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Am J Physiol Heart Circ Physiol 285: H1444-H1452, 2003. First published June 26, 2003; doi:10.1152/ajpheart.00306.2003
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Regulation of SM22{alpha} expression by arginine vasopressin and PDGF-BB in vascular smooth muscle cells

Nihal Kaplan-Albuquerque,1 Chrystelle Garat,1 Vicki Van Putten,1 and Raphael A. Nemenoff1,2

Departments of 1Medicine and 2Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Submitted 3 April 2003 ; accepted in final form 16 June 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Vascular smooth muscle (SM) cells (VSMC) undergo phenotypic modulation in vivo and in vitro. This process involves coordinated changes in expression of multiple SM-specific genes. In cultured VSMC, arginine vasopressin (AVP) increases and PDGF decreases expression of SM {alpha}-actin (SMA), the earliest marker of SM cells (SMC). However, it is unknown whether these agents regulate other SM genes in a similar fashion. SM22{alpha} appears secondary to SMA during development and is also a marker for SMC. This study examined the regulation of SM22{alpha} expression by AVP and PDGF in cultured VSMC. Levels of SM22{alpha} mRNA and protein were increased by AVP and suppressed by PDGF. Consistent with these changes, AVP increased SM22{alpha} promoter activity, whereas PDGF inhibited basal promoter activity and blocked AVP-induced increase. Activation of both JNK and p38 MAPK pathways was necessary for AVP-mediated induction of SM22{alpha} promoter. Expression of constitutively active Ras produced similar suppressions on SM22{alpha} promoter activity as PDGF. Signaling relayed from PDGF/Ras activation involved Raf, or a protein that competes for this site, Ral-GDS, and phosphatidylinositol 3-kinase activation. Truncational analysis showed that the proximal location of three CArG boxes in the promoter was sufficient for AVP stimulation. Mutations in this CArG box reduced basal and AVP-stimulated promoter activity without effecting PDGF suppression. Overexpression of serum response factor enhanced basal and AVP-stimulated promoter activity but had no effect on PDGF-BB-induced suppression. These data indicate that AVP and PDGF initiate specific signaling pathways that control expression of multiple SM genes leading to phenotypic modulation.

signal transduction; transcriptional regulation; mitogen-activated protein kinase; stress response factor; CArG

VASCULAR SMOOTH MUSCLE (SM) cells (VSMC) in mature animal vessels express multiple contractile proteins that are markers for the differentiated contractile phenotype (see Ref. 36 for review). Environmental cues can affect the differentiated state of VSMC, causing them to dedifferentiate into a proliferative phenotype. This occurs in pathophysiological states such as atherosclerotic lesions, in which migration and proliferation of neointimal VSMC lead to vascular remodeling. Certain cues can also induce redifferentiation of this dedifferentiated phenotype, making it relevant to study pathways modulating the differentiated state of these cells (25, 34–36, 39). During dedifferentiation, expression of VSMC contractile proteins such as SM {alpha}-actin (SMA) and myosin heavy chain (MHC) is decreased or lost (22, 48). SMA is the first marker of SM differentiation and appears at embryonic day 2 (34). In adult cultured VSMC, vasoconstrictors such as angiotensin II or arginine vasopressin (AVP) increase SMA expression (13, 14, 46, 47), whereas growth factors such as PDGF decrease protein and message levels of SMA (7, 19, 29). We have previously shown (47) that these effects are mediated largely through transcriptional regulation of SMA promoter activity.

Although the molecular events initiated by AVP or PDGF leading to opposing effects on SMA expression are incompletely understood, we have recently defined specific signal transduction pathways that regulate SMA expression by these agents (12, 29). Activation of JNKs and p38 MAPK was critical for induction of SMA by AVP, whereas activation of Ras was critical for suppression of SMA by PDGF. Possible interactions between signaling pathways, or sensitization of other receptors by AVP (32) or PDGF, could also play a role in the complexity of the signaling pathways leading to the regulation of SM-specific gene expression. The presence of CArG [CC(A/T)6GG] or CArG-like cis-acting elements is necessary for the expression of SM-specific genes (17, 40). The promoters for many VSMC-specific genes contain two to three CArG elements, and these elements represent binding sites for the MCM1, AG, DEFA, and SRF proteins (MADS) box transcription factor, serum response factor (SRF) (41). Truncation mutations (47) or point mutations (12) in individual CArG elements of the SMA promoter abolish both basal and AVP-stimulated activation of the promoter. However, binding of additional transcription factors is necessary for the restricted expression of VSMC-specific genes (44). Sequences flanking the CArG boxes are also necessary for the specificity of the expression (6, 44).

