Rac1 Regulation of Surface Expression of Protease-Activated Receptor-1 and Responsiveness to Thrombin in Vascular Smooth Muscle Cells
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动脉硬化血栓血管生物学 2005年第7期
From the Division of Molecular Cardiology (T.Y., K.H., D.B., M.H., J.N., H.K.), Research Institute of Angiocardiology, the Department of Orthopedics (Y.I.), Graduate School of Medical Sciences, and the Kyushu University COE Program on Lifestyle-Related Diseases (H.K.), Kyushu University, Fukuoka, Japan.
Correspondence to Hideo Kanaide, MD, PhD, Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582. E-mail kanaide@molcar.med.kyushu-u.ac.jp
Abstract
Objective— Protease-activated receptor-1 (PAR1) mediates the thrombin-induced proliferation and hypertrophy of vascular smooth muscle cells. A role of Rac1 in the regulation of PAR1 expression was investigated.
Methods and Results— Treatment with simvastatin, a hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor, for 24 hours attenuated the transient [Ca2+]i elevation induced by thrombin. Immunofluorescence staining revealed that simvastatin decreased the surface expression of PAR1 in a manner dependent on protein geranylgeranylation. Introduction of a Rac1/Cdc42 inhibitory fragment but not a RhoA inhibitory fragment using a cell-penetrating peptide also attenuated the response to thrombin and decreased the surface expression of PAR1. Finally, downregulation of Rac1, but not RhoA, using an RNA interference technique attenuated the thrombin-induced [Ca2+]i elevation. However, the level of PAR1 mRNA and the total amount of PAR1 protein remained unchanged.
Conclusions— Here, we provide for the first time 3 lines of evidence that Rac1 plays a critical role in maintaining the surface expression of PAR1 and the responsiveness to thrombin in vascular smooth muscle cells. Rac1 is suggested to regulate the constitutive trafficking of PAR1 and thereby regulate the surface expression of PAR1.
We provided evidence that Rac1 regulates the surface expression of thrombin receptor PAR1 in vascular smooth muscle. Inhibition of Rac1 by hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor, a Rac1/Cdc42 inhibitory fragment, and an RNA interference technique reduced the surface expression of PAR1 and the responsiveness to thrombin.
Key Words: expression ? protease-activated receptor ? Rac1 ? smooth muscle ? thrombin
Introduction
Protease-activated receptor-1 (PAR1) belongs to a family of G-protein-coupled receptors (GPCRs), and it is known to mediate the cellular effects of thrombin in various types of cells.1–5 In vascular smooth muscle cells, PAR1 regulates the contraction, proliferation, and hypertrophy.1,4,6 The expression of PARs has been reported to either increase or decrease under various pathological conditions, including atherosclerosis, balloon injury of arteries, cerebral ischemia, and neuroinflammatory and neurodegenerative disorders.7–12 Therefore, elucidating the mechanism regulating the expression of PARs is important to understand the pathophysiology of such diseases and also to establish new therapeutic strategies. The nascent PAR1 is considered to be targeted first to the plasma membrane, and then subjected to constitutive internalization, thus resulting in the formation of an intracellular pool.13,14 PAR1 then cycles between the plasma membrane and the intracellular pool under resting conditions. The steady-state level of PAR1 on the cell surface is thus determined by a balance among the rate of de novo synthesis, the receptor internalization, and the recruitment from the intracellular pool.14,15 However, the molecular mechanism regulating the expression of PAR1 remains to be elucidated.
The small G-protein is known to regulate the intracellular vesicle trafficking.16 The Rab family is the most studied small G-protein that regulates GPCR trafficking.17 Rab5a has been shown to be required for the agonist-triggered internalization of PAR2, whereas Rab11a has been shown to contribute to the transport of PAR2 from the Golgi apparatus to the plasma membrane.18 On the other hand, the Rho family of small G-proteins has been suggested recently to play an important role in the internalization of membrane proteins such as thromboxane A2 receptor TP?, epidermal growth factor (EGF) receptor, and E-cadherin, and in the endocytosis of fibroblast growth factor 2.19–22 However, the role of the Rho families in the regulation of PAR1 expression has yet to be elucidated.
In the present study, we investigated the role of Rho proteins in the regulation of expression of PAR1 on the cell surface in vascular smooth muscle cells. We first used hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) to inhibit the protein isoprenylation such as geranylgeranylation and farnesylation, which is essential for small G-proteins to be functional.16,23–25 Second, we introduced the inhibitory proteins of Rho proteins using the cell-penetrating peptide of Tat protein.26–30 The RhoA-binding domain (RB) of Rho kinase and the Rac1/Cdc42-binding domain (PBD) of p21-activated protein kinase-1 was used as inhibitory proteins of Rho proteins, as described previously.26,31 Finally, we downregulated RhoA and Rac1 by RNA interference. As a result, the present study provides 3 lines of evidence supporting the critical role that Rac1 plays in the maintenance of PAR1 expression and the responsiveness to thrombin in cultured vascular smooth muscle cells.
Materials and Methods
The human coronary artery smooth muscle cells (HCASMCs) and the rat aortic smooth muscle cells (RASMCs) in primary culture were used. Cells were treated with HMG-CoA reductase inhibitors (statins), inhibitor proteins of RhoA, and Rac1/Cdc42 tagged with a cell-penetrating peptide of Tat protein or small interfering RNA targeted to RhoA and Rac1. Then, the responsiveness to thrombin and the surface expression of PAR1 were evaluated by fura-2 fluorometry and immunofluorescent staining, respectively. The total expression of PAR1, Rac1, RhoA, and ?-actin mRNA was evaluated by RT-PCR. The total expression of PAR1 protein was evaluated by an immunoblot analysis.
Expanded information for Materials and Methods can be found in the online supplement, available at http://atvb.ahajournals.org.
Results
Long-Term Treatment With Simvastatin Attenuated the Thrombin-Induced [Ca2+]i Elevation in Vascular Smooth Muscle Cells
In HCASMCs, thrombin concentration-dependently induced a transient [Ca2+]i elevation (EC50; 0.17 U/mL), with the maximum response at 1 U/mL (Figure 1A). PAR1-activating peptide (100 μmol/L TFLLR-NH2) induced a [Ca2+]i elevation (47.5±4.5%; n=4), which is comparable to that obtained with 1 U/mL thrombin, whereas PAR4-activating peptide (100 μmol/L GYPGKF-NH2) induced only a small [Ca2+]i elevation (1.0±1.6%; n=4; data not shown). PAR1 is thus suggested to mediate most of the response to thrombin in HCASMCs. The 24-hour treatment with simvastatin concentration-dependently attenuated the [Ca2+]i elevation induced by 1 U/mL thrombin, with a significant inhibition seen at 100 nmol/L and higher concentrations (Figure 1C). Such effective concentrations are similar to the plasma concentrations of simvastatin after the oral administration.32 However, application of 1 μmol/L simvastatin 10 minutes before and during stimulation with 1 U/mL thrombin had no effect on the thrombin-induced [Ca2+]i elevation (data not shown). The similar attenuation of the thrombin-induced [Ca2+]i elevation was observed with fluvastatin at a similar concentration range (data not shown). On the other hand, a hydrophilic statin, pravastatin, at a concentration of up to 1 μmol/L, had no significant effect on the [Ca2+]i elevation induced by 1 U/mL thrombin in HCASMCs (Figure 1A and 1C). A similar attenuation of the response to thrombin by simvastatin was observed in RASMCs (Figure 1B and 1D). In RASMCs, thrombin induced [Ca2+]i elevations with EC50 of 0.11 U/mL, but slightly higher concentrations of simvastatin were required to induce a significant inhibition of the thrombin-induced [Ca2+]i elevations.
Figure 1. Effect of HMG-CoA reductase inhibitors on the response to thrombin in vascular smooth muscle cells. A and B, The concentration-dependent response to thrombin in HCASMCs (A) and RASMCs (B). The resting level of [Ca2+]i and the maximal level of [Ca2+]i elevation induced by ionomycin (50 μmol/L in HCASMCs and 25 μmol/L in RASMCs) were assigned to be 0% and 100%. Data are the mean±SEM (n=3). C and D, The concentration-dependent effect of HMG-CoA reductase inhibitors on the response to 1 U/mL thrombin in HCASMCs (C) and RASMCs (D). Data are the mean±SEM (n=5). *P<0.05 vs the control.
