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Originally published In Press as doi:10.1074/jbc.M201253200 on February 11, 2002

J. Biol. Chem., Vol. 277, Issue 18, 15309-15316, May 3, 2002
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Inhibition of Protein Geranylgeranylation and RhoA/RhoA Kinase Pathway Induces Apoptosis in Human Endothelial Cells*

Xianwu LiDagger , Li LiuDagger , Joan C. TupperDagger , Douglas D. Bannerman§, Robert K. Winn§, Said M. Sebti, Andrew D. Hamilton||, and John M. HarlanDagger **

From the Departments of Dagger  Medicine and § Surgery, University of Washington, Seattle, Washington 98195, the  Department of Biochemistry and Molecular Biology, The University of South Florida, Tampa, Florida 33612, and the || Department of Chemistry, Yale University, New Haven, Connecticut 06520

Received for publication, February 6, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Geranylgeranylation of RhoA small G-protein is essential for its localization to cell membranes and for its biological functions. Many RhoA effects are mediated by its downstream effector RhoA kinase. The role of protein geranylgeranylation and the RhoA pathway in the regulation of endothelial cell survival has not been elucidated. The hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor lovastatin depletes cellular pools of geranylgeranyl pyrophosphate and farnesol pyrophosphate and thereby inhibits both geranylgeranylation and farnesylation. Human umbilical vein endothelial cells (HUVECs) were exposed to lovastatin (3 µM-30 µM) for 48 h, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments as well as caspase-3 activity. The assays showed that lovastatin caused a dose-dependent endothelial cell death. The addition of geranylgeraniol, which restores geranylgeranylation, rescued HUVEC from apoptosis. The geranylgeranyltransferase inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, induced apoptosis in HUVEC. Cell death was also induced by a blockade of RhoA function by exoenzyme C3. In addition, treatment of HUVEC with the RhoA kinase inhibitors Y-27632 and HA-1077 caused dose-dependent cell death. Y-27632 did not inhibit other well known survival pathways, such as NF-kappa B, ERK, and phosphatidylinositol 3-kinase/Akt. However, there was an increase in p53 protein level concomitant with Y-27632-induced cell death. Unlike the apoptosis induced by TNF-alpha , which occurs only with inhibition of new protein synthesis, apoptosis induced by inhibitors of HMG-CoA reductase, geranylgeranyltransferase, or RhoA kinase was blocked by cycloheximide. Our data indicate that inhibition of protein geranylgeranylation and RhoA pathways induce apoptosis in HUVEC and that induction of p53 or other proapoptotic proteins is required for this process.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Apoptosis is a regulated process of programmed cell death, exhibiting characteristic morphological and biochemical hallmarks, which differ from those seen in necrosis. The function of apoptosis is to eliminate excessive cells, as well as those cells that develop improperly or sustain genetic damage. Apoptosis plays an important role both in development and in homeostasis and is a regulatory mechanism in organ growth, wound repair, tumor genesis, and immune response (1).

Endothelial cells (EC)1 line vessels in every organ system and regulate the flow of nutrient substances, diverse biologically active molecules, and blood cells themselves. The regulation of EC survival and death is critical to vascular development and homeostasis. Perturbations of this balance contribute to various vascular diseases (2). Endothelial cell apoptosis is induced by diverse extrinsic and intrinsic signals, such as serum starvation (3), radiation (4), oxidative stress from hypoxia (5), lipopolysaccharide, and cytokines (6). We first reported that human ECs were rendered susceptible to tumor necrosis factor (TNF)-alpha -initiated death by RNA or protein synthesis inhibitors, indicating that TNF-alpha initiates a survival pathway that depends on protein synthesis and a death pathway that does not (6, 7).

Prenylation is an important mechanism of posttranslational modification of proteins. Prenylated proteins are modified by formation of cysteine thioethers with the isoprenoid lipids, farnesyl (C-15), or geranylgeranyl (C-20), at the carboxyl terminus. Geranylgeranylation and farnesylation are catalyzed by the enzymes geranylgeranyltransferase (GGTase) and farnesyltransferase (FTase), respectively. Both enzymes modify cysteines of proteins that end with the motif CAAX (C is Cys, A is an aliphatic amino acid, X is any amino acid) at their carboxyl-terminal. GGTase prefers leucine or isoleucine in the X position, whereas FTase prefers serine or methionine (8, 9). GGTI-298 and FTI-277 are CAAX peptidomimetics that potently and selectively inhibit GGTase I and FTase, respectively (10, 11). Prenylation is required for proper subcellular localization and biological function of these proteins (12, 13). The proteins that undergo these modifications participate in important cell regulatory functions, particularly signal transduction pathways. Protein prenylation has been shown to be involved in cell adhesion (14), cell proliferation (15), malignant transformation (11), and cell survival (16).