SM22{alpha} is the second marker to appear during VSMC differentiation from its progenitor cells. SM22{alpha}, first isolated from the chicken gizzard (26, 27), is a 201-amino acid, calponin-related, SMC-specific protein (31) that may be involved in contraction of SMC due to its interactions with tropomyosin and F-actin (11, 21). On the other hand, SM22{alpha}-deficient mice develop normally and show no vascular or visceral abnormalities in basal conditions (50). However, the functionality of the vessels was not tested under stress conditions. During embryogenesis, it is expressed in smooth, cardiac, and skeletal muscle lineages. However, in adult mammals, expression is restricted to visceral and vascular SM (9, 28, 30, 35, 43). Cloning and sequence analysis of a 1.5-kb region of SM22{alpha} promoter revealed strong and restricted expression in SMC (20). Sequence analysis demonstrated no classical TATAA box but instead a TTTAAA box resembling the one in the MHC promoter. The proximal region of the SM22{alpha} promoter contains three CArG boxes, two of which are overlapping (–275, –283, and –162), an E box (–130), two GC boxes (–123 and –253), a myocyte-enhancing factor-2-like AT-rich region (–912), and several other GC-rich sequences resembling binding sites for the Sp1 family of transcription factors. In addition, a 118-bp truncation mutant of the SM22{alpha} promoter, which has no CArG boxes or GC boxes, was still active in SMC, but not in fibroblasts or COS cells (3, 20). A 445-bp fragment of the SM22{alpha} promoter, which contains all three CArG boxes, is sufficient to drive expression in arterial SMC, but not in visceral SMC (28). This suggested the involvement of differential regulatory mechanisms during development.

Although, the basal SM22{alpha} promoter activity in cultured VSMC and developmental regulation have been extensively studied, effects of hormones and growth factors have not been examined, and signal transduction pathways regulating SM22{alpha} expression are not well defined. It has not been determined whether the signaling pathways regulating SMA expression are specific for this gene, or whether they have a more global effect on VSMC phenotype by controlling multiple SMC markers. This study examines the hormonal regulation of SM22{alpha} promoter activity in VSMC by vasoconstrictors and polypeptide growth factors and seeks to identify signal transduction pathways controlling expression of this gene. Defining these pathways will be critical to understanding phenotypic modulation associated with disease states of adult vessels and lead to new targets for therapy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cell culture. VSMC from the rat thoracic aorta were isolated as described previously (5, 47). Cultured VSMC were grown in Eagle's MEM (EMEM; GIBCO-BRL) containing 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS (Hyclone) in 100-mm dishes. Passages 4 through 12 were used in experiments.

Northern blot analysis. VSMC were made quiescent by incubation in EMEM containing 0.2% FBS for 48 h. Total RNA was isolated from control VSMC or cells stimulated with AVP or PDGF-BB for 24 or 48 h by RNeasy miniprep (Qiagen). Northern blot analysis was performed on 5 µg total RNA with a 478-bp cDNA probe for SM22{alpha}. Membranes were stripped and reprobed for GAPDH as an internal control for equal amounts of RNA loading.

