RhoA-Rho kinase pathway mediates thrombin- and U-46619-induced phosphorylation of a myosin phosphatase inhibitor, CPI-17, in vascular smooth muscle cells

Huan Pang,1 Zhenheng Guo,1 Wen Su,1 Zhongwen Xie,1 Masumi Eto,2 and Ming C. Gong1

1Department of Physiology and Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, Kentucky; and 2Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia

Submitted 10 March 2005 ; accepted in final form 2 April 2005


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Protein kinase C-potentiated phosphatase inhibitor of 17 kDa (CPI-17) mediates some agonist-induced smooth muscle contraction by suppressing the myosin phosphatase in a phosphorylation-dependent manner. The physiologically relevant kinases that phosphorylate CPI-17 remain to be identified. Several previous studies have shown that some agonist-induced CPI-17 phosphorylation in smooth muscle tissues was attenuated by the Rho kinase (ROCK) inhibitor Y-27632, suggesting that ROCK is involved in agonist-induced CPI-17 phosphorylation. However, Y-27632 has recently been found to inhibit protein kinase C (PKC)-{delta}, a well-recognized CPI-17 kinase. Thus the role of ROCK in agonist-induced CPI-17 phosphorylation remains uncertain. The present study was designed to address this important issue. We selectively activated the RhoA pathway using inducible adenovirus-mediated expression of a constitutively active mutant RhoA (V14RhoA) in primary cultured rabbit aortic vascular smooth muscle cells (VSMCs). V14RhoA caused expression level-dependent CPI-17 phosphorylation at Thr38 as well as myosin phosphatase phosphorylation at Thr853. Importantly, we have shown that V14RhoA-induced CPI-17 phosphorylation was not affected by the PKC inhibitor GF109203X but was abolished by Y-27632, suggesting that ROCK but not PKC was involved. Furthermore, we have shown that the contractile agonists thrombin and U-46619 induced CPI-17 phosphorylation in VSMCs. Similarly to V14RhoA-induced CPI-17 phosphorylation, thrombin-induced CPI-17 phosphorylation was not affected by inhibition of PKC with GF109203X, but it was blocked by inhibition of RhoA with adenovirus-mediated expression of exoenzyme C3 as well as by Y-27632. Taken together, our present data provide the first clear evidence indicating that ROCK is responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs.

protein kinase C; signal transduction; adenovirus


AGONISTS INDUCE SMOOTH MUSCLE contraction by increasing cytoplasmic free Ca2+ and the sensitivity of the contractile apparatus to Ca2+. The latter is often referred to as Ca2+ sensitization. Ca2+ sensitization plays an important physiological role in agonist-induced smooth muscle contraction (52). Abnormalities in Ca2+ sensitization have been implicated in the pathophysiology of several cardiovascular disorders, including hypertension, coronary artery spasm, and arterial restenosis (27, 28, 39, 51, 57). While the increase in intracellular free Ca2+ initiates smooth muscle contraction by activating myosin light chain kinase, Ca2+ sensitization mediates smooth muscle contraction by inhibiting myosin light chain phosphatase. Myosin light chain phosphatase is a holoenzyme consisting of three subunits: a 37-kDa catalytic subunit; a 110-kDa regulatory targeting subunit, myosin phosphatase targeting subunit 1 (MYPT1); and a tightly bound 20-kDa subunit of unknown function (26). At least three mechanisms have been proposed to couple agonist stimulation to the inhibition of myosin light chain phosphatase and Ca2+ sensitization (26): direct binding and inhibition by protein kinase C (PKC)-potentiated phosphatase inhibitor of 17 kDa (CPI-17), phosphorylation by Rho kinase (ROCK), and dissociation of phosphatase subunits.

CPI-17 is a phosphatase-inhibitory protein that was first isolated from porcine aorta (14). Accumulating evidence suggests that CPI-17 plays an important role in myosin light chain phosphatase inhibition and Ca2+ sensitization regulation. CPI-17 is highly expressed in smooth muscle tissue, especially in the arteries (15, 59). Agonist-, GTP{gamma}S-, and phorbol ester-induced smooth muscle contraction are associated with an increase in CPI-17 phosphorylation and its phosphatase-inhibitory activity (12, 30, 31, 43). Nitric oxide-induced smooth muscle relaxation is associated with a decrease in CPI-17 phosphorylation and its phosphatase-inhibitory activity (4, 16). In addition, alterations in CPI-17 protein expression level correlate with changes in the Ca2+ sensitization of smooth muscle contraction (32, 44, 46). Phosphorylation of Thr38 in CPI-17 increases its phosphatase inhibitory potency >1,000-fold (15, 50). PKC-{alpha} and PKC-{delta} have been reported to mediate histamine-induced CPI-17 phosphorylation in smooth muscle (12). However, in several recent studies, investigators have found that in an isolated enzyme system, several other kinases can phosphorylate CPI-17. Such kinases include integrin-linked kinase (7, 9), protein kinase N (PKN) (23), MYPT1-associated kinase (37), p21-activated kinase (55), protein kinase A/G (8, 9), and, importantly, ROCK (34).