Many of the prenylated proteins are small G-proteins, which include the Ras, Rho, and Rac families. Prenylation of small G-proteins with farnesyl or geranylgeranyl groups is essential for their localization to cell membranes and hence for their biological functions. RhoA is geranylgeranylated (17), whereas H-Ras is selectively farnesylated (12). Ras family proteins are involved in the regulation of cell growth, differentiation, and apoptosis. The major function of RhoA is to regulate the assembly and organization of the actin cytoskeleton. When cells are stimulated, the activated RhoA binds to specific effectors to exert its biological function. A variety of putative RhoA effectors have been identified (18, 19). Among these effectors, the serine/threonine kinase, named RhoA kinase, has been found to mediate multiple RhoA effects, such as formation of actin stress fibers and focal adhesion (20), EC barrier dysfunction (21), vascular smooth muscle cell DNA synthesis and migration (22), and angiogenesis (23).

Recently, a specific RhoA kinase inhibitor, named Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] was developed (24). Y-27632 has been shown to inhibit the RhoA kinase family 100 times more potently than other kinases, including protein kinase C, cAMP-dependent kinase, and myosin light chain kinase. Subsequently, Y-27632 has been widely used as a RhoA kinase-inhibitor to evaluate the involvement and roles of RhoA kinase in a variety of systems (20-23). In addition, 1-(5-isoquinolinesulfonyl) homopiperazine (HA-1077), which is structurally unrelated to Y-27632, has been recently identified as a potent inhibitor of RhoA kinase (25-27).

Many of the previous studies on the RhoA pathway have focused on adhesion and cytoskeletal reorganization. Much less is known about the participation of RhoA and its effector RhoA kinase on cell survival. The limited published data on this are controversial due to different approaches and cell types (28-31). In the present study, we directed our focus on the role of protein prenylation in general and the RhoA pathway more specifically in the regulation of EC survival. We demonstrate that inhibition of protein geranylgeranylation by lovastatin and GGTI-298 or inhibition of RhoA pathway by exoenzyme C3 and Y-27632 induces apoptosis in HUVEC. Unlike the apoptosis induced by TNF-alpha , which requires the inhibition of new protein synthesis, apoptosis induced by these inhibitors is dependent on de novo protein synthesis. In addition, there is an increase in p53 protein level concomitant with cell death. These results suggest that induction or activation of p53 and other proapoptotic proteins is required for apoptosis induced by inhibition of protein geranylgeranylation or by the RhoA/RhoA kinase pathway.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Cell culture media and supplies were obtained from BioWhittaker (Walkersville, MD). Lovastatin, exoenzyme C3, and HA-1077 were purchased from Calbiochem (La Jolla, CA). RhoA kinase inhibitor, Y-27632, was kindly provided by Welfide Corporation (Osaka, Japan). Ac-DEVD-AMC was purchased from BACHEM (Torrance, CA). Anti-p53 antibody was purchased from BD PharMingen (San Diego, CA). Anti-Ikappa Balpha was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho-Akt, anti-phospho-ERK, anti-phospho-Ikappa Balpha , anti-Akt, and anti-ERK antibodies were purchased from New England Biolabs, Inc. (Beverly, MA).

Cell Culture-- Human umbilical vein endothelial cells (HUVECs) were isolated and cultured as previously described (32). Cells were used in passages two through five.

Immunoblot Analysis-- After experimental treatment, the confluent HUVEC monolayers were washed with ice-cold phosphate-buffered saline, lysed with ice-cold RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, protease inhibitor mixture tablet (Roche Molecular Biochemicals), 1 µg/ml pepstatin, 1 µg/ml type I DNase, 1 mM vanadate, 50 mM NaF). The protein was collected by microcentrifugation at 12,000 rpm for 15 min. The supernatants were resolved by SDS-PAGE on a 4-20% Tris glycine gradient gel (Invitrogen Inc., Carlsbad, CA) and transferred to nitrocellulose membrane. Blots were blocked overnight with 5% skim milk in TBST (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20) and then incubated with primary antibodies at a 1:1000 dilution for 1 h. The blots were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin. Immunoreactive proteins were detected with enhanced chemiluminescence (Amersham Biosciences) and exposed to Kodak X-Omat Film.