Involvement of Protein Geranylgeranylation in the Statin-Induced Attenuation of the Response to Thrombin in Vascular Smooth Muscle Cells
The involvement of protein isoprenylation in the simvastatin-induced attenuation of the response to thrombin was investigated (Figure 2). HCASMCs were cotreated with simvastatin and geranylgeranyl pyrophosphate (GGPP) or farnesyl pyrophosphate (FPP) for 24 hours at the concentrations indicated in Figure 2. Cotreatment with GGPP prevented the attenuation of the response to thrombin by simvastatin in a concentration-dependent manner, with a complete prevention seen with 1 μmol/L GGPP (Figure 2A). However, cotreatment with FPP did not show any preventive effect in HCASMCs (Figure 2A). In RASMCs, GGPP (> 3 μmol/L) completely prevented the simvastatin-induced attenuation of the response to thrombin, whereas FPP partially prevented it even at 30 μmol/L (Figure 2B). The combination of 10 μmol/L GGPP and 10 μmol/L FPP showed the effect similar to that seen with GGPP alone. However, the 24 hour treatment of RASMCs with 10 μmol/L GGTI-298, a geranylgeranyl transferase inhibitor, attenuated the response to 1 U/mL thrombin to the extent similar to that obtained with 10 μmol/L simvastatin, whereas 10 μmol/L FTI-277, a farnesyl transferase inhibitor (FTI), had no effect on the response to thrombin (Figure 2B).
Figure 2. Involvement of protein geranylgeranylation in the simvastatin-induced attenuation of the response to thrombin in vascular smooth muscle cells. The concentration-dependent effect of GGPP and FPP on the simvastatin-induced attenuation of the [Ca2+]i elevation induced by 1 U/mL thrombin in HCASMCs (A) and RASMCs (B). Cells were cotreated for 24 hours with simvastatin (1 μmol/L for HCASMCs and 10 μmol/L for RASMCs) and GGPP or FPP at the indicated concentrations. B also shows the effect of GGTI and FTI on the response to thrombin (n=4). The resting level of [Ca2+]i and the maximal level of the [Ca2+]i elevation induced by ionomycin (50 μmol/L in HCASMCs and 25 μmol/L in RASMCs) were assigned to be 0% and 100%. Data are the mean±SEM (n=4). *P<0.05 vs the control.
Downregulation of the Surface Expression of PAR1 by Simvastatin in Vascular Smooth Muscle Cells
The surface expression of PAR1 was evaluated by immunofluorescence staining with a monoclonal antibody WEDE15 without fixation or permeabilization of the cells, as described previously.33 The untreated control HCASMCs exhibited a spotted pattern of fluorescence mainly on the cell periphery under fluorescence microscopy (Figure 3A). The fluorescence intensity at the peak of the fluorescence distribution was 856.7±290.1 arbitrary units (n=3) in control cells (Figure 3B). The treatment with simvastatin reduced the fluorescence staining and shifted the fluorescence distribution to the left (Figure 3A). The fluorescence intensity at the peak distribution (386.7±75.7 arbitrary units; n=3) was significantly lower than the control (Figure 3B). We reported previously that trypsin removes the epitope of WEDE15 (residues 51 to 64) of PAR1.33 Treatment with trypsin removed most of the staining in control and the simvastatin-treated cells and caused a leftward shift of the fluorescence distribution, thus resulting in a similar peak intensity (55.0±17.3 arbitrary units for control; 55.3±13.1 arbitrary units for simvastatin-treated cells; n=3; Figure 3A and 3B).
Figure 3. The effect of simvastatin and TATHA-PBD on the surface expression of PAR1 and the total cellular expression of PAR1 mRNA and protein in vascular smooth muscle cells. A, The representative microscopic images of immunofluorescence staining with phycoerythrine-conjugated anti-PAR1 antibody and the representative data of flow cytometry obtained with HCASMCs either untreated (control) or treated with 1 μmol/L simvastatin for 24 hours. Cells were stained before (intact) and after trypsin treatment (trypsin). The dashed lines in the histogram indicate the peak of the fluorescence distribution. Bar=50 μm. B, Summary of the fluorescence intensity at the peak of the fluorescence distribution in the cells untreated (control) and treated with 1 μmol/L simvastatin before and after trypsin treatment. Data are the mean±SEM (n=3). C, RT-PCR analysis of PAR1 mRNA expression in RASMCs untreated (control) and treated with 10 μmol/L simvastatin with and without 10 μmol/L GGPP for 24 hours. D, Immunoblot detection of the total amount of PAR1 in HCASMCs untreated (control) and treated with 1 μmol/L simvastatin in the presence and absence of 1 μmol/L GGPP, 3 μmol/L TATHA-PBD and 3 μmol/L (His)6-PBD. A densitometric analysis of electrophoresis was shown bellow the representative photos.
Effect of Rac1 Inhibition on the Expression of PAR1 mRNA and Protein in Vascular Smooth Muscle Cells
An RT-PCR analysis revealed that the level of PAR1 mRNA did not change after the 24-hour treatment of RASMCs with 10 μmol/L simvastatin, either in the presence or absence of 10 μmol/L GGPP (Figure 3C). On the other hand, a Western blot analysis revealed that the level of PAR1 protein did not change after the 24-hour treatment of HCASMCs with 1 μmol/L simvastatin either in the presence or absence of 1 μmol/L GGPP, 3 μmol/L TATHA-PBD (PBD conjugated to a cell-penetrating peptide of human immunodeficiency viral Tat protein and a hemagglutinin tag), or 3 μmol/L hexahistidine-tagged PBD ((His)6-PBD; Figure 3D). Immunofluorescence staining after fixation and permeabilization of the cells could detect PAR1 in the intracellular pool as well as on the plasma membrane.33 The intracellular staining was much higher than the surface staining in HCASMCs, as we reported previously in endothelial cells.33 Treatment with simvastatin or TATHA-PBD had no apparent effect on the intracellular staining of PAR1 (data not shown).
The Effect of Inhibition of Rho Signaling on the Response to Thrombin and the Expression of PAR1
The 24-hour treatment with TATHA-PBD concentration-dependently (EC50; 0.85 μmol/L) inhibited the [Ca2+]i elevation induced by 1 U/mL thrombin (Figure 4A and 4B). The inhibition seen with 3 μmol/L concentrations of TATHA-PBD was similar to that seen with 1 μmol/L simvastatin (Figure 4B). However, the application of 3 μmol/L TATHA-PBD 10 minutes before and during stimulation with thrombin had no significant effect on the [Ca2+]i elevation induced by thrombin (data not shown). The removal of the cell-penetrating peptide (His)6-PBD abolished the inhibitory effect of TATHA-PBD (Figure 4A and 4B). On the other hand, TATHA-RB, a RhoA inhibitory protein, had no effect on the response to thrombin (Figure 4A and 4B). In line with this, 24-hour treatment with 1 μmol/L Y27632, a Rho kinase inhibitor, also had no significant effect on the response to thrombin (Figure 4B). The protein transduction was confirmed by an immunoblot analysis as described previously.26,27 The extract of the cells exposed to the recombinant protein for 24 hours was subjected to the immunoblot detection with an anti-(His)6 antibody. This antibody detected all recombinant proteins (Figure 4C). However, PBD and RB were detected in the cell extract only when they were conjugated with Tat peptide (Figure 4C).
Figure 4. Effect of the transduction of inhibitors of Rho proteins on the response to thrombin in HCASMCs. A, The [Ca2+]i elevations induced by 1 U/mL thrombin in the HCASMCs untreated (control) and treated with simvastatin, TATHA-PBD, (His)6-PBD, TATHA-RB, and Y27632. The resting level of [Ca2+]i and the maximal level of [Ca2+]i elevation induced by 50 μmol/L ionomycin were assigned to be 0% and 100%, respectively. Data are the mean±SEM (n=3). B, Immunoblot detection of recombinant proteins in the extracts of the cells treated with 3 μmol/L of TATHA-PBD, (His)6-PBD, TATHA-RB, and (His)6-RB (lanes C). The purified proteins (100 ng) were loaded as a positive control (lanes P). Actin was detected with naphtol blue black staining after immunodetection to validate the equal loading of the cell extracts.