Immunoblotting. VSMC were lysed with ice-cold RIPA buffer, pH 7.4 [50 mM Tris · HCl (pH 7.0), 150 mM NaCl, 1% NP-40, 1% Na deoxycholate, 0.1% SDS, 50 mM sodium fluoride, 2 mM EDTA, 200 µMNa3VO4, and protease inhibitors]. Solubilized proteins were centrifuged at 14,000 g in a microfuge (4°C) for 10 min. Supernatants were separated by using 15% SDS-PAGE and transferred to Immobilon P membranes (Millipore, Bedford, MA). Membranes were blocked for 1 h at room temperature in Tris-buffered saline (10 mM Tris · HCl, pH 7.4, and 140 mM NaCl) containing 0.1% Tween 20 (TTBS) and 5% milk and then incubated with a monoclonal antibody against SM22{alpha} (a gift of Dr. Angela Chiavegato, University of Padua, Padua, Italy) in blocking buffer for 12–16 h at 4°C. Membranes were washed in TTBS, and bound antibodies were visualized with horseradish peroxidase-coupled anti-mouse and enhanced chemiluminescence (New England Nuclear-Life Science Products, Boston, MA) according to the manufacturer's directions.

DNA constructs and cell transfections. The following constructs derived from the SM22{alpha} promoter were used: pcB55, –1,500 to +65; pcB80, –303 to +65; pcB81, –193 to +65; and pcB83, –118 to +65. All truncations and mutants were inserted into a promoterless pCATBasic vector (20). Mutation in the near-CArG box was made by using pcB81 (–193 to +65) and pcB55 (–1,500 to +65) by site-directed mutagenesis (QuikChange; Stratagene, La Jolla, CA) as –162 CArG box mutation: 5'-CCAAATATGG-3' to 5'-CCAAAGATGG-3'.

VSMC were transiently transfected by using electroporation (geneZAPPER; IBI) with 2–10 µg of SM22{alpha} promoter constructs (gift of Dr. Paul Kemp, Oxford University, Oxford, UK) along with 2 µg of cytomegalovirus (CMV)-{beta}-galactosidase vector to normalize for transfection efficiency as previously described (12). Transfections were performed in 100-µl suspensions of 2 million VSMC in complete media and plated in 60-mm dishes. After an overnight incubation, media were changed to EMEM containing 0.2% FBS with or without AVP (10–6 M) and/or PDGF-BB (20 ng/ml) for 48 h. Cotransfection experiments were done with 5 µg of other DNA. The volume of the DNA mixture was kept between 10 and 12 µl in Tris · HCl per 100-µl transfection, and total DNA was kept constant by the addition of plasmid lacking an insert. Cells were lysed, and chloramphenicol acetyltransferase activity and {beta}-galactosidase activity were determined as previously described (47). Results are expressed as the fold change compared with basal normalized promoter activity.

Mutations in the Hras V12-pcDNA construct were made based on published results (49) by using the QuikChange site-directed mutagenesis kit from Stratagene according to the manufacturer's directions. Oligonucleotides (sense) used for the mutations were as follows: for the T35S mutation (binds only Raf), (118–152) 5'-G GAC GAA TAC GAC CCC TCT ATA GAG GAT TCC TAC C-3'; for the E37G mutation (binds only Ral-GDS), (127–154) 5'-C GAC CCC ACT ATA GGG GAT TCC TAC CGG-3'; and for the Y40C mutation [binds only phosphatidylinositol 3-kinase (PI3-kinase)], (133–166) 5'-C ACT ATA GAG GAT TCC TGC CGC AAG CAG GTC GTC-3'. After transformation and purification from XL2 blue competent cells, the constructs were sequenced to verify the resulting mutations.