It is well recognized that the RhoA-ROCK pathway plays a critical role in agonist-induced Ca2+ sensitization of smooth muscle contraction (52). A variety of agonists can activate RhoA, a small GTPase, in vascular smooth muscle. The activated RhoA then turns on downstream effectors, including ROCK. ROCK directly phosphorylates MYPT1 at Thr696/Thr853 (17, 18, 29) and consequently inhibits phosphatase activity. In addition, in isolated enzyme systems, it was reported that ROCK can phosphorylate CPI-17 (34). This raises the possibility that phosphorylating CPI-17 is another mechanism by which the RhoA-ROCK pathway inhibits myosin light chain phosphatase in smooth muscle. In agreement with this possibility, inhibition of ROCK by Y-27632 partially but significantly inhibited histamine-, endothelin-, and phenylephrine-induced CPI-17 phosphorylation in permeabilized smooth muscle tissue (30, 31, 43). However, recently it was found that at the concentration used in the studies that showed inhibition of agonist-induced CPI-17 phosphorylation, Y-27632 also inhibited a well-recognized CPI-17 kinase, PKC-{delta} (12), and acetylcholine-induced activation of PKC (40). Thus the inhibition of agonist-induced CPI-17 phosphorylation by Y-27632 may reflect the ability of Y-27632 to inhibit PKC-{delta} rather than indicating the involvement of ROCK in CPI-17 phosphorylation. Indeed, PMA-induced CPI-17 phosphorylation by selectively activating PKC was significantly suppressed by Y-27632 (12). Therefore, whether ROCK mediates agonist-induced CPI-17 phosphorylation in intact vascular smooth muscle cells (VSMCs) remains an open question.

To address this unresolved but important issue, we used primary cultured VSMCs and combined molecular biological, biochemical, and pharmacological approaches in the current study. The hypothesis that ROCK activated by RhoA serves as an endogenous kinase mediating some agonist-induced CPI-17 phosphorylation was tested. Our data provide the first solid evidence that the RhoA-ROCK pathway is responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in VSMCs.


    EXPERIMENTAL PROCEDURES
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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Materials. New Zealand White rabbits (male, 2–3 kg) were purchased from Myrtle's Rabbitry (Thompson Station, TN). The cDNA of V14 RhoA, wild-type RhoA, and PRK5-C3 were kindly provided by Dr. Alan Hall (University College London, London, UK). The antibodies for CPI-17 and Thr38-phosphorylated CPI-17 were produced against peptides 12–36 or 33–43 and affinity purified as described previously (30). Anti-RhoA-, anti-p-MYPT1 (Thr853), anti-{alpha}-actin, anti-calponin, anti-caldesmon, and anti-smooth muscle myosin heavy chain were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-myosin phosphatase (total) was purchased from Babco (Richmond, CA). Dulbecco's modified Eagle's medium (DMEM) was obtained from Invitrogen (Carlsbad, CA). Phorbol-12,13-dibutyrate (PDBu), phorbol 12-myristate 13-acetate (PMA), and bisindolylmaleimide I hydrochloride (GF109203X) were purchased from Sigma (St. Louis, MO). U-46619 and thrombin were purchased from Calbiochem (San Diego, CA). Collagenase I and II were obtained from Worthington Biochemical (Lakewood, NJ). Other chemicals and reagents were purchased from Fisher Scientific. Y-27632 was a gift from Yoshotomi Pharmaceutical Industries (Osaka, Japan).

Primary cell culture. Rabbit VSMCs were isolated by performing enzymatic dissociation as described previously (47). Briefly, thoracic aortas were removed and washed in Hanks' balanced salt solution containing 100 U/ml penicillin and 100 µg/ml streptomycin. After being cut into four pieces, the vessels were digested in 2 mg/ml collagenase I and then in 2 mg/ml collagenase II plus 0.5 mg/ml elastase for 30 min each at 37°C. The isolated cells were cultured in DMEM supplemented with 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cultures were maintained at 37°C in a 95% air-5% CO2 humidified atmosphere.