Cell Death Detection Enzyme-linked Immunosorbent Assay-- We used a cell death detection enzyme-linked immunosorbent assay kit (Roche Diagnostics, Indianapolis, IN) to quantitatively determine cytoplasmic histone-associated DNA oligonucleosome fragments associated with apoptotic cell death. Briefly, after treatment, cells were lysed with 200 µl of lysis buffer and incubated for 30 min at room temperature. Then 20 µl of supernatant was transferred into the streptavidin-coated microtiter plate, and 80 µl of the immunoreagent was added to each well. After incubation at room temperature for 2 h, the solution was decanted, and each well was rinsed three times with incubation buffer. Color development was carried out by adding 100 µl of ABTS solution, and absorbency was measured at 405 nm against ABTS solution as a blank.

Caspase-3 Activity Assay-- Caspase-3 activity was measured by the method of Enari et al. (33) with some modification. In brief, after 1 × 106 cells were treated as indicated, cytosolic extracts were prepared by adding 300 µl of incubation buffer (Caspase-3 activity assay Kit, Roche Diagnostics). 100 µl of cell lysates were transferred into a 96-well plate, and additional 100 µl of incubation buffer with 100 µM Ac-DEVD-AMC were added to each well. After 2.5 h of incubation at 37 °C, the fluorescence of the cleaved substrate was measured by a spectrofluorometer (Cytofluor 4000) with an excitation wavelength at 360 nm and an emission wavelength at 460 nm.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lovastatin Induces HUVEC Death-- Previous studies showed that lovastatin as well as other HMG-CoA reductase inhibitors induced apoptosis in various cell types (15, 16). To determine whether lovastatin had a similar effect on EC, HUVECs were cultured in medium containing various concentrations of lovastatin for 48 h, and cell death was then assayed by cytoplasmic histone-associated DNA fragments. As shown in Fig. 1A, lovastatin caused a dose-dependent increase in cell death with a 9-fold increase compared with control at 30 µM. Because lovastatin inhibits protein geranylgeranylation by depleting cellular pools of geranylgeranyl pyrophosphate, we next determined whether the addition of geranylgeraniol (GGOH), which restores geranylgeranylation, would rescue cells from apoptosis. HUVECs were coincubated with 30 µM lovastatin and 5 µM GGOH for 48 h. As shown in Fig. 1, B and C, treatment of HUVECs with GGOH almost completely reversed cell death induced by lovastatin. However, farnesol, which restores farnesylation, did not rescue cells from apoptosis (data not shown). These results suggest that inhibition of protein geranylgeranylation accounts for lovastatin-induced cell death.


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  		Fig. 1.   Lovastatin induces endothelial cell death. A, HUVECs were treated with various concentrations of lovastatin for 48 h, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments. B, HUVECs were incubated with 30 µM lovastatin in the presence or absence of 5 µM GGOH for 48 h, and cell death was determined as above. C, cells were treated as in B and then visualized by phase contrast microscopy. Magnification was ×400, medium control (panel A), lovastatin (panel B), GGOH (panel C), and lovastatin and GGOH (panel D). Results are the means ± S.D. of triplicate wells in a single experiment and are representative of three separate experiments.

Inhibition of Protein Geranylgeranylation, but Not Farnesylation, Induces HUVEC Death-- To confirm that lovastatin induced apoptosis by inhibiting protein geranylgeranylation, we next examined whether the GGTase inhibitor, GGTI-298, mimicked the effect of lovastatin. HUVECs were treated with a range of concentrations of GGTI-298 or the FTase inhibitor FTI-277, and histone-associated DNA fragmentation was measured after 48 h. As shown in Fig. 2A, treatment of these cells with GGTI-298 caused a significant increase of cell death at 10 and 30 µM. In contrast, cells treated with FTI-277 at the same concentrations showed very little increase in cell death compared with control cells. The above results indicate that inhibition of protein geranylgeranylation but not farnesylation induces cell death in HUVEC.