EMSA. After stimulation, nuclear extracts were prepared by using a variation of the procedure of Dignam et al. (8) as described previously (12). A double-stranded 94-bp DNA piece (–184 to –92), which included the near-CArG and GC boxes, was made by PCR amplification. Primers used for PCR amplifications included a 3' MluI site for the purpose of fill-in labeling with [32P]dCTP by using Klenow DNA polymerase. Unincorporated [32P]dCTP was removed by PAGE. Binding was performed for 30 min at 4°C with 2 µg nuclear binding proteins and 1 ng of labeled DNA probe in 20 µl of total volume containing (in mM) 12 HEPES-KOH (pH 7.9), 150 KCl, 1.0 EDTA, and 0.3 dithiothreitol, and 0.125 mg poly(dI/dC), 12% glycerol, and protease inhibitors. Competition experiments were performed by using a 50-fold excess of unlabeled DNA. For supershift experiments, 2 µg SRF antibody was added at 4°C 30 min before the addition of radiolabeled probe. Protein-DNA complexes were resolved on a 24-cm 5% acrylamide gel (29:1 acrylamide-bisacrylamide; Fisher) at 25 mAmp/gel in 1x TGE (25 mM Tris, 1.0 mM EDTA, and 190 mM glycine, pH 8.0) for 2.5 h. Gels were dried and autoradiographed.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Regulation of SM22{alpha} expression by AVP and PDGF-BB. To investigate the possible role of hormonal factors in the regulation of SM22{alpha} expression in VSMC, the effects of AVP and PDGF-BB on the expression of SM22{alpha} mRNA were determined by Northern blot analysis. Previous studies (33) reported two transcripts of SM22{alpha} in tissues, 999 and 1,186 nt. We were able to detect both transcripts, the shorter transcript being the most abundant in cultures of VSMC. Stimulation of quiescent cells with AVP increased SM22{alpha} mRNA levels approximately twofold after either 24 or 48 h of exposure (Fig. 1A). Conversely, exposure to PDGF-BB decreased SM22{alpha} mRNA levels by 50%. To confirm that changes in mRNA levels were reflected at the protein level, extracts from VSMC exposed to AVP or PDGF were immunoblotted with a specific antibody. Exposure to AVP increased expression approximately two- to threefold, whereas exposure to PDGF-BB decreased the protein expression levels of SM22{alpha} (Fig. 1B). We then investigated the ability of these agents to regulate SM22{alpha} promoter activity. Transient transfection of a 1.5-kb fragment of the SM22{alpha} promoter (pCB55) into VSMC exhibited high basal activity (Fig. 1C) compared with the SM {alpha}-actin promoter (data not shown). Stimulation of cells with AVP caused a significant two- to fourfold increase in the promoter activity. PDGF-BB decreased basal promoter activity and blocked the increase seen in AVP-stimulated cells (Fig. 1C).



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  		Fig. 1. Regulation of smooth muscle (SM)22{alpha} expression by AVP and PDGF. A: Northern blot analysis of SM22{alpha} mRNA (top) in vascular SM cells (VSMC) stimulated with AVP and PDGF-BB. VSMC were made quiescent for 48 h and then stimulated with AVP (10–6 M) or PDGF-BB (20 ng/ml) for 24 or 48 h. Northern blot analysis using a cDNA probe of SM22{alpha} was done with 5 µg total RNA. Bottom, GAPDH mRNA from the same blot as a control for RNA loading. Expression was quantitated by densitometry. These results are representative of 3 independent experiments. B: immunoblot analysis of SM22{alpha} protein in VSMC stimulated with AVP and PDGF-BB. VSMC were made quiescent for 24 h and then stimulated with AVP (10–6 M) or PDGF-BB (20 ng/ml) for 96 h. Western blot analysis was done with an antibody against SM22{alpha} as described in MATERIALS AND METHODS. C: SM22{alpha} promoter activity in VSMC stimulated with AVP or PDGF-BB. VSMC were transfected with a 1.5-kb fragment of the SM22{alpha} promoter along with CMV-{beta}-galactosidase plasmid to normalize for transfection efficiency as described in MATERIALS AND METHODS. Cells were stimulated with AVP, PDGF-BB, or AVP + PDGF-BB for 48 h in 0.2% serum-containing media. Promoter activities are represented as fold change compared with the basal promoter activity. Results represent means ± SE of 4 experiments. *P = 0.05 vs. basal activity; #P = 0.05 vs. AVP stimulated activity.

 