Measurement of VSMC contraction. VSMC contraction was measured by analyzing the thrombin-stimulated transvascular horseradish peroxidase (HRP) diffusion according to a previously published protocol (2, 10, 48). Briefly, VSMCs were plated on collagen-coated Transwell inserts (6 x 104 cells/cm2, 3.0-µm pore size; Costar) and cultured for 5 days in DMEM supplemented with 10% serum. Subsequently, the cells were incubated in serum-free DMEM for 2 days. To establish basal HRP diffusion, HRP (1.5 µg/ml, type VI-A, 44,000 Mr; Sigma) was added to the upper chamber. Medium (60 µl) was collected from the lower chamber after 3 min and kept on ice until the enzymatic activity of HRP had been assayed. After being washed twice for total of 2 h, the cells were stimulated with thrombin (1 U/ml) for 20 min and then HRP was added to the upper chamber to determine the thrombin-stimulated HRP diffusion. To assay HRP enzymatic activity, the collected 60 µl of lower-chamber medium was added to 840 µl of reaction buffer (50 mM NaH2PO4 and 5 mM guaiacol). The reaction was initiated by addition of 100 µl of freshly made H2O2 solution (60 mM in H2O). The absorbance was measured at 470 nm after 15-min incubation at room temperature.

Immunoblot analysis. Proteins from primary cultured rabbit aortic VSMCs were denatured using trichloroacetic acid and separated by SDS-polyacrylamide gel electrophoresis. Specific proteins were detected using immunoblot analysis as described previously (22). The dilutions of specific antibodies were anti-phosphorylated MYPT1 antibody [p-MYPT1 (Thr853)], 1:1,000; total MYPT1, 1:10,000; anti-phosphorylated CPI-17 [p-CPI-17 (Thr38)], 1:2,500; anti-total-CPI-17 protein, 1:20,000; and anti-RhoA, 1:1,000. CPI-17 and MYPT1 phosphorylation levels were quantified using the ratios of phosphorylated vs. total CPI-17 and MYPT1 signals from two sets of parallel immunoblots. Special care was taken to ensure that the immunoblot signals were within linear range.

Recombinant adenovirus construction. The BamHI-EcoRI fragment of pBK-CMV/V14 was generated by performing PCR. This V14 RhoA fragment was then ligated into a modified version of pBluescript KS (pFLAG BamHI) containing an NH2-terminal FLAG epitope tag (DYKDDDDK). The FLAG-V14 RhoA fragment was excised by SpeI and XhoI and ligated into a modified adenoviral shuttle vector (pAdtracgfptre) containing two expression cassettes. One cassette uses the cytomegalovirus (CMV) promoter to drive green fluorescent protein (GFP) expression, and the other uses the tetracycline response element promoter to drive RhoA expression.

C3 DNA was excised from the PRK5-C3 vector using double digestion with EcoRI and ClaI. The cohesive ends of the C3 DNA fragment were filled in by a large fragment of DNA polymerase I (Klenow fragment). The blunt ends of the C3 DNA were then ligated into the Zero Blunt PCR vector (Invitrogen). After double digestion with SpeI and XhoI, the C3 DNA fragment was cloned into the pShuttle-IRES-hrGFP1 vector (Stratagene). This adenoviral vector contains a dicistronic expression cassette in which both C3 and a recombinant GFP are under the common control of a CMV promoter.

The V14RhoA and C3 adenoviruses were constructed using the Adeasy system (4) as described previously (22). Expression of FLAG V14 RhoA was examined using immunoblot analysis with anti-FLAG and anti-RhoA antibodies, respectively. Large quantities of recombinant adenovirus were produced by sequentially infecting human embryonic kidney-293 cells in 100-mm2 dishes and purified using CsCl gradient ultracentrifugation (22). The viral particle infectious units (pfu) were determined using an Adeno-X rapid titer kit (Clontech).

Adenoviral infection. VSMCs were starved in serum-free medium for 24 h and then incubated with Ad/V14 RhoA [multiplicity of infection (MOI) of 500] plus Ad/Tet-on (encoding a tetracycline-regulatory transcription factor) or Ad/C3 (MOI of 200) viruses for 12 h. Subsequently, the cells were cultured in serum-free medium for 36 h to allow V14RhoA and C3 expression. Different concentrations of doxycycline were present for the total 48-h period. The adenoviral transfection conditions were optimized to maximize RhoA or C3 expression and to minimize the cytopathic effect of the adenovirus.

Statistical analysis. Each experiment was repeated a minimum of three times. Data are expressed as means ± SE. Statistical analysis was performed using an unpaired t-test.


    RESULTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Primary cultured aortic VSMCs express smooth muscle marker proteins and contract in response to thrombin stimulation. To test whether primary cultures of aortic VSMCs express smooth muscle cell marker proteins, we examined the expression of smooth muscle {alpha}-actin, smooth muscle myosin heavy chain, caldesmon, and calponin in mature aortic smooth muscle tissue, primary cultures of aortic VSMCs, and A10 smooth muscle cells. We found that within the first five passages, these smooth muscle marker proteins were expressed at levels comparable to those of mature smooth muscle tissue (Fig. 1A). In contrast, the expression of osteopontin, a marker protein of the synthetic smooth muscle phenotype, was not detected in the primary cultured VSMCs but was detected in the A10 smooth muscle cell line (Fig. 1A). Moreover, as measured using an established transvascular HRP diffusion method, primary cultures of aortic VSMCs exhibited significant contraction in response to thrombin stimulation (Fig. 1B). These results suggest that primary cultured aortic VSMCs retain their ability to contract for at least five passages. Therefore, primary cultured cells were used within five passages in all subsequent experiments.



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Fig. 1. Primary cultured rabbit aortic vascular smooth muscle cells (VSMCs) express normal levels of contractile proteins (A) and contract in response to thrombin stimulation (B). Total cellular protein was isolated from rabbit aortic tissue, primary cultured rabbit aortic smooth muscle cells at either passage 3 (p3) or passage 5 (p5), or A10 smooth muscle cells. Equal amounts of all proteins were resolved using SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted for various contractile proteins (A). The primary antibodies used were {alpha}-actin (1:10,000 dilution), calponin (1:20,000 dilution), caldesmon (1:8,000 dilution), smooth muscle myosin heavy chain (SM-MHC; 1:8,000 dilution), and osteopontin (1:2,000 dilution). Immunoblots representative of three independent experiments are shown. B: VSMC contraction in response to thrombin (1 U/ml, 20 min) was measured using a horseradish peroxidase (HRP) diffusion method as described in EXPERIMENTAL PROCEDURES. OD, optical density at 470 nm. n = 5. *P < 0.05.

 
Selective activation of RhoA induces CPI-17 phosphorylation in primary cultured VSMCs. To test whether the activation of RhoA causes CPI-17 phosphorylation in intact VSMCs, we selectively activated the RhoA-ROCK pathway by expressing a constitutively active RhoA mutant, V14RhoA. A tetracycline-dependent, inducible adenoviral vector was used for the ectopic V14RhoA expression. It allowed nearly 100% transfection efficiency and the adjustment of the V14RhoA expression level by varying doxycycline concentrations. As shown in Fig. 2, doxycycline induced concentration-dependent V14RhoA protein expression in primary cultured rabbit aortic VSMCs. The upward mobility shift of the expressed V14RhoA compared with endogenous RhoA was probably caused by the FLAG tag added in the expressed protein. The absence of immunodetectable FLAG-tagged V14RhoA without added doxycycline (Fig. 2A, lane 1) indicated that V14RhoA expression in VSMCs was tightly controlled by doxycycline.



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Fig. 2. Adenovirus-mediated, doxycycline (Dox) dose-dependent V14RhoA expression in primary cultured rabbit aortic VSMCs. Confluent VSMCs were incubated with Ad-V14RhoA [multiplicity of infection (MOI) of 500] for 12 h and then in serum-free Dulbecco's modified Eagle's medium (DMEM; without virus) for 36 h. Various concentrations of doxycycline were added during and after the viral incubation to induce V14RhoA expression. Cells subsequently were lysed, and V14RhoA expression levels were determined using immunoblot analysis. A: representative immunoblots showing doxycycline concentration-dependent expression of V14RhoA in VSMCs. B: summary of densitometric analysis of V14RhoA immunoblots shown in A. Data are expressed as means ± SE from three independent experiments.

 
To investigate whether the selective activation by V14RhoA of the RhoA-ROCK pathway caused CPI-17 phosphorylation, we used immunoblot analysis with two CPI-17 antibodies to determine the CPI-17 phosphorylation level. One antibody selectively recognizes CPI-17 that is phosphorylated at Thr38, and another antibody recognizes total CPI-17 protein, including nonphosphorylated and phosphorylated protein. Our results showed that V14RhoA induced expression level-dependent CPI-17 phosphorylation (Fig. 3A, CPI-17-P) without affecting the total CPI-17 protein level (Fig. 3A, CPI-17-T). The CPI-17 phosphorylation level increased to 189.5 ± 25.2% of the control level (n = 8) in the presence of 0.5 µg/ml doxycycline and to 387.2 ± 61.3% of the control level (n = 8) in the presence of 2 µg/ml doxycycline. In a control experiment, up to 4 µg/ml doxycycline in the absence of an adenovirus did not cause any detectable CPI-17 phosphorylation (data not shown). Because ROCK has been shown to phosphorylate myosin phosphatase directly at Thr853, we investigated whether V14RhoA caused MYPT1 phosphorylation by activation of ROCK. The results showed that in parallel with CPI-17 phosphorylation, ectopically expressed V14RhoA induced expression level-dependent MYPT1 phosphorylation at Thr853 (Fig. 3, B and D). In contrast, the basal Thr696 phosphorylation was high, and no consistent increase by V14RhoA expression was detected (data not shown).