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  		Fig. 2.   Blockade of the RhoA/RhoA kinase pathway induces cell death in HUVEC. HUVECs were treated with the various inhibitors, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments. Results are the means ± S.D. of triplicate wells in a single experiment and are representative of three separate experiments. A, GGTI-298 and FTI-277 for 48 h. B, 30 µg/ml exoenzyme C3 for 24 h. C, Y-27632 for 24 h. D, HA-1077 for 24 h.

Inhibition of the RhoA/RhoA Kinase Pathway Induces HUVEC Death-- Because RhoA is a geranylgeranylated protein, the inhibition of protein geranylgeranylation should affect the RhoA pathway. We hypothesized that the apoptosis induced by lovastatin and GGTI-298 was mediated by RhoA and its effector, RhoA kinase. Clostridium botulinum exoenzyme C3 catalyzes the specific ADP-ribosylation and inactivation of RhoA, RhoB, or RhoC and has been used to probe Rho function. Treatment of HUVECs with 30 µg/ml C3 for 24 h caused a 2-3-fold increase in histone-associated DNA fragmentation (Fig. 2B). We next examined whether inhibition of RhoA kinase, the immediate downstream effector of RhoA, caused HUVEC death. Two structurally different RhoA inhibitors, Y-27632 and HA-1077, were used to address this question. HUVECs were incubated in the absence or presence of inhibitors at concentrations ranging from 3 to 30 µM for 24 h. Fig. 2, C and D show that treatment of HUVECs for 24 h with either Y-27632 or HA-1077 induced a cell death in a concentration-dependent manner with a 5-7-fold increase in cell death at the concentration of 30 µM for both inhibitors. These data show that inhibition of RhoA or RhoA kinase reduced endothelial cell survival.

Activation of Caspase-3 by Lovastatin, GGTI-298, and Y-27632 Treatment in HUVEC-- Because caspase-3 plays a key role in various forms of apoptosis, we investigated whether caspase-3 was involved in the cell death induced by these inhibitors. HUVECs were treated with lovastatin, GGTI-298, or Y-27632 at 30 µM for 24 h, and caspase-3 activity was then determined by use of a fluorogenic tetrapeptide substrate, Ac-DEVD-AMC. Fig. 3 shows that 24 h of treatment with the inhibitors resulted in a 6-fold increase in caspase-3 activity. The same result was obtained when cells were treated with 10 ng/ml TNF-alpha and 1 µg/ml cycloheximide. Treatment of HUVECs with 30 µM FTI-277 showed no substantial increase in caspase-3 activity (data not shown). Thus, apoptosis induced by these inhibitors involved caspase-3.


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  		Fig. 3.   Caspase-3 activation by lovastatin, GGTI-298, and Y-27632. HUVECs were treated with 30 µM lovastatin, 30 µM GGTI-298, 30 µM Y-27632, or 10 ng/ml TNF-alpha plus 1 µg/ml cycloheximide for 24 h. Caspase-3 activity was measured by using Ac-DEVD-AMC as a substrate. Results are the means ± S.D. of triplicate wells in a single experiment and are representative of three separate experiments.

De Novo Protein Synthesis Is Required for the Apoptosis Induced by Lovastatin, GGTI-298, and Y-27632 but Not for TNF-alpha -- Apoptosis is affected differentially by protein synthesis inhibition depending on the cell type and stimulus. Our previous studies showed that TNF-alpha induced HUVEC cell death only in the presence of cycloheximide or actinomycin D (6, 7). These results were confirmed as shown in Fig. 4. The role of new protein synthesis in lovastatin-induced apoptosis was examined. HUVECs were coincubated with cycloheximide and lovastatin for 48 h. In contrast to TNF-alpha , lovastatin-induced cell death, demonstrated by both cell death enzyme-linked immunosorbent assay (Fig. 4A) and microscopic examination (Fig. 4B), was completely abrogated by cycloheximide at the concentration of 1 µg/ml. Cycloheximide also prevented cell death induced by GGTI-298 and Y-27632 (Fig. 4).