Role of MAPK pathways in regulation of SM22{alpha} expression. Previously, we (12, 18) have shown that JNK and p38 MAPKs but not ERK1/2 are involved in induction of SMA promoter activity by AVP. To assess whether these pathways also mediated regulation of SM22{alpha} expression, VSMC were treated with a novel JNK MAPK inhibitor, SP-600125 (1, 16). At concentrations that inhibited c-jun phosphorylation by AVP (10 µM; not shown), this agent blocked AVP-induced increases in promoter activity without significant effects on basal levels (Fig. 2A). Treatment with a p38 MAPK inhibitor, SB-203580 (24), inhibited both basal and AVP-induced increases in the promoter activity without having an effect on the PDGF-BB-induced suppression (Fig. 2A). However, as seen with the SMA promoter, blocking the ERK pathway with a specific MEK inhibitor, PD-98059 (10), had no effect on either induction of SM22{alpha} promoter activity by AVP or suppression by PDGF-BB. To confirm a role for the JNK pathway, cells were cotransfected with gain of function MKK7/JNK fusions (45). Expression of gain of function JNKs increased basal and AVP-stimulated promoter activity (Fig. 2B), similar to what was observed with the SMA promoter (12). The expression of the constitutively active JNK fusions did not significantly affect the ability of PDGF-BB to suppress SM22{alpha} promoter activity. These results indicate the involvement of p38 and JNK MAPKs on AVP-induced activation of the SM22{alpha} promoter.



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  		Fig. 2. Signaling pathways mediating induction of SM22{alpha} promoter activity. A: VSMC were transfected with a 1.5-kb fragment of the SM22{alpha} promoter along with a plasmid encoding {beta}-galactosidase as described in MATERIALS AND METHODS. Transfected cells treated with vehicle (0.1% DMSO), SP-600125 (10 µM), SB-203580 (10 µM), or PD-98059 (50 µM) were stimulated with AVP, PDGF-BB, or AVP + PDGF-BB for 48 h in 0.2% serum-containing media. Promoter activity normalized to {beta}-galactosidase was determined. The promoter activity is shown as fold change over basal promoter activity in cells treated with the vehicle. Results represent the means ± SE of 3 experiments. *P = 0.05 vs. basal activity; #P = 0.05 vs. vehicle-treated group. B: VSMC were cotransfected with a 1.5-kb fragment of the SM22{alpha} promoter, plasmids encoding for MAPK kinase (MKK) 7/JNK fusions (MKK7/JNK3, MKK7/JNK1{alpha}), or empty vector (control). Cells were stimulated with AVP, PDGF-BB, or AVP + PDGF-BB for 48 h in 0.2% serum-containing media, and promoter activity was determined. Activity is shown as fold change over basal promoter activity in cells cotransfected with empty vector. Data represent means ± SE of 3 experiments. *P = 0.05 vs. basal activity; #P = 0.05 vs. transfection with empty vector.

 

We have previously shown that activation of Ras is critical for suppression of SMA promoter activity by PDGF (29). To assess the effects of Ras on SM22{alpha} expression, cells were transiently transfected with gain of function Ras (H-Ras-V12) along with the SM22{alpha} promoter construct. Expression of gain of function H-Ras suppressed both basal and AVP-stimulated SM22{alpha} promoter activity to similar levels as PDGF-BB (Fig. 3A). Activated Ras engages multiple downstream effectors (4). Employing expression constructs containing point mutations in the effector domain of Ras, three major effectors have been defined (see Ref. 2 for review): the Raf/MEK/ERK pathway, the PI3-kinase pathway, and a guanine nucleotide exchange factor for small G-protein Ral (RalGDS). H-Ras(V12,S35) selectively couples to Raf, H-Ras(V12,G37) couples to RalGDS, and H-Ras(V12, C40) couples to PI3-kinase. We examined the effects of these constructs on regulation of SM22{alpha} promoter activity. Each construct was cotransfected into VSMC along with the SM22{alpha} promoter. Expression of H-Ras(V12,S35), H-Ras(V12,G37), or H-Ras(V12,C40) caused minimal but significant inhibition on promoter activity (Fig. 3B). Expression of H-ras mutants in dual combination caused further inhibition, and expression of all three mutant forms of H-ras recovered the inhibitory effect of H-ras on the promoter activity (Fig. 3B). These data suggest that signaling through all three signaling pathways may be critical for Ras and PDGF-mediated regulation of muscle gene expression.