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Fig. 3. Constitutively active V14RhoA induces expression level-dependent CPI-17 and MYPT1 phosphorylation. V14RhoA was expressed at various levels in confluent VSMCs by adenovirus-mediated gene transfer as described in Fig. 1. To determine CPI-17 and MYPT1 phosphorylation levels, cells were collected by addition of ice-cold 10% trichloroacetic acid (TCA) and then frozen in liquid nitrogen. After being washed three times in acetone, the samples were grounded in homogenization buffer. Proteins were separated using SDS-PAGE. Phosphorylated CPI-17 or MYPT1 and total CPI-17 or MYPT1 were detected using phosphorylation-specific and total antibodies, respectively. A and B: representative immunoblots showing doxycycline induced concentration-dependent CPI-17 and MYPT1 Thr-853 phosphorylation without affecting total CPI-17 and MYPT1 protein expression. C and D: summary of the experiments shown in A and B. Phosphorylated CPI-17 and MYPT1 were normalized to their respective total proteins. Data are expressed as means ± SE. n = 5 experiments. **P < 0.01.

 
ROCK mediates V14RhoA-induced CPI-17 phosphorylation. It has been shown that activation of RhoA directly activates ROCK (27) and PKN (23). In addition, activation of RhoA stimulates phospholipase D, resulting in the accumulation of diacylglycerol and activation of PKC (38). Therefore, ROCK, PKN, and PKC are the candidate kinases that may mediate V14RhoA-induced CPI-17 phosphorylation. To identify the kinase that mediates V14RhoA-induced CPI-17 phosphorylation, we determined the effects of GF109203X and Y-27632 on V14RhoA-induced CPI-17 phosphorylation. GF109203X is a potent and selective inhibitor of conventional and novel PKCs (56). As shown in Fig. 4, inhibition of PKCs by GF109203X (3 µM) had no significant effect on V14RhoA-induced CPI-17 phosphorylation. As a control, GF109203X abolished PDBu-induced CPI-17 phosphorylation (Fig. 4), suggesting that at the concentration used, GF109203X was able to inhibit PKC effectively in our system. Thus we concluded that PKC is not involved in V14RhoA-induced CPI-17 phosphorylation.



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Fig. 4. Y-27632, but not GF109203X, inhibits V14RhoA-induced CPI-17 phosphorylation. The VSMCs with or without ectopic expression of V14RhoA were incubated with GF109203X (3 µM) or Y-27632 (10 µM) for 30 min and then stimulated with vehicle (DMSO) or phorbol-12,13-dibutyrate (PDBu) (1 µM) for 30 min. Reactions were terminated using 10% TCA, and samples were processed as described in Fig. 2. Note that V14RhoA-induced CPI-17 phosphorylation was inhibited by Y-27632 but not by GF109203X, whereas PDBu-induced CPI-17 phosphorylation was inhibited by GF109203X but not by Y-27632. n = 3. **P < 0.01 compared with V14RhoA only or PDBu only, respectively.

 
Because Y-27632 does not inhibit PKN (36), and because we have shown that PKC is not involved in V14RhoA-induced CPI-17 phosphorylation, we next used Y-27632 to identify the role of ROCK in V14RhoA-induced CPI-17 phosphorylation. Our results show that Y-27632 (10 µM) abolished V14RhoA-induced CPI-17 phosphorylation, suggesting a predominant role of ROCK in V14RhoA-induced CPI-17 phosphorylation.