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  		Fig. 4.   Effect of protein synthesis inhibition on apoptosis in HUVEC. A, HUVECs were treated with 30 µM lovastatin, 30 µM GGTI-298, and 30 µM Y-27632 for 48 h or 10 ng/ml TNF-alpha for 24 h in the presence or absence of 1 µg/ml cycloheximide, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments. Results are the means ± S.D. of triplicate wells in a single experiment and are representative of three independent experiments. B, cells were treated as in A and then visualized by phase contrast microscopy. Magnification was ×400, medium control (panel A), 30 µM lovastatin (panel B), 30 µM GGTI-298 (panel C), 30 µM Y-27632 (panel D), 10 ng/ml TNF-alpha (panel E), 1 µg/ml cycloheximide (panel F), 30 µM lovastatin + 1 µg/ml cycloheximide (panel G), 30 µM GGTI-298 + 1 µg/ml cycloheximide (panel H), 30 µM Y-27632 + 1 µg/ml cycloheximide (panel I), 10 ng/ml TNF-alpha  + 1 µg/ml cycloheximide (panel J).

Y-27632 Increases p53 Protein Level-- The p53 tumor suppressor gene is crucial in some forms of apoptosis. Several studies have shown that p53 is involved in RhoA-regulated survival pathways (34, 35). We examined whether p53 protein level was increased in HUVEC after inhibition of RhoA/RhoA kinase pathway. Fig. 5 shows that a minimal level of p53 protein was present in untreated HUVECs. However, treatment of HUVECs with Y-27632 at 30 µM caused a significant increase in p53 protein level. Cycloheximide at 1 µg/ml abrogated apoptosis induced by Y-27632 and prevented the induction of p53 (Fig. 5). These results show a correlation between cell death induced by RhoA kinase blockade and p53 induction.


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  		Fig. 5.   Induction of p53 by Y-27632 is abrogated by cycloheximide. HUVECs were incubated with 30 µM Y-27632 in the presence or absence of 1 µg/ml cycloheximide for 24 h, and lysates were then subjected to immunoblotting with anti-p53 antibody.

RhoA Kinase Inhibitor Y-27632 Does Not Affect the Activation of NF-kappa B, ERK, and Akt-- NF-kappa B, ERK, and PI 3-kinase/Akt are well known survival pathways in EC as well as in other cell types (6, 36-38). We next investigated whether Y-27632 inhibited activation of these pathways. The activation of NF-kappa B was evaluated by immunoblot using phospho-Ikappa Balpha and Ikappa Balpha antibody. As shown in Fig. 6A, the cytoplasmic Ikappa Balpha , but not phospho-Ikappa Balpha , was detected in untreated HUVEC. Treatment with TNF-alpha for 15 min resulted in phosphorylation of Ikappa Balpha as well as Ikappa Balpha degradation. However, pretreatment with Y-27632 for 30 min did not block Ikappa Balpha phosphorylation and degradation induced by TNF-alpha . The activation of ERK and Akt by TNF-alpha was assessed by immunoblot using phosphoprotein-specific antibodies. The phospho-ERK and phospho-Akt protein levels were dramatically increased after stimulation by TNF-alpha for 15 min, and preincubation with Y-27632 had no effect on level of these phosphorylated proteins (Fig. 6, B and C). These data suggest that Y-27632 induced-apoptosis is not mediated by inhibition of well identified survival pathways, including NF-kappa B, ERK, and PI 3-kinase/Akt pathways.


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  		Fig. 6.   Y-27632 does not inhibit the activation of NF-kappa B, ERK, or Akt. HUVECs were cultured overnight in serum-free medium, then pretreated with 30 µM Y-27632 for 30 min, and then treated with 10 ng/ml TNF-alpha for 15 min. Cell lysates were immunoblotted with antibodies against: phosphorylated Ikappa Balpha and Ikappa Balpha (A), phosphorylated ERK and ERK (B), and phosphorylated Akt and Akt (C).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Prenylation of protein is an important posttranslational modification, which is required for cellular localization and biological function of small G-proteins. The addition of isoprenoid lipid farnesyl or geranylgeranyl is mediated by the enzymes FTase and GGTase, respectively. Lovastatin and related drugs are inhibitors of HMG-CoA reductase, an early and rate-limiting enzyme in the sterol synthesis pathway. The inhibitors reduce the level of isoprenoids including geranylgeranyl pyrophosphate and farnesyl pyrophosphate by depleting cellular pools of the precursors, which are substrates for GGTase and FTase, respectively. Several studies have shown that lovastatin, as well as other HMG-CoA reductase inhibitors, inhibit both geranylgeranylation and farnesylation in various cell types. In this study, we found that lovastatin caused dose-dependent cell death in HUVEC (Fig. 1A), which is consistent with other reports showing cell death induced by lovastatin (15, 16). GGOH, but not farnesol, reversed the effect of lovastatin, suggesting that a protein(s) modified by a geranylgeranyl group is required for EC survival (Fig. 1, B and C).