The effect of the transduction of TATHA-PBD on the expression of PAR1 was investigated by immunofluorescence staining, as shown in Figure 3 (Figure 5). Treatment of HCASMCs with 3 μmol/L TATHA-PBD caused a leftward shift in the fluorescence distribution to an extent similar to that seen with 1 μmol/L simvastatin (Figure 5). However, (His)6-PBD had no significant effect (Figure 5). Trypsin caused a marked reduction in fluorescence staining as shown in Figure 3.
Figure 5. Effect of the transduction of TATHA-PBD on the surface expression of PAR1 in HCASMCs. A, Representative data of a flow cytometric analysis of the immunofluorescence staining with phycoerythrine-conjugated anti-PAR1 antibody in the cells untreated (control) and treated with 1 μmol/L simvatatin, 3 μmol/L TATHA-PBD, 3 μmol/L (His)6-PBD. Control cells were subjected to immunofluorescence staining before and after trypsin digestion as shown in Figure 3. Dashed lines indicate the peak of the fluorescence distribution. B, Summary of the fluorescence intensity at the peak fluorescence distribution in the cells treated as indicated. Data are the mean±SEM (n=3). *P<0.05 vs control.
Effect of Downregulation of RhoA and Rac1 on the Response to Thrombin
RhoA and Rac1 were downregulated by an RNA interference technique. The transfection of control small interfering RNA had no effect on the responsiveness to thrombin in HCASMCs (Figure 6). On the other hand, downregulation of Rac1 but not RhoA significantly attenuated the [Ca2+]i elevation induced by 1 U/mL thrombin.
Figure 6. Effect of downregulation of RhoA and Rac1 on the response to thrombin in HCASMCs. A, RT-PCR analysis of Rac1, RhoA, and ?-actin mRNA expression in the cells untreated (control) and treated with control small interfering RNA (siRNA; control interference) and siRNA targeted against RhoA (RhoA knockdown) and Rac1 (Rac1 knockdown). B, The response to 1 U/mL thrombin in the cells untreated (control) and treated with 1 μmol/L simvastatin, control siRNA, and siRNA targeted against RhoA and Rac1. The resting level of [Ca2+]i and the maximal level of [Ca2+]i elevation induced by 50 μmol/L ionomycin were assigned to be 0% and 100%. Data are the mean±SEM (n=3); P<0.05.
Discussion
We herein demonstrated for the first time that Rac1 plays a crucial role in the maintenance of the surface expression of PAR1 and the responsiveness to thrombin in vascular smooth muscle cells. This conclusion is supported by 3 lines of evidence. First, the inhibition of HMG-CoA reductase reduced the expression of PAR1 on the cell surface and attenuated the response to thrombin. The attenuation of the thrombin response was prevented by cotreatment with GGPP in human and rat smooth muscle cells. In RASMCs, FPP also prevented the simvastatin-induced attenuation of the responsiveness to thrombin, whereas GGTI but not FTI mimicked the effect of simvastatin. These observations suggested that protein geranylgeranylation is a critical step involved in the expression of PAR1 on the cell surface. However, these observations could not specify which proteins are geranylgeranylated and regulate the expression of PAR1. Second, the transduction of the inhibitory protein of Rac1/Cdc42 reduced the surface expression of PAR1 and attenuated the responsiveness to thrombin, whereas the inhibitory protein of RhoA had no significant effect. These observations provided direct evidence that Rac1/Cdc42 but not RhoA is involved in the maintenance of the surface expression of PAR1 and the responsiveness to thrombin. Third, the knockdown of Rac1 but not RhoA by the RNA interference technique inhibited the responsiveness to thrombin. All these observations are consistent with the hypothesis that Rac1, but not Cdc42 or RhoA, plays a crucial role in the maintenance of the surface expression of PAR1 and the responsiveness to thrombin in vascular smooth muscle cells. The possibility that the inhibition of Rac1 directly inhibited the thrombin-induced [Ca2+]i elevation was ruled out on the basis of observations in the short-term treatment with simvastatin and TATHA-PBD.
The mechanism regarding how Rac1 regulates the expression of PAR1 on the cell surface remains to be elucidated. The downregulation of the surface expression of PAR1 by simvastatin was not associated with the downregulation of PAR1 mRNA. The change in the transcription of the PAR1 gene or the stability of PAR1 mRNA is thus not suggested to play a major role in the downregulation of PAR1 induced by inactivation of Rac1. Treatment with simvastatin or inhibition of Rac1 signaling had little effect on the total amount of PAR1 protein. It is thus likely that the alteration of the trafficking of PAR1 plays a critical role in the downregulation of PAR1 induced by the inactivation of Rac1.
PAR1 exhibits both constitutive and agonist-triggered internalization.2,4,14 The 2 modes of PAR1 internalization have been demonstrated to require the distinct residues in C-terminal region of PAR1, and they are thus suggested to be differently regulated by distinct mechanisms.13,15,34 The agonist-activated PAR1 has been shown to be internalized via a clathrin- and dynamin-dependent but arrestin-independent pathway, and then it mainly targets to lysosome degradation.14,15 On the other hand, the constitutive internalization takes place under resting conditions without receptor stimulation, and it has been reported to be necessary for the formation and the maintenance of the intracellular PAR1 pool.13 Therefore, the resting level of the surface expression of PAR1 is suggested to be dynamically maintained by continuous cycling between the cell surface and the intracellular pool. Our finding suggests that Rac1 regulates such constitutive trafficking of PAR1. The inhibition of Rac1 activity may either increase the constitutive internalization or inhibit the membrane targeting, thereby decreasing the level of PAR1 expressed on the cell surface in cultured smooth muscle cells.
PAR1 has been reported to be upregulated in smooth muscle cells seen in the human atherosclerotic lesions and in a rat carotid artery balloon injury model.10–12 The level of PAR1 expression has been shown to correlate to the degree of proliferation of smooth muscle cells.10,12 Growth factors such as platelet-derived growth factor, EGF or insulin, phorbol ester, and some GPCR agonists such as bombesin and lysophosphatidic acid were shown to activate Rac1.35 Rac1 is thus speculated to contribute to the upregulation of PAR1 in the vascular lesions. Our observations suggest that cultured vascular smooth muscle cells have some basal activity of Rac1, which contributes to the maintenance of surface expression of PAR1 under resting conditions. In the present study, we cultured and maintained the cells in the media containing 5% serum, 0.5 ng/mL EGF, 2 ng/mL fibroblast growth factor-B and 5 μg/mL insulin. Thus, it is conceivable that some growth factors in the media contributed to the basal activity of Rac1 in the cultured smooth muscle cells. Thus, our observations may be relevant to situations that are related to atherosclerotic lesions. However, such a possibility remains to be evaluated.
The preventive effect of FPP differed between HCASMCs and RASMCs. GGPP and FPP prevented the simvastatin-induced attenuation of the thrombin response in RASMCs, whereas only GGPP was effective in HCASMCs. Because GGTI but not FTI mimicked the effect of simvastatin in RASMCs, the findings suggested that geranylgeranylation but not farnesylation was involved in the effect of simvastatin in HCASMCs and RASMCs. We speculate that FPP was converted to GGPP in RASMCs, thereby exerting its preventive effect. GGPP is synthesized by a condensation of FPP and isopentenyl pyrophosphate.24,36 The residual amount of isopentenyl pyrophosphate after the inhibition of HMG-CoA reductase by simvastatin may be high enough to convert the exogenously added FPP to GGPP and to restore protein geranylgeranylation in RASMCs. However, this was not the case in HCASMCs, in which the amount of isopentenyl pyrophosphate was not sufficient to convert FPP to GGPP. The degree of inhibition of HMG-CoA reductase by statins or metabolism of isopentenyl pyrophosphate may differ between HCASMCs and RASMCs.