Minimal role of PKC in thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs. To determine the kinase that mediates agonist-induced CPI-17 phosphorylation, we stimulated VSMCs with thrombin and U-46619. Thrombin and U-46619 induce Ca2+ sensitization in smooth muscle by activating specific receptors: protease-activated receptors and thromboxane A2 receptors, respectively (6, 42). As shown in Fig. 5, we found that thrombin and U-46619 induced marked CPI-17 phosphorylation. We next tried to identify the endogenous kinase that mediates agonist-induced CPI-17 phosphorylation in smooth muscle cells. Because PKC-{alpha} and PKC-{delta} are well recognized to mediate some agonists, such as histamine-induced CPI-17 phosphorylation in vascular smooth muscle cells, we first tested the role of PKC in thrombin- and U-46619-induced CPI-17 phosphorylation. Interestingly, inhibition of conventional and novel PKCs with GF109203X affected neither thrombin-induced nor U-46619-induced CPI-17 phosphorylation (Fig. 5). As a control, the same GF109203X treatment nearly abolished PMA-induced CPI-17 phosphorylation. Taken together, these results suggest that in contrast to a prominent role in histamine-induced CPI-17 phosphorylation, PKC-{alpha} and PKC-{delta} play a minimal role in thrombin- and U-46619-induced CPI-17 phosphorylation.



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Fig. 5. GF109203X does not inhibit thrombin- and U-46619-induced CPI-17 phosphorylation. Serum-starved VSMCs were pretreated with 3 µM GF109203X for 30 min and then stimulated with thrombin (1 U/ml, 20 min) or U-46619 (1 µM, 10 min) or PKC activator phorbol 12-myristate 13-acetate (PMA; 1 µM, 5 min). Cells were then harvested by the addition of 10% TCA, and samples were processed as described in Fig. 2. A: representative immunoblots showing the lack of effect of GF109203X on thrombin- and U-46619-induced CPI-17 phosphorylation. B: summary of the densitometric analysis of results shown in A. n = 4–6. **P < 0.01.

 
Critical role of RhoA-ROCK pathway in thrombin- and U-46619-induced CPI-17 phosphorylation. We next proceeded to test the role of the RhoA-ROCK pathway in thrombin- and U-46619-induced CPI-17 phosphorylation. We determined the effects of inhibiting RhoA and ROCK on thrombin- and U-46619-induced CPI-17 phosphorylation.

To inhibit RhoA function by ADP-ribosylation, VSMCs were infected with recombinant adenovirus-encoding exoenzyme C3 and then stimulated with thrombin. As shown in Fig. 6, A and B, thrombin-induced CPI-17 phosphorylation was significantly decreased by prior treatment with C3. As a control, a parallel treatment with an empty adenoviral vector did not affect thrombin-induced CPI-17 phosphorylation (Fig. 6A). This suggests that the inhibition of thrombin-induced CPI-17 phosphorylation by C3-containing recombinant adenovirus is not due to a nonspecific effect of the adenovirus. In addition, the upshift of RhoA mobility in SDS-PAGE gel (Fig. 6C) indicated effective ADP ribosylation of RhoA under our experimental conditions (1). In addition to inhibition of thrombin-induced CPI-17 phosphorylation, ADP ribosylation of RhoA inhibited thrombin-induced MYPT1 Thr853 phosphorylation (data not shown).



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Fig. 6. ADP ribosylation of RhoA inhibits thrombin-induced CPI-17 phosphorylation. VSMCs were infected with adenovirus encoding exoenzyme C3 or empty vector at a MOI of 200 for 48 h and then stimulated with thrombin (1 U/ml) for 20 min. Reactions were terminated by the addition of 10% TCA, and samples were processed as described in Fig. 2. A: representative immunoblots showing the inhibition of thrombin-induced CPI-17 phosphorylation by C3 treatment. B: summary of densitometric analysis of results shown in A. C: representative RhoA immunoblot showing RhoA mobility shift after C3 expression. n = 5. ***P < 0.001.

 
To further test the role of ROCK in agonist-induced CPI-17 phosphorylation, VSMCs were incubated with Y-27632 and then stimulated with thrombin or U-46619. As shown in Fig. 7, Y-27632 abolished thrombin- and U-46619-induced CPI-17 phosphorylation. In contrast, Y-27632 only slightly inhibited PMA-induced CPI-17 phosphorylation (Fig. 7). Although Y-27632 inhibits both PKC-{delta} and ROCK, its inhibition of thrombin- and U-46619-induced CPI-17 phosphorylation was likely mediated by inhibiting ROCK, because we have shown that PKC plays a minimal role in thrombin- and U-46619-induced CPI-17 phosphorylation (Fig. 5). In addition, our results showing that 10 µM Y-27632 inhibits V14RhoA-induced, but not PMA-induced, CPI-17 phosphorylation demonstrate the selectivity of this inhibitor for ROCK relative to PKC in cultured VSMCs (Figs. 4 and 5).