Next, we used an alternative approach to demonstrate that protein geranylgeranylation is required for EC survival. FTI-277 and GGTI-298 are CAAX peptidomimetics that potently and selectively inhibit FTase and GGTase, respectively. These inhibitors have been widely used to evaluate the biological functions of prenylated proteins and have been shown the specific inhibition of protein farnesylation or geranylgeranylation in various cell types (10, 11, 14, 15). We used these inhibitors to assess the importance of protein farnesylation and geranylgeranylation in HUVEC survival. Consistent with a selective effect on protein prenylation, the inhibitors also showed a different effect on EC viability. GGTI-298 induced cell death in a dose-dependent manner as measured by histone assay, whereas FTI-277 had little effect on cell viability (Fig. 2A). Caspase-3 activity was also increased after 24 h of treatment with GGTI-298 (Fig. 3), correlating with cell death measured by histone assay. These results suggest that the inhibition of protein geranylgeranylation induces caspase-3-mediated apoptosis. Protein geranylgeranylation has been shown to be involved in survival in vascular smooth muscle cells (15) and rat cortical neurons (16), as well as human lung adenocarcinoma A549 cells (39). Our results are consistent with these other studies.

Rho subfamily proteins require geranylgeranylation for their function, and several have been implicated in the regulation of cell survival (28, 30). Our studies showed that 48 h of treatment with lovastatin or GGTI-298 induced EC cell death, which is consistent with the half-life of Rho proteins. We further show that cell death was induced by exoenzyme C3 transferase (Fig. 2B), which is a highly specific inhibitor of RhoA, RhoB, and RhoC, whereas other Rho subfamily members, Rac and Cdc42, are poor substrates (40, 41). These results indicate that the Rho pathway is involved in lovastatin- and GGTI-298-induced cell death.

RhoA kinase is an immediate effector of RhoA, and it has been implicated in most of the identified functions of RhoA (18). Recently, a new synthetic compound, Y-27632, has been widely used as a specific inhibitor of RhoA kinase to identify the roles of RhoA pathway in a variety of systems (21-24). Here we used Y-27632 to determine the role of RhoA kinase in cell viability. Treatment with Y-27632 at concentrations from 3 to 30 µM induced EC death (Figs. 2C and 4). Y-27632 also increased caspase-3 activity, consistent with caspase-mediated apoptosis (Fig. 3). Another RhoA kinase inhibitor HA-1077, which is structurally unrelated to Y-27632, was used to confirm the effect of inhibition of RhoA kinase on cell viability. HA-1077 has been demonstrated to inhibit purified RhoA kinase with an IC50 value of ~2 µM and to prevent RhoA kinase-mediated inhibition of myosin phosphatase in smooth muscle cells (25). Increased synthesis of phosphatidylinositol 4,5-bisphosphate induced by overexpression of RhoA and constitutively active RhoA kinase was reversed by HA-1077 (42), and HA-1077 specifically blocked the stress fiber and focal adhesion formation induced by the active form of RhoA or RhoA kinase in NIH 3T3 cells (43). Like Y-27632, treatment of HUVECs with HA-1077 induced dose-dependent cell death (Fig. 2D). Thus, a blockade of RhoA/RhoA kinase pathway by multiple reagents induced EC apoptosis, demonstrating that the RhoA/RhoA kinase pathway is a survival pathway in EC. Of note, the fact that Y-27632 did not promote TNF-alpha -induced EC death suggests that the RhoA/RhoA kinase pathway is not a component of the TNF-alpha -induced survival pathway in EC (data not shown).

