GGPP, a downstream interme-
volume of 13 ml with distilled water. The stock solution diate of mevalonate in the presence of lovastatin and
(10 mM) was stored at −20°C [12]. At least three separate measured tPA production. The effect of lovastatin to
cell culture experiments were performed in duplicate (using increase tPA production was overcome by addition of
two wells), and each experiment gave similar results. GGPP, indicating that lovastatin-induced inhibition of
GGPP synthesis is critical for the increase of tPA (Fig. 2).
tPA protein Lovastatin might increase tPA production through interven-
tion in the GGPP pathway.
Met5A cells (1×105) were plated into 12-well plate and Rho is known to be one of the most important
treated with the appropriate concentration of the chemicals isoprenylated proteins. To determine whether lovastatin
to be tested for 18 h (overnight interval) where cells were in effects on tPA result from the inhibition of Rho proteins, we
sub-confluent. Cells were lysed in phosphate buffer incubated Met5A cells with C3 exoenzyme, a specific
solution by freeze-thawing followed by brief sonication. inhibitor for RhoGTPase. As shown in Fig. 2, C3
The tPA concentration in cell lysate was determined with completely mimicked the effect of lovastatin on tPA
Gynecol Surg (2010) 7:189–193 191

production and also blocked the reversion of lovastatin

effects by GGPP. These results suggest that the effect of the
lovastatin on tPA production was through the reduction of
* RhoA geranylgeranylation.
To demonstrate that the effects of lovastatin to increase
the level of medium tPA were not due to stimulation of their
cellular secretion, tPA mRNA level was examined. The RT-
PCR analysis revealed higher levels of tPA mRNA level in
lovastatin-treated Met5A cells (Fig. 3). GGPP totally
abolished the effect of lovastatin on tPA mRNA expression.
The addition of exoenzyme C3 transferase reversed tPA
mRNA level to control from those observed in lovastatin—
or GGPP-treated cells. These results were good agreeable
with protein level changes. Lovastatin had no effects on the
levels of plasminogen activator inhibitor (PAI) protein (data
not shown).
Rho protein's cycle between an active (GTP-bound) form
Fig. 1 Dose-dependency of lovastatin-stimulated tissue-type plasmin-
ogen activator (tPA) production. Met5A cells were treated with 0 and an inactive (guanosine diphosphate (GDP)-bound) form
(vehicle), 0.1, 0.3, or 1µM lovastatin for 18 h. tPA protein content of is important for Rho's interaction with upstream regulators
cell lysate was measured by tPA enzyme-linked immunosorbent assay and downstream effectors. In Fig. 2, we showed C3
kit. Values are expressed as means optical density (450 nm)±SD of
reversed the effect of lovastatin on tPA production. To
three independent experiments performed in duplicate determination.
*P<0.05 versus control determine whether lovastatin decreases the RhoA active
form, we measured directly RhoA active form by using
ELISA. As shown in Fig. 4, lovastatin decreased the active
form of RhoA. This lovastatin-induced change was atten-
uated by the addition of GGPP. A RhoA protein inhibitor
C3 transferase mimicked the effects of lovastatin to reduce
RhoA active form in the Met5A cells. These results indicate
that geranylgeranylation of Rho to be active GTP-form is
required for reversion of the effect of lovastatin by GGPP.
* * *
tPA mRNA(relative to control)

* *

Fig. 2 Tissue-type plasminogen activator (tPA) protein level in

Met5A cells exposed to geranylgeranyl pyrophosphate (GGPP) or Fig. 3 Tissue-type plasminogen activator mRNA level in Met5A cells
C3 transferase. Met5A cells were exposed to lovastatin (1µM), exposed to lovastatin, geranylgeranyl pyrophosphate (GGPP), or C3.
lovastatin and GGPP (1µM), C3 (1 µg/ml), or lovastatin, GGPP, and Total RNA was extracted for real-time polymerase chain reaction
C3 for 18 h. tPA protein content of cell lysate was measured by tPA analysis from the cells exposed to lovastatin (1µM), lovastatin and
enzyme-linked immunosorbent assay kit. Values are expressed as GGPP (1 µM), C3 (1 µg/ml), or lovastatin, GGPP, and C3 for 18 h. As
means optical density (450 nm)±SD of three independent experiments relative to control±SD of three independent experiments performed in
performed in duplicate determination. *P<0.05 versus control duplicate determination. *P<0.05 versus control
192 Gynecol Surg (2010) 7:189–193

bound form and an active membrane GTP-bound form.