In conclusion, the present study demonstrated for the first time that Rac1 plays a critical role in the maintenance of the surface expression of PAR1 and the responsiveness to thrombin in the cultured vascular smooth muscle cells. The inactivation of Rac1 by HMG-CoA reductase inhibitor, the introduction of the inhibitor protein and downregulation by RNA interference attenuated the responsiveness to thrombin by reducing the level of the surface expression of PAR1. Because the level of PAR1 mRNA and the total amount of PAR1 protein remained unchanged, Rac1 is suggested to regulate the constitutive trafficking of PAR1.
Acknowledgments
This study was supported in part by the grant from the 21st Century COE Program and grants-in-aid for scientific research (No. 15590758 and 16590695) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Brian Quinn for linguistic comments and help with this manuscript.
References
Coughlin SR. Thrombin signaling and protease-activated receptors. Nature. 2000; 407: 258–264.
Dery O, Corvera CU, Steinhoff M, Bunnett NW. Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. Am J Physiol. 1998; 274: C1429–C1452.
Hollenberg MD, Compton SJ. International Union of Pharmacology. XXVIII. Proteinase-activated receptors. Pharmacol Rev. 2002; 54: 203–217.
Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. Proteinase-activated receptors. Pharmacol Rev. 2001; 53: 245–282.
Schmidlin F, Bunnett NW. Protease-activated receptors: how proteases signal to cells. Curr Opin Pharmacol. 2001; 1: 575–582.
Hirano K, Kanaide H. Role of protease-activated receptors in the vascular system. J Atheroscler Thromb. 2003; 10: 211–225.
Noorbakhsh F, Vergnolle N, Hollenberg MD, Power C. Proteinase-activated receptors in the nervous system. Nat Rev Neurosci. 2003; 4: 981–990.
Ossovskaya VS, Bunnett NW. Protease-activated receptors: contribution to physiology and disease. Physiol Rev. 2004; 84: 579–621.
Rohatgi T, Henrich-Noack P, Sedehizade F, Goertler M, Wallesch CW, Reymann KG, Reiser G. Transient focal ischemia in rat brain differentially regulates mRNA expression of protease-activated receptors 1 to 4. J Neurosci Res. 2004; 75: 273–279.
Ku DD, Dai J. Expression of thrombin receptors in human atherosclerotic coronary arteries leads to an exaggerated vasoconstrictory response in vitro. J Cardiovasc Pharmacol. 1997; 30: 649–657.
Nelken NA, Soifer SJ, O’Keefe J, Vu TK, Charo IF, Coughlin SR. Thrombin receptor expression in normal and atherosclerotic human arteries. J Clin Invest. 1992; 90: 1614–1621.
Wilcox J, Rodriguez J, Subramanian R, Ollerenshaw J, Zhong C, Hayzer D, Horaist C, Hanson S, Lumsden A, Salam T. Characterization of thrombin receptor expression during vascular lesion formation. Circ Res. 1994; 75: 1029–1038.
Shapiro MJ, Coughlin SR. Separate signals for agonist-independent and agonist-triggered trafficking of protease-activated receptor 1. J Biol Chem. 1998; 273: 29009–29014.
Trejo J. Protease-activated receptors: new concepts in regulation of G-protein-coupled receptor signaling and trafficking. J Pharmacol Exp Ther. 2003; 307: 437–442.
Marchese A, Chen C, Kim YM, Benovic JL. The ins and outs of G-protein-coupled receptor trafficking. Trends Biochem Sci. 2003; 28: 369–376.
Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev. 2001; 81: 153–208.
Seachrist JL, Ferguson SS. Regulation of G-protein-coupled receptor endocytosis and trafficking by Rab GTPases. Life Sci. 2003; 74: 225–235.
Roosterman D, Schmidlin F, Bunnett NW. Rab5a and rab11a mediate agonist-induced trafficking of protease-activated receptor 2. Am J Physiol. 2003; 284: C1319–C1329.
Izumi G, Sakisaka T, Baba T, Tanaka S, Morimoto K, Takai Y. Endocytosis of E-cadherin regulated by Rac and Cdc42 small G-proteins through IQGAP1 and actin filaments. J Cell Biol. 2004; 166: 237–248.
Rochdi MD, Laroche G, Dupre E, Giguere P, Lebel A, Watier V, Hamelin E, Lepine M-C, Dupuis G, Parent J-L. Nm23–H2 interacts with a G-protein-coupled receptor to regulate its endocytosis through an Rac1-dependent mechanism. J Biol Chem. 2004; 279: 18981–18989.
Tkachenko E, Lutgens E, Stan R-V, Simons M. Fibroblast growth factor 2 endocytosis in endothelial cells proceed via syndecan-4-dependent activation of Rac1 and a Cdc42-dependent macropinocytic pathway. J Cell Sci. 2004; 117: 3189–3199.
Wherlock M, Gampel A, Futter C, Mellor H. Farnesyltransferase inhibitors disrupt EGF receptor traffic through modulation of the RhoB GTPase. J Cell Sci. 2004; 117: 3221–3231.
Bellosta S, Ferri N, Bernini F, Paoletti R, Corsini A. Non-lipid-related effects of statins. Ann Med. 2000; 32: 164–176.
Laufs U, Liao JK. Direct vascular effects of HMG-CoA reductase inhibitors. Trends Cardiovasc Med. 2000; 10: 143–148.
Lefer AM, Scalia R, Lefer DJ. Vascular effects of HMG CoA-reductase inhibitors (statins) unrelated to cholesterol lowering: new concepts for cardiovascular disease. Cardiovasc Res. 2001; 49: 281–287.
Hirano K, Hirano M, Nishimura J, Kanaide H. A critical period requiring Rho proteins for cell cycle progression uncovered by reversible protein transduction in endothelial cells. FEBS Lett. 2004; 570: 149–154.
Hirano K, Derkach DN, Hirano M, Nishimura J, Takahashi S, Kanaide H. Transduction of the N-terminal fragments of MYPT1 enhances myofilament Ca2+ sensitivity in an intact coronary artery. Arterioscler Thromb Vasc Biol. 2004; 24: 464–469.
Koga M, Hirano K, Hirano M, Nishimura J, Nakano H, Kanaide H. Akt plays a central role in the anti-apoptotic effect of estrogen in endothelial cells. Biochem Biophys Res Commun. 324: 321–325.
Lindgren M, Hallbrink M, Prochiantz A, Langel U. Cell-penetrating peptides. Trends Pharmacol Sci. 2000; 21: 99–103.
Schwarze SR, Hruska KA, Dowdy SF. Protein transduction: unrestricted delivery into all cells? Trends Cell Biol. 2000; 10: 290–295.
Welsh CF, Roovers K, Villanueva J, Liu Y, Schwartz MA, Assoian RK. Timing of cyclin D1 expression within G1 phase is controlled by Rho. Nat Cell Biol. 2001; 3: 950–957.
Bergman AJ, Murphy G, Burke J, Zhao JJ, Valesky R, Liu L, Lasseter KC, He W, Prueksaritanont T, Qiu Y, Hartford A, Vega JM, Paolini JF. Simvastatin does not have a clinically significant pharmacokinetic interaction with fenofibrate in humans. J Clin Pharmacol. 2004; 44: 1054–1062.
Nakayama T, Hirano K, Hirano M, Nishimura J, Kuga H, Nakamura K, Takahashi S, Kanaide H. Inactivation of protease-activated receptor-1 by proteolytic removal of the ligand region in vascular endothelial cells. Biochem Pharmacol. 2004; 68: 23–32.
Shapiro MJ, Trejo J, Zeng D, Coughlin SR. Role of the thrombin receptor’s cytoplasmic tail in intracellular trafficking. Distinct determinants for agonist-triggered versus tonic internalization and intracellular localization. J Biol Chem. 1996; 271: 32874–32880.
Burridge K, Wennerberg K. Rho and Rac take center stage. Cell. 2004; 116: 167–179.