Support for a role for RhoA/RhoA kinase as cell survival signaling comes from studies in other cell types. Overexpression of a constitutively active form of RhoA protein enhanced resistance of HeLa cells to the cytotoxic effects of Clostridium difficle toxins (44). Activation of RhoA by cytotoxic necrotizing factor-1 (CNF-1) inhibited epithelial cell apoptosis induced by UVB (30). Bobak et al. created a novel system that could rapidly and efficiently produce high intracellular levels of C3 transferase in intact cells and found that the inactivation of RhoA by C3 exoenzyme induced apoptosis in murine L929 cells (28). Thymocytes from C3 transferase-transgenic mice that lack RhoA function showed cell survival defects (45). Treatment of rats with Y-27632 also significantly increased apoptotic smooth muscle cells in neointima (46). In contrast to these studies demonstrating a survival function for RhoA/RhoA kinase, Lacal and co-workers showed that the overexpression of constitutively active forms of RhoA in NIH 3T3 cells induced apoptosis upon serum deprivation but not in the presence of serum (29). There are several possible reasons for the conflicting results of Lacal and co-workers and our studies. First, they overexpressed constitutively active RhoA, whereas we studied the endogenous enzyme. Second, in our system, HUVECs were cultured in serum-containing medium, which is a physiological condition, whereas they studied serum withdrawal. However, we observed that serum deprivation increased apoptosis induced by both Y-27632 and HA-1077 (data not shown). Finally, the regulation of cell survival by the RhoA/RhoA kinase pathway may be dependent upon the cell type. NIH 3T3 cells, which were used by Lacal and co-workers, are an immortalized murine cell line, whereas HUVECs examined in our study are human primary cells.

RhoA/RhoA kinase plays a critical role in the regulation of the assembly and organization of the actin cytoskeleton (18), which are required for cell spreading and focal adhesion. A previous study showed that increased endothelial cell spreading promoted cell survival and growth (47). Therefore, inhibition of cell spreading as well as adhesion may account for the reduction of cell survival produced by disrupting the RhoA/RhoA kinase pathway. Consistent with this notion, Y-27632 has been reported to block formation of stress fibers and cell spreading in various cell types including EC (21). In addition, Y-27632 inhibited phosphorylation of focal adhesion kinase and the assembly of focal contacts (21, 26, 48). Moreover, activation of RhoA by CNF-1 increased the expression of focal adhesion kinase and promoted cell spreading (49). Recently, RhoA kinase activation was showed to be required for apoptotic membrane blebbing in NIH 3T3 and Jurkat cells (50, 51). Notably, although Y-27632 treatment inhibited membrane blebbing induced by the apoptosis stimulus, it did not prevent the biochemical processes of apoptosis, such as caspase activation, release of cytochrome c from mitochondria, or exposure of phosphatidylserine on the outer plasma membrane. Thus, membrane blebbing appears to be a concomitant morphological change rather than a cause of apoptosis. Consequently, it does not appear that RhoA kinase is a proapoptotic pathway in these cells. Although our results indicate that RhoA is involved in the regulation of HUVEC survival, we cannot rule out the possibility that, in addition to RhoA, other geranylgeranylated proteins are also involved.

The tumor suppressor gene p53 has been implicated in apoptosis in response to direct genomic damage (radiation and chemotherapeutic agents) as well as physiological stimuli (hypoxia and oxidative stress) (52). p53-induced apoptosis is mediated by both transcription-dependent and -independent pathways. Several studies demonstrated that p53 plays a critical role in cell death induced by inhibition of the RhoA pathway or protein geranylgeranylation. Thymocytes from p53-/- mice were shown to be resistant to C3 transferase-induced death (34). While inhibition of Rac1 and Cdc42 by dominant-negative mutants efficiently triggered apoptosis in adherent fibroblasts, cell death was not observed in p53-/- cells (35). In the present study, we found that Y-27632 increased endogenous p53 protein levels. In addition, the induction of p53 protein as well as cell death induced by Y-27632 was abrogated by 1 µg/ml cycloheximide (Figs. 4 and 5). Induction of p53 by blockade of the RhoA/RhoA kinase pathway may be due to inhibition of cell adhesion signaling. Ligation of integrin alpha vbeta 3 in EC suppressed p53 activity and increased the Bcl-2:Bax ratio, thereby promoting cell survival (53). In contrast, blocking integrin alpha vbeta 3 ligation with integrin antagonists induced p53 activation and inhibited Bcl-2 expression (53). Both CNF-1, an activator of RhoA, and constitutively active RhoA induced Bcl-2 expression (54, 55). Our previous study also showed that endogenous p53 level correlated with cell death in hypoxic HUVEC, and over-expression of p53 protein via adenoviral vector was sufficient to induce apoptosis in HUVEC (5). Taken together, we conclude that induction of p53 or other proapoptotic proteins is required for EC death induced by inhibition of RhoA/RhoA kinase pathway.