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  		Fig. 3. Signaling pathways mediating suppression of SM22{alpha} promoter activity. A: VSMC were cotransfected with a 1.5-kb fragment of the SM22{alpha} promoter and a plasmid encoding gain of function H-ras or control empty vector. Cells were stimulated with AVP or vehicle (basal) for 48 h. Promoter activity was determined and is shown as fold change over basal promoter activity in cells cotransfected with empty vector. Data represent means ± SE of 4 experiments. *P = 0.05, AVP-treated group vs. basal activity; #P = 0.05 vs. transfection with empty vector. B: VSMC were cotransfected with a 1.5-kb fragment of the SM22{alpha} promoter, plasmids encoding gain of function H-ras (H-RasV12), gain of function H-ras with either a T35S, E37G, or Y40C mutation, or empty vector (control). Promoter activity was determined and is shown as 26-fold change over basal promoter activity in cells cotransfected with empty vector. *P = 0.05 vs. transfection with empty vector; #P = 0.05 vs. H-rasV12; $P = 0.05 vs. H-rasV12 mutants alone; &P = 0.05 vs. H-rasV12 mutants in dual combinations.

 

Regulatory elements of the SM22{alpha} promoter. To define critical regulatory elements necessary for hormonal regulation of the promoter, a series of truncation mutants of the SM22{alpha} promoter were employed (20). Transfection of cells with a 303-base truncation mutant of the promoter (pCB80) containing all three of the CArG boxes had similar basal activity to that seen with the 1.5-kb promoter construct. Stimulation by AVP and inhibition by PDGF-BB was also qualitatively similar to that seen with pCB55 (Fig. 4A). A shorter fragment containing only one of the CArG boxes (pCB81) had lower basal and AVP-stimulated activity, but fold stimulation by AVP and suppression by PDGF-BB was similar to that seen with pCB80. Finally, a shorter fragment that contains no CArG boxes (pCB83) had low basal activity in VSMC, and no stimulation by AVP was detected. However, promoter activity of the pCB83 fragment was still suppressed by PDGF-BB (Fig. 4A). These results suggested that the near-CArG box is important for AVP-induced stimulation of the promoter, but PDGF-BB suppression does not require the near-CArG box or GC-rich sequences. To confirm the role of the near-CArG box, a single nucleotide mutation [–166, T(G)] was made. This point mutation significantly decreased AVP-mediated stimulation of SM22{alpha} promoter activity (Fig. 4B). However, PDGF-BB still suppressed promoter activity. Mutation of this near-CArG box in the context of the full-length 1.5-kb promoter (pCB55) also inhibited basal and AVP-induced promoter activities (data not shown).



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  		Fig. 4. Near-CArG and GC boxes are necessary for AVP-induced promoter activity. A: VSMC were transfected with the indicated fragments of the SM22{alpha} promoter along with CMV-{beta}-galactosidase plasmid to normalize for transfection efficiency as described in MATERIALS AND METHODS. Cells were stimulated with AVP, PDGF-BB, or AVP + PDGF-BB for 48 h in 0.2% serum-containing media. Promoter activities are represented as fold change compared with the basal activity of the 1.5-kb promoter fragment. Results represent means ± SE of 4 experiments. *P <= 0.05 vs. basal activity. B: VSMC were transiently transfected with either WT 193-bp (pCB81) SM22{alpha} promoter fragment or the same fragment containing point mutation in the CArG box (–162). Cells were treated with AVP, PDGF-BB, or AVP + PDGF-BB for 48 h in 0.2% serum-containing media. Normalized promoter activity was determined as in Fig. 1 and is presented as fold change over basal activity of the wild-type (WT) promoter. The graph represents means ± SE of 3 experiments. *P = 0.05 vs. basal activity; $ P = 0.05 vs. WT. C: a 193-bp (pCB81) SM22{alpha} fragment was cotransfected with an expression plasmid encoding WT-serum response factor (WT-SRF) or a plasmid lacking an insert. Cells were stimulated with AVP, PDGF-BB, or AVP + PDGF-BB for 48 h in 0.2% serum-containing media, and promoter activity normalized to {beta}-galactosidase was determined. Activity is shown as fold change over basal promoter activity cotransfected with empty vector. Data represent means ± SE of 3 experiments. *P = 0.05 vs. basal activity; #P = 0.05 vs. cells transfected with the empty vector treated with the same agonist.