Hirai A, Nakamura S, Noguchi Y, Yasuda T, Kitagawa M, Tatsuno I, Oeda T, Tahara K, Terano T, Narumiya S, Kohn LD, Saito Y. Geranylgeranylated Rho small GTPase(s) are essential for the degradation of p27Kip1 and facilitate the progression from G1 to S phase in growth-stimulated rat FRTL-5 cells. J Biol Chem. 1997; 272: 13–16.(Tatsuya Yufu; Katsuya Hir)
Correspondence to Hideo Kanaide, MD, PhD, Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582. E-mail kanaide@molcar.med.kyushu-u.ac.jp
Abstract
Objective— Protease-activated receptor-1 (PAR1) mediates the thrombin-induced proliferation and hypertrophy of vascular smooth muscle cells. A role of Rac1 in the regulation of PAR1 expression was investigated.
Methods and Results— Treatment with simvastatin, a hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor, for 24 hours attenuated the transient [Ca2+]i elevation induced by thrombin. Immunofluorescence staining revealed that simvastatin decreased the surface expression of PAR1 in a manner dependent on protein geranylgeranylation. Introduction of a Rac1/Cdc42 inhibitory fragment but not a RhoA inhibitory fragment using a cell-penetrating peptide also attenuated the response to thrombin and decreased the surface expression of PAR1. Finally, downregulation of Rac1, but not RhoA, using an RNA interference technique attenuated the thrombin-induced [Ca2+]i elevation. However, the level of PAR1 mRNA and the total amount of PAR1 protein remained unchanged.
Conclusions— Here, we provide for the first time 3 lines of evidence that Rac1 plays a critical role in maintaining the surface expression of PAR1 and the responsiveness to thrombin in vascular smooth muscle cells. Rac1 is suggested to regulate the constitutive trafficking of PAR1 and thereby regulate the surface expression of PAR1.
We provided evidence that Rac1 regulates the surface expression of thrombin receptor PAR1 in vascular smooth muscle. Inhibition of Rac1 by hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor, a Rac1/Cdc42 inhibitory fragment, and an RNA interference technique reduced the surface expression of PAR1 and the responsiveness to thrombin.
Key Words: expression ? protease-activated receptor ? Rac1 ? smooth muscle ? thrombin
Introduction
Protease-activated receptor-1 (PAR1) belongs to a family of G-protein-coupled receptors (GPCRs), and it is known to mediate the cellular effects of thrombin in various types of cells.1–5 In vascular smooth muscle cells, PAR1 regulates the contraction, proliferation, and hypertrophy.1,4,6 The expression of PARs has been reported to either increase or decrease under various pathological conditions, including atherosclerosis, balloon injury of arteries, cerebral ischemia, and neuroinflammatory and neurodegenerative disorders.7–12 Therefore, elucidating the mechanism regulating the expression of PARs is important to understand the pathophysiology of such diseases and also to establish new therapeutic strategies. The nascent PAR1 is considered to be targeted first to the plasma membrane, and then subjected to constitutive internalization, thus resulting in the formation of an intracellular pool.13,14 PAR1 then cycles between the plasma membrane and the intracellular pool under resting conditions. The steady-state level of PAR1 on the cell surface is thus determined by a balance among the rate of de novo synthesis, the receptor internalization, and the recruitment from the intracellular pool.14,15 However, the molecular mechanism regulating the expression of PAR1 remains to be elucidated.
The small G-protein is known to regulate the intracellular vesicle trafficking.16 The Rab family is the most studied small G-protein that regulates GPCR trafficking.17 Rab5a has been shown to be required for the agonist-triggered internalization of PAR2, whereas Rab11a has been shown to contribute to the transport of PAR2 from the Golgi apparatus to the plasma membrane.18 On the other hand, the Rho family of small G-proteins has been suggested recently to play an important role in the internalization of membrane proteins such as thromboxane A2 receptor TP?, epidermal growth factor (EGF) receptor, and E-cadherin, and in the endocytosis of fibroblast growth factor 2.19–22 However, the role of the Rho families in the regulation of PAR1 expression has yet to be elucidated.
In the present study, we investigated the role of Rho proteins in the regulation of expression of PAR1 on the cell surface in vascular smooth muscle cells. We first used hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) to inhibit the protein isoprenylation such as geranylgeranylation and farnesylation, which is essential for small G-proteins to be functional.16,23–25 Second, we introduced the inhibitory proteins of Rho proteins using the cell-penetrating peptide of Tat protein.26–30 The RhoA-binding domain (RB) of Rho kinase and the Rac1/Cdc42-binding domain (PBD) of p21-activated protein kinase-1 was used as inhibitory proteins of Rho proteins, as described previously.26,31 Finally, we downregulated RhoA and Rac1 by RNA interference. As a result, the present study provides 3 lines of evidence supporting the critical role that Rac1 plays in the maintenance of PAR1 expression and the responsiveness to thrombin in cultured vascular smooth muscle cells.
Materials and Methods
The human coronary artery smooth muscle cells (HCASMCs) and the rat aortic smooth muscle cells (RASMCs) in primary culture were used. Cells were treated with HMG-CoA reductase inhibitors (statins), inhibitor proteins of RhoA, and Rac1/Cdc42 tagged with a cell-penetrating peptide of Tat protein or small interfering RNA targeted to RhoA and Rac1. Then, the responsiveness to thrombin and the surface expression of PAR1 were evaluated by fura-2 fluorometry and immunofluorescent staining, respectively. The total expression of PAR1, Rac1, RhoA, and ?-actin mRNA was evaluated by RT-PCR. The total expression of PAR1 protein was evaluated by an immunoblot analysis.
Expanded information for Materials and Methods can be found in the online supplement, available at http://atvb.ahajournals.org.
Results
Long-Term Treatment With Simvastatin Attenuated the Thrombin-Induced [Ca2+]i Elevation in Vascular Smooth Muscle Cells
In HCASMCs, thrombin concentration-dependently induced a transient [Ca2+]i elevation (EC50; 0.17 U/mL), with the maximum response at 1 U/mL (Figure 1A). PAR1-activating peptide (100 μmol/L TFLLR-NH2) induced a [Ca2+]i elevation (47.5±4.5%; n=4), which is comparable to that obtained with 1 U/mL thrombin, whereas PAR4-activating peptide (100 μmol/L GYPGKF-NH2) induced only a small [Ca2+]i elevation (1.0±1.6%; n=4; data not shown). PAR1 is thus suggested to mediate most of the response to thrombin in HCASMCs. The 24-hour treatment with simvastatin concentration-dependently attenuated the [Ca2+]i elevation induced by 1 U/mL thrombin, with a significant inhibition seen at 100 nmol/L and higher concentrations (Figure 1C). Such effective concentrations are similar to the plasma concentrations of simvastatin after the oral administration.32 However, application of 1 μmol/L simvastatin 10 minutes before and during stimulation with 1 U/mL thrombin had no effect on the thrombin-induced [Ca2+]i elevation (data not shown). The similar attenuation of the thrombin-induced [Ca2+]i elevation was observed with fluvastatin at a similar concentration range (data not shown). On the other hand, a hydrophilic statin, pravastatin, at a concentration of up to 1 μmol/L, had no significant effect on the [Ca2+]i elevation induced by 1 U/mL thrombin in HCASMCs (Figure 1A and 1C). A similar attenuation of the response to thrombin by simvastatin was observed in RASMCs (Figure 1B and 1D). In RASMCs, thrombin induced [Ca2+]i elevations with EC50 of 0.11 U/mL, but slightly higher concentrations of simvastatin were required to induce a significant inhibition of the thrombin-induced [Ca2+]i elevations.
Figure 1. Effect of HMG-CoA reductase inhibitors on the response to thrombin in vascular smooth muscle cells. A and B, The concentration-dependent response to thrombin in HCASMCs (A) and RASMCs (B). The resting level of [Ca2+]i and the maximal level of [Ca2+]i elevation induced by ionomycin (50 μmol/L in HCASMCs and 25 μmol/L in RASMCs) were assigned to be 0% and 100%. Data are the mean±SEM (n=3). C and D, The concentration-dependent effect of HMG-CoA reductase inhibitors on the response to 1 U/mL thrombin in HCASMCs (C) and RASMCs (D). Data are the mean±SEM (n=5). *P<0.05 vs the control.