Apoptosis is an active process, and protein synthesis is required in certain types of apoptosis in certain cell types. Consistent with our previous studies, we found that 10 ng/ml TNF-alpha alone did not induce HUVEC cell death. However, coincubation of cells with TNF-alpha and 1 µg/ml cycloheximide resulted in substantial EC death, indicating that inducible or constitutive cytoprotective proteins are required for HUVEC survival during exposure to TNF-alpha (6, 7). In contrast, apoptosis induced by lovastatin, GGTI-298, or Y-27632 was prevented by the same concentration of cycloheximide. These results suggest that de novo protein synthesis is required for cell death induced by inhibition of protein geranylgeranylation or RhoA/RhoA kinase signaling. The mechanisms of apoptosis could be different in the same cell type, depending on different apoptotic stimuli. One simple explanation for the protective effects of cycloheximide is that it prevents the expression of proapoptotic genes, such as p53 or others. Additionally, treatment of cells with inhibitors of protein prenylation such as lovastatin and GGTI-298 leads to depletion of the prenylated forms of proteins and accumulation of the non-prenylated forms of proteins. The non-prenylated proteins may exert a dominant-negative effect due to sequestration of functionally prenylated proteins (10). Therefore, an alternative interpretation by which cycloheximide inhibits apoptosis is by preventing the accumulation of non-prenylated proteins. In support of this, cycloheximide at 1 µg/ml has been shown to effectively prevent the incorporation of [14C]mevalonolactone into proteins in J744 cells, thus preventing the synthesis of proteins that would normally be prenylated (56). Cycloheximide has also been shown to prevent other effects associated with inhibition of prenylation, such as breakdown of the actin cytoskeleton after treatment of the cells with lovastatin (57, 58). The exact mechanism of the protective effect of cycloheximide observed in our study needs to be further elucidated.

Y-27632 is an inhibitor of RhoA kinase, which is a serine/threonine kinase. Phosphorylation of serine/threonine residues is involved in the activation of other kinases, such as NF-kappa B, ERK, and PI 3-kinase/Akt. Moreover, activation of these pathways is antiapoptotic in various cell types, including endothelial cells (6, 36-38). A previous study demonstrated that activation of NF-kappa B is regulated by RhoA (59). We therefore investigated whether Y-27632 inhibited activation of these serine/threonine kinases. Fig. 6 shows that the phosphorylation of Ikappa Balpha , ERK, and Akt by TNF-alpha was not inhibited by Y-27632. These results suggest that Y-27632 specifically inhibits RhoA kinase rather than other serine/threonine kinases activated by TNF-alpha . It is therefore unlikely that NF-kappa B, ERK, and PI 3-kinase/Akt are the downstream targets of the RhoA/RhoA kinase survival pathway.

In summary, we demonstrate a role of geranylgeranylated proteins, specifically RhoA, and RhoA kinase in the regulation of EC survival. Unlike the apoptosis induced by TNF-alpha , which requires the inhibition of de novo protein synthesis, apoptosis induced by inhibition of protein geranylgeranylation and RhoA/RhoA kinase requires de novo protein synthesis. Apoptosis induced by inhibition of this pathway may involve the induction of p53 or other proapoptotic proteins. These findings further demonstrate the different mechanisms involved in the regulation of EC survival and apoptosis.

    ACKNOWLEDGEMENTS

We thank the Welfide Corporation for providing Y-27632 and Kristine Eiting for cell culture.

    FOOTNOTES

* This work was supported by United States Public Health Service Grants HL 18645 and 03174 (to J. M. H.) and a Parker B. Francis Fellowship (to L. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Div. of Hematology, University of Washington, Box 359756, Harborview Medical Center, Seattle, WA 98104-2499. Tel.: 206-341-5314; Fax: 206-341-5312; E-mail: jharlan@u.washington.edu.

Published, JBC Papers in Press, February 11, 2002, DOI 10.1074/jbc.M201253200

    ABBREVIATIONS

The abbreviations used are: EC, endothelial cell; TNF, tumor necrosis factor; GGTase, geranylgeranyltransferase; HMG, 3-hydroxy-3-methylglutaryl; FTase, farnesyltransferase; HUVEC(s), human umbilical vein endothelial cell(s); GGOH, geranylgeraniol; ERK, extracellular signal-regulated kinase; PI, phosphatidylinositol; CNF-1, cytotoxic necrotizing factor-1; FTI, farnesyltransferase inhibitor; GGTI, geranylgeranyltransferase inhibitor; NF-kappa B, nuclear factor kappa B.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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