GTP-bound Rho recognizes and interacts with its cofactor
to initiate a downstream response [13]. Our study demon-
strated that the effect of lovastatin on tPA was reproduced
OD490nm

*
* * by the C3 exoenzyme, a specific inhibitor of the Rho
proteins. We also observed that lovastatin reduced the level
of active RhoA. Furthermore, C3 blocked the reversion of
lovastatin effects induced by GGPP, thus suggesting the
involvement of the RhoA activation in the signaling
pathway of lovastatin on tPA.
These results indicated that by preventing membrane
interaction, statins rapidly inactivate RhoA, leading to
increased tPA expression (protein and mRNA levels) and
activity in peritoneal mesothelial cells. The mechanism
linking Rho proteins to fibrinolysis is not fully elucidated.
Fig. 4 RhoA activity in Met5A cells exposed to lovastatin. Met5A An involvement of the cytoskeleton could be assumed
cells were exposed to lovastatin (1µM), lovastatin and geranylgeranyl because Rho proteins are known to regulate the organiza-
pyrophosphate (GGPP; 1 µM), C3 (1 µg/ml), or lovastatin, GGPP,
and C3 for 18 h. Values are expressed as means optical density
tion of the cytoskeleton and the formation. In the course of
(490 nm)± SD of three independent experiments performed in examination of statin's effects on balance of tPA-PA
duplicate determination. *P<0.05 versus control inhibitor (PAI), we found actin skeleton perturbation and
morphology alterations of peritoneal cells in response to
statin (data not shown). Statin acts on peritoneal cells by the
Discussion possible mechanism involving RhoA-modified actin fila-
ment reorganization [3, 14, 15].
Statins are synthetic HMG-CoA reductase inhibitors that are In summary, our study describes that lovastatin increased
potent suppressors of cholesterol biosynthesis in humans. tPA production by a mechanism involving geranylgeranyl-
Recently, increasing evidence suggests that statins exert modified intermediates in Met5A cells in culture. The
pleiotropic effects independent of cholesterol reduction, in proposed mechanism is summarized in Fig. 5. RhoA would
particular, in animal models [3, 7]. For postoperative adhesion play a central role in signal transduction for peritoneal cells
prevention, several in vitro studies indicated that statins
increased fibrinolytic activity in human peritoneal cells Lovastatin
independent of cholesterol lowering [3, 7]. In this study, we
showed that lovastatin caused a dose-dependent increase in Plasma membrane

tPA production within a range of concentrations 0.1–1µM Rho kinase

using Met5A cells; lovastatin had no effect on PAI levels. HMG-CoA
More than 5µM, lovastatin had possibility of a cytotoxic Lovastatin
Reductase
effect of lovastatin in the Met-5A cells, as previously reported (-) C3 (-)
[8]. By inhibition of HMG-CoA reductase, statins block the
reduction of HMG-CoA to mevalonate which is the precursor PA
RhoA
molecule for the generation of the isoprenoid intermediates
farnesylpyrophosphate and GGPP. The effect of lovastatin to Cholesterol
induce tPA production could be suppressed by GGPP. (+)
The use of this cell line differs in many ways from GGPP
primary cultured mesothelial cells, for example, from RhoA
omental tissues or cells derived from peritoneal fluid. They
differ in the release of fibrinolytic factors upon stimulation. Fig. 5 The proposed effects of lovastatin on plasminogen activator
(PA) upregulation. Hydroxymethylglutaryl (HMG)-CoA reductase
However, consideration of poor knowledge regarding stimulates the synthesis of mevalonate and isoprenoid intermediate
mechanism and treatment against postoperative peritoneal geranylgeranyl pyrophosphate (GGPP). GGPP catalyzes the geranyl-
adhesion led us to study the Met5A cells for fibrinolytic PA geranylation of RhoA, a key step in the translocation of RhoA from
activity because of no information using Met5A cells. cytoplasm to the plasma membrane. The active membrane-bound
RhoA initiates downstream signaling via Rho kinase to downregulate
The Rho family is one of the most important geranylger- PA. HMG-CoA inhibitor lovastatin reduces RhoA activation and
anylated proteins. The Rho belongs to the Ras superfamily fibrinolytic activity by preventing the membrane localization and
of GTPases that cycle between an inactive cytosolic GDP- activation of PA production
Gynecol Surg (2010) 7:189–193 193

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