 

SRF is a MADS box transcription factor binding to CArG boxes and regulating the transcriptional activity of many muscle-specific genes (see Ref. 41 for review). We tested the effects of SRF overexpression on the regulation of SM22{alpha} promoter by AVP and PDGF-BB. Cotransfection with an expression plasmid encoding wild-type (WT)-SRF enhanced basal promoter activity significantly with either pCB55 (not shown) or pCB81 fragments (Fig. 4C) of SM22{alpha}. AVP stimulation of promoter activity was even further stimulated. However, in the setting of SRF overexpression, PDGF-BB still suppressed basal promoter activity and completely blocked the increase seen with AVP.

EMSA analysis. To confirm that the inhibitory effects of CArG mutation on the induction by AVP was due to diminished binding of SRF, nuclear extracts were prepared from VSMC, and EMSA analysis was performed by using a 94-bp probe consisting of the near-CArG box and the GC box (–184 to –92). A number of specific complexes were identified, which could be displaced by excess unlabeled oligonucleotide probe (Fig. 5A). The intensity of these complexes was not affected by either AVP or PDGF-BB stimulation. Incubation with an anti-SRF resulted in the disappearance of two complexes (a and d) and the appearance of two slower migrating bands (b and c). An oligonucleotide containing a point mutation in the CArG box eliminated SRF binding as indicated by the disappearance of band a. However, the intensity of band d, which was also shifted by SRF antibody, did not change (Fig. 5B).



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  		Fig. 5. Binding of SRF to the near-CArG box and effects of point mutations on protein-DNA binding. A: labeled 94-bp WT-probe (–184 to –92) was incubated with 2 µg nuclear binding proteins prepared from untreated VSMC or cells stimulated with AVP or PDGF-BB for 6 h and analyzed by EMSA. For supershift assays, 2 µg of antibody against SRF were incubated with nuclear extracts for 30 min at 4°C before the addition of the probe. For competition assays (last 3 lanes), binding was performed in the presence of 50-fold excess unlabeled probe. In the presence of SRF antibody, specific bands a and d were shifted to give bands a and c. These data are representative of 3 independent experiments. B: EMSA were performed with WT probe or probe containing a mutation in the CArG box (–162). Incubation used 2 µg of nuclear binding proteins from untreated VSMC or cells stimulated with AVP or PDGF-BB for 6 h. Mutation of the CArG box eliminated band a. These data are representative of 2 independent experiments.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
VSMC regulate expression of SM contractile proteins during development and in response to vascular injury or atherosclerosis (35, 37, 39). The differentiated phenotype of VSMC is characterized by increased amounts of proteins such as SMA, MHC, and SM22{alpha}, whereas the dedifferentiated phenotype is associated with proliferation of VSMC and is characterized by reduced amounts of these SM-specific proteins (42). In cultured VSMC, vasoconstrictors such as AVP (47) and angiotensin II (17) increase the level of SMA expression through transcriptional activation of the promoter. Conversely, PDGF-BB suppresses expression of SMA (29) and is able to block the inductive effects of vasoconstrictors. However, it is not clear whether this regulation is limited to SMA or reflects a coordinated regulation of multiple SM markers, resulting in a more contractile phenotype in response to vasoconstrictors and a more proliferative phenotype in response to PDGF. In this study, we demonstrated that expression of a second well-defined SM marker, SM22{alpha}, is regulated in a similar fashion in VSMC through common signal transduction pathways. To our knowledge, this is the first study showing the effects of hormones and signaling pathways on the regulation of the SM22{alpha} gene in cultured VSMC. On the basis of these data, we would propose that phenotypic modulation of VSMC involves "master" signaling pathways that simultaneously act on a large number of genes. Although these individual pathways can control a variety of biological processes, they act in a combinatorial fashion to control SM-gene expression. This suggests that regulating these pathways may be a potent mechanism for modulating SM phenotype in vascular disease.