Involvement of Protein Geranylgeranylation in the Statin-Induced Attenuation of the Response to Thrombin in Vascular Smooth Muscle Cells
The involvement of protein isoprenylation in the simvastatin-induced attenuation of the response to thrombin was investigated (Figure 2). HCASMCs were cotreated with simvastatin and geranylgeranyl pyrophosphate (GGPP) or farnesyl pyrophosphate (FPP) for 24 hours at the concentrations indicated in Figure 2. Cotreatment with GGPP prevented the attenuation of the response to thrombin by simvastatin in a concentration-dependent manner, with a complete prevention seen with 1 μmol/L GGPP (Figure 2A). However, cotreatment with FPP did not show any preventive effect in HCASMCs (Figure 2A). In RASMCs, GGPP (> 3 μmol/L) completely prevented the simvastatin-induced attenuation of the response to thrombin, whereas FPP partially prevented it even at 30 μmol/L (Figure 2B). The combination of 10 μmol/L GGPP and 10 μmol/L FPP showed the effect similar to that seen with GGPP alone. However, the 24 hour treatment of RASMCs with 10 μmol/L GGTI-298, a geranylgeranyl transferase inhibitor, attenuated the response to 1 U/mL thrombin to the extent similar to that obtained with 10 μmol/L simvastatin, whereas 10 μmol/L FTI-277, a farnesyl transferase inhibitor (FTI), had no effect on the response to thrombin (Figure 2B).
Figure 2. Involvement of protein geranylgeranylation in the simvastatin-induced attenuation of the response to thrombin in vascular smooth muscle cells. The concentration-dependent effect of GGPP and FPP on the simvastatin-induced attenuation of the [Ca2+]i elevation induced by 1 U/mL thrombin in HCASMCs (A) and RASMCs (B). Cells were cotreated for 24 hours with simvastatin (1 μmol/L for HCASMCs and 10 μmol/L for RASMCs) and GGPP or FPP at the indicated concentrations. B also shows the effect of GGTI and FTI on the response to thrombin (n=4). The resting level of [Ca2+]i and the maximal level of the [Ca2+]i elevation induced by ionomycin (50 μmol/L in HCASMCs and 25 μmol/L in RASMCs) were assigned to be 0% and 100%. Data are the mean±SEM (n=4). *P<0.05 vs the control.
Downregulation of the Surface Expression of PAR1 by Simvastatin in Vascular Smooth Muscle Cells
The surface expression of PAR1 was evaluated by immunofluorescence staining with a monoclonal antibody WEDE15 without fixation or permeabilization of the cells, as described previously.33 The untreated control HCASMCs exhibited a spotted pattern of fluorescence mainly on the cell periphery under fluorescence microscopy (Figure 3A). The fluorescence intensity at the peak of the fluorescence distribution was 856.7±290.1 arbitrary units (n=3) in control cells (Figure 3B). The treatment with simvastatin reduced the fluorescence staining and shifted the fluorescence distribution to the left (Figure 3A). The fluorescence intensity at the peak distribution (386.7±75.7 arbitrary units; n=3) was significantly lower than the control (Figure 3B). We reported previously that trypsin removes the epitope of WEDE15 (residues 51 to 64) of PAR1.33 Treatment with trypsin removed most of the staining in control and the simvastatin-treated cells and caused a leftward shift of the fluorescence distribution, thus resulting in a similar peak intensity (55.0±17.3 arbitrary units for control; 55.3±13.1 arbitrary units for simvastatin-treated cells; n=3; Figure 3A and 3B).
Figure 3. The effect of simvastatin and TATHA-PBD on the surface expression of PAR1 and the total cellular expression of PAR1 mRNA and protein in vascular smooth muscle cells. A, The representative microscopic images of immunofluorescence staining with phycoerythrine-conjugated anti-PAR1 antibody and the representative data of flow cytometry obtained with HCASMCs either untreated (control) or treated with 1 μmol/L simvastatin for 24 hours. Cells were stained before (intact) and after trypsin treatment (trypsin). The dashed lines in the histogram indicate the peak of the fluorescence distribution. Bar=50 μm. B, Summary of the fluorescence intensity at the peak of the fluorescence distribution in the cells untreated (control) and treated with 1 μmol/L simvastatin before and after trypsin treatment. Data are the mean±SEM (n=3). C, RT-PCR analysis of PAR1 mRNA expression in RASMCs untreated (control) and treated with 10 μmol/L simvastatin with and without 10 μmol/L GGPP for 24 hours. D, Immunoblot detection of the total amount of PAR1 in HCASMCs untreated (control) and treated with 1 μmol/L simvastatin in the presence and absence of 1 μmol/L GGPP, 3 μmol/L TATHA-PBD and 3 μmol/L (His)6-PBD. A densitometric analysis of electrophoresis was shown bellow the representative photos.
Effect of Rac1 Inhibition on the Expression of PAR1 mRNA and Protein in Vascular Smooth Muscle Cells
An RT-PCR analysis revealed that the level of PAR1 mRNA did not change after the 24-hour treatment of RASMCs with 10 μmol/L simvastatin, either in the presence or absence of 10 μmol/L GGPP (Figure 3C). On the other hand, a Western blot analysis revealed that the level of PAR1 protein did not change after the 24-hour treatment of HCASMCs with 1 μmol/L simvastatin either in the presence or absence of 1 μmol/L GGPP, 3 μmol/L TATHA-PBD (PBD conjugated to a cell-penetrating peptide of human immunodeficiency viral Tat protein and a hemagglutinin tag), or 3 μmol/L hexahistidine-tagged PBD ((His)6-PBD; Figure 3D). Immunofluorescence staining after fixation and permeabilization of the cells could detect PAR1 in the intracellular pool as well as on the plasma membrane.33 The intracellular staining was much higher than the surface staining in HCASMCs, as we reported previously in endothelial cells.33 Treatment with simvastatin or TATHA-PBD had no apparent effect on the intracellular staining of PAR1 (data not shown).
The Effect of Inhibition of Rho Signaling on the Response to Thrombin and the Expression of PAR1
The 24-hour treatment with TATHA-PBD concentration-dependently (EC50; 0.85 μmol/L) inhibited the [Ca2+]i elevation induced by 1 U/mL thrombin (Figure 4A and 4B). The inhibition seen with 3 μmol/L concentrations of TATHA-PBD was similar to that seen with 1 μmol/L simvastatin (Figure 4B). However, the application of 3 μmol/L TATHA-PBD 10 minutes before and during stimulation with thrombin had no significant effect on the [Ca2+]i elevation induced by thrombin (data not shown). The removal of the cell-penetrating peptide (His)6-PBD abolished the inhibitory effect of TATHA-PBD (Figure 4A and 4B). On the other hand, TATHA-RB, a RhoA inhibitory protein, had no effect on the response to thrombin (Figure 4A and 4B). In line with this, 24-hour treatment with 1 μmol/L Y27632, a Rho kinase inhibitor, also had no significant effect on the response to thrombin (Figure 4B). The protein transduction was confirmed by an immunoblot analysis as described previously.26,27 The extract of the cells exposed to the recombinant protein for 24 hours was subjected to the immunoblot detection with an anti-(His)6 antibody. This antibody detected all recombinant proteins (Figure 4C). However, PBD and RB were detected in the cell extract only when they were conjugated with Tat peptide (Figure 4C).
Figure 4. Effect of the transduction of inhibitors of Rho proteins on the response to thrombin in HCASMCs. A, The [Ca2+]i elevations induced by 1 U/mL thrombin in the HCASMCs untreated (control) and treated with simvastatin, TATHA-PBD, (His)6-PBD, TATHA-RB, and Y27632. The resting level of [Ca2+]i and the maximal level of [Ca2+]i elevation induced by 50 μmol/L ionomycin were assigned to be 0% and 100%, respectively. Data are the mean±SEM (n=3). B, Immunoblot detection of recombinant proteins in the extracts of the cells treated with 3 μmol/L of TATHA-PBD, (His)6-PBD, TATHA-RB, and (His)6-RB (lanes C). The purified proteins (100 ng) were loaded as a positive control (lanes P). Actin was detected with naphtol blue black staining after immunodetection to validate the equal loading of the cell extracts.