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Fig. 7. Y-27632 inhibits thrombin- and U-46619-induced CPI-17 phosphorylation. Serum-starved VSMCs were pretreated with Y-27632 (10 µM) for 30 min and then stimulated with thrombin (1 U/ml, 20 min), U-46619 (1 µM, 10 min), or PKC activator PMA (1 µM, 5 min). Cells were harvested by the addition of 10% TCA, and samples were processed as described in Fig. 2. A: representative immunoblots showing Y-27632 significantly inhibited thrombin- and U-46619-induced CPI-17 phosphorylation. B: summary of the densitometric analysis of results shown in A. n = 4–6. **P < 0.01.

 

    DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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 REFERENCES
 
Accumulating evidence indicates an important role for CPI-17 in agonist-induced inhibition of myosin phosphatase and Ca2+ sensitization of contraction in smooth muscle. Multiple kinases, including ROCK, have been shown to phosphorylate CPI-17 in isolated enzyme systems. However, whether ROCK can phosphorylate CPI-17 in intact smooth muscle cells and mediate agonist-induced CPI-17 phosphorylation in intact smooth muscle cells remains to be verified. The major finding of the present study is that ROCK mediates thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs.

CPI-17 is a phosphorylation-dependent myosin phosphatase-inhibitory protein. PKC-{alpha} and PKC-{delta} have been shown to phosphorylate CPI-17 in vitro and in situ (12, 30, 33, 40). Depleting CPI-17 protein by skinning smooth muscle cells eliminates PKC activator-induced Ca2+ sensitization of smooth muscle contraction (12, 36). The response can be reconstituted by the addition of recombinant CPI-17 protein and PKC (36). Furthermore, the inhibition of PKC by GF109203X abolishes histamine-induced CPI-17 phosphorylation in smooth muscle tissue (12). However, in several recent studies, investigators have reported that multiple kinases, including ROCK (34), integrin-linked kinase (7, 9), PKN (23), MYPT1-associated kinase (37), p21-activated kinase (55), and protein kinase A/G (8, 9), can phosphorylate CPI-17 in an isolated enzyme system. This raises the possibility that, in addition to PKC-{alpha} and PKC-{delta}, other kinases may be involved in CPI-17 phosphorylation. Consistent with this possibility, inhibition of ROCK by Y-27632 partially inhibits histamine-, endothelin-, and phenylephrine-induced CPI-17 phosphorylation (30, 31, 43). However, the following facts prevent the establishment of a role of ROCK in agonist-induced CPI-17 phosphorylation. First, there is no evidence that selective activation of the RhoA-ROCK pathway indeed phosphorylates CPI-17 in intact smooth muscle cells. Second, at the concentration used in the studies that showed inhibition of agonist-induced CPI-17 phosphorylation, Y-27632 recently was found to inhibit PKC-{delta}, which is well known to phosphorylate CPI-17 (12). In addition, Y-27632 inhibited acetylcholine-induced PKC activation by 45% in smooth muscle cells (40).

To clarify the role of the RhoA-ROCK pathway in agonist-induced CPI-17 phosphorylation, we used molecular biological and biochemical approaches in addition to pharmacological approaches in the present study. Our data clearly show that ROCK is responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs. First, we selectively activated the RhoA-ROCK pathway by ectopic expression of a constitutively active mutant V14RhoA in VSMCs and found that it was sufficient to induce CPI-17 phosphorylation in a V14RhoA expression level-dependent manner (Fig. 3). Second, V14RhoA-induced CPI-17 phosphorylation was not affected by inhibition of PKC with GF109203X but was abolished by Y-27632 (Fig. 4), suggesting that selective activation of RhoA is sufficient to phosphorylate CPI-17 by activating ROCK in intact smooth muscle cells. Third, we have shown that some agonists, such as thrombin and U-46619, induced CPI-17 phosphorylation in VSMCs (Fig. 5) and that inhibition of RhoA by adenovirus-mediated expression of exoenzyme C3 in VSMCs significantly inhibited thrombin-induced CPI-17 phosphorylation (Fig. 6). Fourth, we have shown that Y-27632, similarly to exoenzyme C3, also significantly diminished thrombin- and U-46619-induced CPI-17 phosphorylation in VSMCs (Fig. 7).