AVP increased mRNA and protein levels for SM22{alpha}, whereas PDGF-BB suppressed mRNA and protein expression, suggesting transcriptional regulation. Basal promoter activity of SM22{alpha} in quiescent VSMC appeared to be higher than SMA promoter activity. Exposure of cells to AVP increased promoter activity, and this appeared to be mediated through activation of the JNK and p38 family of MAPKs, similar to what has been observed for SMA (12). Although in neonatal VSMC thrombin-mediated increases of MHC were shown to signal through the Ras/Raf/MEK/ERK pathway (38), we were unable to demonstrate any involvement of ERK1/2 pathway in the regulation of SM22{alpha} promoter by AVP by use of a specific inhibitor of the ERK1/2 pathway, PD-98059 (Fig. 2A), similar to the regulation of SMA promoter activity by AVP in adult VSMC (12). These results likely reflect the operation of different signaling pathways controlling SM-specific genes in adult compared with neonatal cells. We have also observed differences in the regulation of gene expression by extracellular matrix (data not shown).

Similar to other SM-specific proteins, the SM22{alpha} promoter contains multiple CArG boxes within the first 300 bp (20). On the basis of truncation mutations, maximal increases in promoter activity in response to AVP required all of the CArG boxes. Promoter truncations lacking a CArG box showed no response to AVP, indicating that CArG boxes are necessary for AVP-regulated transcription (Fig. 4B). Mutation in the near-CArG box, which eliminates SRF binding (Fig. 5, A and B), blunted promoter activity. SRF bound to the CArG boxes, as has been shown for other muscle gene promoters and overexpression of SRF, increased basal and AVP-stimulated promoter activity.

The presence of the CArG box does not appear to be necessary for PDGF-BB-induced suppression of promoter activity. In fact, suppression was still detected by using a short region of the promoter (–118 to +65) or a fragment that contained the mutated form of this element (Fig. 4, A and B). Consistent with this observation, SRF overexpression was not sufficient to overcome the repressive effect of PDGF-BB on the promoter activity (Fig. 4C). These results might suggest that the effects of PDGF-BB are not directly on the promoter but involve inhibition of upstream signaling pathways required for basal activity or activation by AVP. However, the two signaling pathways that we have shown to be critical for induction by AVP, JNKs and p38 MAPK, are also activated by PDGF, suggesting that other signaling pathways, which are selectively engaged by PDGF, mediate suppression. One example of such a pathway is activation of Ras, which is observed in response to PDGF but not AVP (29). Expression of constitutively active H-Ras suppressed SM22{alpha} promoter activity. To identify the downstream targets of the suppressive pathway, we employed the mutated forms of H-rasV12, which selectively couple to a single downstream effector (see Ref. 2 for a review). Our results using these mutants indicate that all three effector pathways (Raf, RalGDS, and PI3-kinase) cooperate to suppress SM22{alpha} expression. Only when Ras engaged all three signaling pathways was the inhibitory effect complete. Because activation of the ERK pathway does not regulate SM22{alpha} expression, the ability of the Raf selective mutant to mediate suppression suggests that other effectors may compete with Raf for binding to this region of Ras. Recently, Tiam1, a GEF for Rac, has been shown (23) to have a Ras binding site that shares significant sequence similarity with the Ras binding domain of Raf. Further examination of this pathway in control of SM gene expression will be required. A role for the RalGDS pathway in control of muscle gene expression has not been previously demonstrated, but recent studies (15) have shown that this effector is critical for the transforming effects of gain of function Ras in human cells.

In summary, modulation of VSMC is characterized by coordinated changes in a number of muscle-specific genes. Earlier studies have postulated that vasoconstrictors and PDGF can modulate the phenotype of these cells, largely based on studies examining the regulation of SMA. This study demonstrates that these agents have a similar effect on a second SM marker, SM22{alpha}. The promoters for these two genes have similar elements, and binding of SRF to CArG boxes is critical for basal and regulated expression. Furthermore, both the inductive signaling pathways (JNKs and p38), and the suppressive pathways (Ras), which regulate SM22{alpha} expression also are critical for SMA. On the basis of these data, we propose that vasoconstrictors and PDGF engage a limited number of signaling pathways that can act on multiple genes, thus supporting a key role in phenotypic modulation for these factors.


   DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-19928 and DK-39902 and National Heart, Lung, and Blood Institute Grant HL-62824.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Nemenoff, Div. of Renal Diseases and Hypertension, Box C-281, Univ. of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262 (E-mail: Raphael.Nemenoff{at}UCHSC.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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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