The effect of the transduction of TATHA-PBD on the expression of PAR1 was investigated by immunofluorescence staining, as shown in Figure 3 (Figure 5). Treatment of HCASMCs with 3 μmol/L TATHA-PBD caused a leftward shift in the fluorescence distribution to an extent similar to that seen with 1 μmol/L simvastatin (Figure 5). However, (His)6-PBD had no significant effect (Figure 5). Trypsin caused a marked reduction in fluorescence staining as shown in Figure 3.
Figure 5. Effect of the transduction of TATHA-PBD on the surface expression of PAR1 in HCASMCs. A, Representative data of a flow cytometric analysis of the immunofluorescence staining with phycoerythrine-conjugated anti-PAR1 antibody in the cells untreated (control) and treated with 1 μmol/L simvatatin, 3 μmol/L TATHA-PBD, 3 μmol/L (His)6-PBD. Control cells were subjected to immunofluorescence staining before and after trypsin digestion as shown in Figure 3. Dashed lines indicate the peak of the fluorescence distribution. B, Summary of the fluorescence intensity at the peak fluorescence distribution in the cells treated as indicated. Data are the mean±SEM (n=3). *P<0.05 vs control.
Effect of Downregulation of RhoA and Rac1 on the Response to Thrombin
RhoA and Rac1 were downregulated by an RNA interference technique. The transfection of control small interfering RNA had no effect on the responsiveness to thrombin in HCASMCs (Figure 6). On the other hand, downregulation of Rac1 but not RhoA significantly attenuated the [Ca2+]i elevation induced by 1 U/mL thrombin.
Figure 6. Effect of downregulation of RhoA and Rac1 on the response to thrombin in HCASMCs. A, RT-PCR analysis of Rac1, RhoA, and ?-actin mRNA expression in the cells untreated (control) and treated with control small interfering RNA (siRNA; control interference) and siRNA targeted against RhoA (RhoA knockdown) and Rac1 (Rac1 knockdown). B, The response to 1 U/mL thrombin in the cells untreated (control) and treated with 1 μmol/L simvastatin, control siRNA, and siRNA targeted against RhoA and Rac1. The resting level of [Ca2+]i and the maximal level of [Ca2+]i elevation induced by 50 μmol/L ionomycin were assigned to be 0% and 100%. Data are the mean±SEM (n=3); P<0.05.
Discussion
We herein demonstrated for the first time that Rac1 plays a crucial role in the maintenance of the surface expression of PAR1 and the responsiveness to thrombin in vascular smooth muscle cells. This conclusion is supported by 3 lines of evidence. First, the inhibition of HMG-CoA reductase reduced the expression of PAR1 on the cell surface and attenuated the response to thrombin. The attenuation of the thrombin response was prevented by cotreatment with GGPP in human and rat smooth muscle cells. In RASMCs, FPP also prevented the simvastatin-induced attenuation of the responsiveness to thrombin, whereas GGTI but not FTI mimicked the effect of simvastatin. These observations suggested that protein geranylgeranylation is a critical step involved in the expression of PAR1 on the cell surface. However, these observations could not specify which proteins are geranylgeranylated and regulate the expression of PAR1. Second, the transduction of the inhibitory protein of Rac1/Cdc42 reduced the surface expression of PAR1 and attenuated the responsiveness to thrombin, whereas the inhibitory protein of RhoA had no significant effect. These observations provided direct evidence that Rac1/Cdc42 but not RhoA is involved in the maintenance of the surface expression of PAR1 and the responsiveness to thrombin. Third, the knockdown of Rac1 but not RhoA by the RNA interference technique inhibited the responsiveness to thrombin. All these observations are consistent with the hypothesis that Rac1, but not Cdc42 or RhoA, plays a crucial role in the maintenance of the surface expression of PAR1 and the responsiveness to thrombin in vascular smooth muscle cells. The possibility that the inhibition of Rac1 directly inhibited the thrombin-induced [Ca2+]i elevation was ruled out on the basis of observations in the short-term treatment with simvastatin and TATHA-PBD.
The mechanism regarding how Rac1 regulates the expression of PAR1 on the cell surface remains to be elucidated. The downregulation of the surface expression of PAR1 by simvastatin was not associated with the downregulation of PAR1 mRNA. The change in the transcription of the PAR1 gene or the stability of PAR1 mRNA is thus not suggested to play a major role in the downregulation of PAR1 induced by inactivation of Rac1. Treatment with simvastatin or inhibition of Rac1 signaling had little effect on the total amount of PAR1 protein. It is thus likely that the alteration of the trafficking of PAR1 plays a critical role in the downregulation of PAR1 induced by the inactivation of Rac1.
PAR1 exhibits both constitutive and agonist-triggered internalization.2,4,14 The 2 modes of PAR1 internalization have been demonstrated to require the distinct residues in C-terminal region of PAR1, and they are thus suggested to be differently regulated by distinct mechanisms.13,15,34 The agonist-activated PAR1 has been shown to be internalized via a clathrin- and dynamin-dependent but arrestin-independent pathway, and then it mainly targets to lysosome degradation.14,15 On the other hand, the constitutive internalization takes place under resting conditions without receptor stimulation, and it has been reported to be necessary for the formation and the maintenance of the intracellular PAR1 pool.13 Therefore, the resting level of the surface expression of PAR1 is suggested to be dynamically maintained by continuous cycling between the cell surface and the intracellular pool. Our finding suggests that Rac1 regulates such constitutive trafficking of PAR1. The inhibition of Rac1 activity may either increase the constitutive internalization or inhibit the membrane targeting, thereby decreasing the level of PAR1 expressed on the cell surface in cultured smooth muscle cells.
PAR1 has been reported to be upregulated in smooth muscle cells seen in the human atherosclerotic lesions and in a rat carotid artery balloon injury model.10–12 The level of PAR1 expression has been shown to correlate to the degree of proliferation of smooth muscle cells.10,12 Growth factors such as platelet-derived growth factor, EGF or insulin, phorbol ester, and some GPCR agonists such as bombesin and lysophosphatidic acid were shown to activate Rac1.35 Rac1 is thus speculated to contribute to the upregulation of PAR1 in the vascular lesions. Our observations suggest that cultured vascular smooth muscle cells have some basal activity of Rac1, which contributes to the maintenance of surface expression of PAR1 under resting conditions. In the present study, we cultured and maintained the cells in the media containing 5% serum, 0.5 ng/mL EGF, 2 ng/mL fibroblast growth factor-B and 5 μg/mL insulin. Thus, it is conceivable that some growth factors in the media contributed to the basal activity of Rac1 in the cultured smooth muscle cells. Thus, our observations may be relevant to situations that are related to atherosclerotic lesions. However, such a possibility remains to be evaluated.
The preventive effect of FPP differed between HCASMCs and RASMCs. GGPP and FPP prevented the simvastatin-induced attenuation of the thrombin response in RASMCs, whereas only GGPP was effective in HCASMCs. Because GGTI but not FTI mimicked the effect of simvastatin in RASMCs, the findings suggested that geranylgeranylation but not farnesylation was involved in the effect of simvastatin in HCASMCs and RASMCs. We speculate that FPP was converted to GGPP in RASMCs, thereby exerting its preventive effect. GGPP is synthesized by a condensation of FPP and isopentenyl pyrophosphate.24,36 The residual amount of isopentenyl pyrophosphate after the inhibition of HMG-CoA reductase by simvastatin may be high enough to convert the exogenously added FPP to GGPP and to restore protein geranylgeranylation in RASMCs. However, this was not the case in HCASMCs, in which the amount of isopentenyl pyrophosphate was not sufficient to convert FPP to GGPP. The degree of inhibition of HMG-CoA reductase by statins or metabolism of isopentenyl pyrophosphate may differ between HCASMCs and RASMCs.
In conclusion, the present study demonstrated for the first time that Rac1 plays a critical role in the maintenance of the surface expression of PAR1 and the responsiveness to thrombin in the cultured vascular smooth muscle cells. The inactivation of Rac1 by HMG-CoA reductase inhibitor, the introduction of the inhibitor protein and downregulation by RNA interference attenuated the responsiveness to thrombin by reducing the level of the surface expression of PAR1. Because the level of PAR1 mRNA and the total amount of PAR1 protein remained unchanged, Rac1 is suggested to regulate the constitutive trafficking of PAR1.