We found that in contrast to the predominant role of PKC in histamine-induced CPI-17 phosphorylation in mature vascular smooth muscle tissue (12), the RhoA-ROCK pathway plays a predominant role in thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs. That different kinases mediate CPI-17 phosphorylation may reflect the different systems used: primary cultured cells vs. mature smooth muscle tissue. In line with this possibility, recent reports have shown that the contractility of cultured VSMCs display a greater dependency than fresh tissue on the RhoA-ROCK pathway (3). Moreover, the phosphorylation site on MYPT1 changes upon culturing (53). Mature VSMCs are in a quiescent contractile phenotype under physiological conditions. They shift to a synthetic phenotype under certain pathological conditions such as restenosis, wound healing, and atherosclerosis. Cultured VSMCs resemble the synthetic phenotype in many aspects. Alternatively, on the basis of the comparable expression levels of smooth muscle marker genes and the ability of the primary cultures of aortic smooth muscle to contract in response to thrombin stimulation, it is also possible that the fact that different kinases mediate CPI-17 phosphorylation may reflect the use of different agonists: thrombin and U-46619 vs. histamine.

The physiological significance of the RhoA-ROCK pathway in the regulation of vascular smooth muscle contraction has been shown in numerous studies. We (21) and others (24, 35) have shown that recombinant RhoA or ROCK induces Ca2+ sensitization of smooth muscle contraction. Inhibiting endogenous RhoA by ADP ribosylation (20, 21, 24) or glycosylation (45), or inhibiting ROCK by Y-27632 (19, 25, 57) or HA-1077 (41, 49), inhibits various G protein-coupled receptor agonist-induced vascular smooth muscle contractions. Moreover, recent data indicate a significant pathological role of the RhoA-ROCK pathway in several cardiovascular diseases, including hypertension, coronary artery spasm, effort-induced angina, atherosclerosis, and restenosis (for review, see Refs. 5, 52).

The molecular mechanisms underlying RhoA-ROCK pathway-induced inhibition of myosin phosphatase remain to be elucidated completely. Our data underscore the significance of phosphorylating CPI-17 as one mechanism mediating ROCK-induced inhibition of myosin phosphatase. ROCK can inhibit myosin phosphatase by at least two mechanisms: phosphorylation of MYPT1 (17, 18) and phosphorylation of CPI-17 (Ref. 34 and the present study). ROCK has been reported to phosphorylate MYPT1 on Thr696 in isolated enzyme systems and in smooth muscle tissue, platelets, and endothelial cells (54), and Thr696 phosphorylation inhibits phosphatase activity (57). Therefore, Thr696 phosphorylation has been thought to be the mechanism mediating RhoA-ROCK pathway-induced phosphatase inhibition. However, this view has been challenged recently on the basis of the failure to detect an increase in Thr696 phosphorylation during the agonist-induced Ca2+ sensitization of smooth muscle contraction (31, 43). Indeed, with the ectopic expression of V14RhoA in smooth muscle, we failed to observe consistent stimulation of MYPT1 phosphorylation on Thr696. In contrast, we consistently observed V14RhoA expression level-dependent MYPT1 phosphorylation at Thr853. Furthermore, inhibition of ROCK by Y-27632 is associated with the inhibition of Thr853 phosphorylation. Although phosphorylation of MYPT1 at Thr853 does not directly affect phosphatase activity (17, 18), Thr853 phosphorylation may cause MYPT1 dissociation from its substrate myosin (58), resulting in the decrease of phosphatase activity toward myosin. Indeed, Thr853 is located at the myosin binding site on MYPT1 (18). In addition, our results clearly show that activation of the RhoA-ROCK pathway causes CPI-17 phosphorylation. Phosphorylated CPI-17 specifically binds and inhibits myosin phosphatase in cells (11). This suggests that at least two mechanisms, directly phosphorylating MYPT1 at Thr853 and phosphorylating CPI-17, can mediate ROCK-induced myosin phosphatase inhibition. Because both the MYPT1 Thr853 phosphorylation and the CPI-17 phosphorylation are observed in parallel using V14RhoA expression or thrombin or U-46619 stimulation, further experiments are required to assess the exact contribution by each of the two mechanisms to the RhoA-ROCK pathway-mediated inhibition of phosphatase activity.

In conclusion, the selective activation of the RhoA-ROCK pathway can phosphorylate CPI-17 in primary culture rabbit aortic smooth muscle cells, and the RhoA-ROCK pathway is primarily responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs.


    GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-67284, by a Career Development Award from the American Diabetes Association (to M. C. Gong), and by Scientist Development Grants from the American Heart Association (to M. Eto and Z. Guo).


    ACKNOWLEDGMENTS
 
We thank the University of Kentucky Cardiovascular Research Group for invaluable advice and assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. Gong, Dept. of Physiology, Univ. of Kentucky, 509 Wethington Bldg., 900 S. Limestone, Lexington, KY 40536 (e-mail: mcgong2{at}uky.edu)

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.


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