Acknowledgments
This study was supported in part by the grant from the 21st Century COE Program and grants-in-aid for scientific research (No. 15590758 and 16590695) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Brian Quinn for linguistic comments and help with this manuscript.
References
Coughlin SR. Thrombin signaling and protease-activated receptors. Nature. 2000; 407: 258–264.
Dery O, Corvera CU, Steinhoff M, Bunnett NW. Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. Am J Physiol. 1998; 274: C1429–C1452.
Hollenberg MD, Compton SJ. International Union of Pharmacology. XXVIII. Proteinase-activated receptors. Pharmacol Rev. 2002; 54: 203–217.
Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. Proteinase-activated receptors. Pharmacol Rev. 2001; 53: 245–282.
Schmidlin F, Bunnett NW. Protease-activated receptors: how proteases signal to cells. Curr Opin Pharmacol. 2001; 1: 575–582.
Hirano K, Kanaide H. Role of protease-activated receptors in the vascular system. J Atheroscler Thromb. 2003; 10: 211–225.
Noorbakhsh F, Vergnolle N, Hollenberg MD, Power C. Proteinase-activated receptors in the nervous system. Nat Rev Neurosci. 2003; 4: 981–990.
Ossovskaya VS, Bunnett NW. Protease-activated receptors: contribution to physiology and disease. Physiol Rev. 2004; 84: 579–621.
Rohatgi T, Henrich-Noack P, Sedehizade F, Goertler M, Wallesch CW, Reymann KG, Reiser G. Transient focal ischemia in rat brain differentially regulates mRNA expression of protease-activated receptors 1 to 4. J Neurosci Res. 2004; 75: 273–279.
Ku DD, Dai J. Expression of thrombin receptors in human atherosclerotic coronary arteries leads to an exaggerated vasoconstrictory response in vitro. J Cardiovasc Pharmacol. 1997; 30: 649–657.
Nelken NA, Soifer SJ, O’Keefe J, Vu TK, Charo IF, Coughlin SR. Thrombin receptor expression in normal and atherosclerotic human arteries. J Clin Invest. 1992; 90: 1614–1621.
Wilcox J, Rodriguez J, Subramanian R, Ollerenshaw J, Zhong C, Hayzer D, Horaist C, Hanson S, Lumsden A, Salam T. Characterization of thrombin receptor expression during vascular lesion formation. Circ Res. 1994; 75: 1029–1038.
Shapiro MJ, Coughlin SR. Separate signals for agonist-independent and agonist-triggered trafficking of protease-activated receptor 1. J Biol Chem. 1998; 273: 29009–29014.
Trejo J. Protease-activated receptors: new concepts in regulation of G-protein-coupled receptor signaling and trafficking. J Pharmacol Exp Ther. 2003; 307: 437–442.
Marchese A, Chen C, Kim YM, Benovic JL. The ins and outs of G-protein-coupled receptor trafficking. Trends Biochem Sci. 2003; 28: 369–376.
Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev. 2001; 81: 153–208.
Seachrist JL, Ferguson SS. Regulation of G-protein-coupled receptor endocytosis and trafficking by Rab GTPases. Life Sci. 2003; 74: 225–235.
Roosterman D, Schmidlin F, Bunnett NW. Rab5a and rab11a mediate agonist-induced trafficking of protease-activated receptor 2. Am J Physiol. 2003; 284: C1319–C1329.
Izumi G, Sakisaka T, Baba T, Tanaka S, Morimoto K, Takai Y. Endocytosis of E-cadherin regulated by Rac and Cdc42 small G-proteins through IQGAP1 and actin filaments. J Cell Biol. 2004; 166: 237–248.
Rochdi MD, Laroche G, Dupre E, Giguere P, Lebel A, Watier V, Hamelin E, Lepine M-C, Dupuis G, Parent J-L. Nm23–H2 interacts with a G-protein-coupled receptor to regulate its endocytosis through an Rac1-dependent mechanism. J Biol Chem. 2004; 279: 18981–18989.
Tkachenko E, Lutgens E, Stan R-V, Simons M. Fibroblast growth factor 2 endocytosis in endothelial cells proceed via syndecan-4-dependent activation of Rac1 and a Cdc42-dependent macropinocytic pathway. J Cell Sci. 2004; 117: 3189–3199.
Wherlock M, Gampel A, Futter C, Mellor H. Farnesyltransferase inhibitors disrupt EGF receptor traffic through modulation of the RhoB GTPase. J Cell Sci. 2004; 117: 3221–3231.
Bellosta S, Ferri N, Bernini F, Paoletti R, Corsini A. Non-lipid-related effects of statins. Ann Med. 2000; 32: 164–176.
Laufs U, Liao JK. Direct vascular effects of HMG-CoA reductase inhibitors. Trends Cardiovasc Med. 2000; 10: 143–148.
Lefer AM, Scalia R, Lefer DJ. Vascular effects of HMG CoA-reductase inhibitors (statins) unrelated to cholesterol lowering: new concepts for cardiovascular disease. Cardiovasc Res. 2001; 49: 281–287.
Hirano K, Hirano M, Nishimura J, Kanaide H. A critical period requiring Rho proteins for cell cycle progression uncovered by reversible protein transduction in endothelial cells. FEBS Lett. 2004; 570: 149–154.
Hirano K, Derkach DN, Hirano M, Nishimura J, Takahashi S, Kanaide H. Transduction of the N-terminal fragments of MYPT1 enhances myofilament Ca2+ sensitivity in an intact coronary artery. Arterioscler Thromb Vasc Biol. 2004; 24: 464–469.
Koga M, Hirano K, Hirano M, Nishimura J, Nakano H, Kanaide H. Akt plays a central role in the anti-apoptotic effect of estrogen in endothelial cells. Biochem Biophys Res Commun. 324: 321–325.
Lindgren M, Hallbrink M, Prochiantz A, Langel U. Cell-penetrating peptides. Trends Pharmacol Sci. 2000; 21: 99–103.
Schwarze SR, Hruska KA, Dowdy SF. Protein transduction: unrestricted delivery into all cells? Trends Cell Biol. 2000; 10: 290–295.
Welsh CF, Roovers K, Villanueva J, Liu Y, Schwartz MA, Assoian RK. Timing of cyclin D1 expression within G1 phase is controlled by Rho. Nat Cell Biol. 2001; 3: 950–957.
Bergman AJ, Murphy G, Burke J, Zhao JJ, Valesky R, Liu L, Lasseter KC, He W, Prueksaritanont T, Qiu Y, Hartford A, Vega JM, Paolini JF. Simvastatin does not have a clinically significant pharmacokinetic interaction with fenofibrate in humans. J Clin Pharmacol. 2004; 44: 1054–1062.
Nakayama T, Hirano K, Hirano M, Nishimura J, Kuga H, Nakamura K, Takahashi S, Kanaide H. Inactivation of protease-activated receptor-1 by proteolytic removal of the ligand region in vascular endothelial cells. Biochem Pharmacol. 2004; 68: 23–32.
Shapiro MJ, Trejo J, Zeng D, Coughlin SR. Role of the thrombin receptor’s cytoplasmic tail in intracellular trafficking. Distinct determinants for agonist-triggered versus tonic internalization and intracellular localization. J Biol Chem. 1996; 271: 32874–32880.
Burridge K, Wennerberg K. Rho and Rac take center stage. Cell. 2004; 116: 167–179.
Hirai A, Nakamura S, Noguchi Y, Yasuda T, Kitagawa M, Tatsuno I, Oeda T, Tahara K, Terano T, Narumiya S, Kohn LD, Saito Y. Geranylgeranylated Rho small GTPase(s) are essential for the degradation of p27Kip1 and facilitate the progression from G1 to S phase in growth-stimulated rat FRTL-5 cells. J Biol Chem. 1997; 272: 13–16.(Tatsuya Yufu; Katsuya Hir)