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J. Biol. Chem., Vol. 282, Issue 14, 10133-10137, April 6, 2007

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Response Gene to Complement 32, a Novel Regulator for Transforming Growth Factor--induced Smooth Muscle Differentiation of Neural Crest Cells^*

Fengmin Li, Zaiming Luo, Wenyan Huang, Quansheng Lu, Christopher S. Wilcox, Pedro A. Jose, and Shiyou Chen¹

From the Departments of Pediatrics, and Medicine, Georgetown University Medical Center, Washington, D. C. 20057

Received for publication, August 23, 2006 , and in revised form, February 26, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We previously developed a robust in vitro model system for vascular smooth muscle cell (VSMC) differentiation from neural crest cell line Monc-1 upon transforming growth factor- (TGF-) induction. Further studies demonstrated that both Smad and RhoA signaling are critical for TGF--induced VSMC development. To identify downstream targets, we performed Affymetrix cDNA array analysis of Monc-1 cells and identified a gene named response gene to complement 32 (RGC-32) to be important for the VSMC differentiation. RGC-32 expression was increased 5-fold after 2 h and 50-fold after 24 h of TGF- induction. Knockdown of RGC-32 expression in Monc-1 cells by small interfering RNA significantly inhibited the expression of multiple smooth muscle marker genes, including SM -actin (-SMA), SM22, and calponin. Of importance, the inhibition of RGC-32 expression correlated with the reduction of -SMA while not inhibiting smooth muscle-unrelated c-fos gene expression, suggesting that RGC-32 is an important protein factor for VSMC differentiation from neural crest cells. Moreover, RGC-32 overexpression significantly enhanced TGF--induced -SMA, SM22, and SM myosin heavy chain promoter activities in both Monc-1 and C3H10T1/2 cells. The induction of VSMC gene promoters by RGC-32 appears to be CArG-dependent. These data suggest that RGC-32 controls VSMC differentiation by regulating marker gene transcription in a CArG-dependent manner. Further studies revealed that both Smad and RhoA signaling are important for RGC-32 activation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Alterations in the differentiated state of the VSMC² are known to contribute to a number of major cardiovascular diseases in humans including atherosclerosis, systematic and pulmonary hypertension, and restenosis (1–7). In many of these diseases, it is the ability of VSMCs to modulate their phenotype that leads to pathology. Unlike skeletal or cardiac muscle, VSMCs exhibit a wide variation of phenotypes both in vitro and in vivo (8). The ability of VSMCs to change from a contractile to a synthetic (proliferative) phenotype, while entering the cell cycle and proliferating, may underlie some of the pathological findings in the vascular proliferative disorders mentioned above (9). These dedifferentiated cells display a phenotype closely resembling that of developing VSMCs observed during embryonic angiogenesis (10). Therefore, understanding the molecular mechanisms that control VSMC development and differentiation will provide fundamental insights into several pathological processes, including atherosclerosis, and eventually will lead to the identification of targets for therapeutic intervention.

VSMCs are derived from at least three precursor cell types, depending on the timing and location of formation (11, 12). The three major sources of VSMCs outside of the cardiac circulation in the vertebrate embryo are the neural crest, mesodermal precursors, and endothelium or endothelial stem cells. The other sources of vascular SMC include proepicardial organ and the dermamyotome of the somite (13, 14). Neural crest stem cells are the primary precursor cells that form VSMCs in parts of the cranial circulation, cardiac outflow tracts, and great vessels (15, 16). Mouse mutants defective in neural crest formation exhibit severe defects in cardiac development reminiscent of DiGeorge syndrome (17), underscoring the importance in understanding how VSMCs develop from these precursors. Several factors have been shown to serve as intrinsic and extrinsic fate determinants for neural crest cells (18–25), but the molecular mechanisms controlling their migration and differentiation remain largely unknown. The in vivo signals that specify neural crest commitment to the VSMC lineage have not been fully identified, but TGF- plays an important role both in vivo and in vitro (26–28).

TGF- family members transmit signals through transmembrane serine-threonine kinase receptors (29). The TGF- type II receptor (TRII) binds ligand and recruits and phosphorylates the type I receptor, activating its kinase activity. Activated type I receptor then binds and phosphorylates downstream signaling intermediates called Smads. Activated Smads translocate to the nucleus where they regulate target gene transcription (30).

Recent studies have shown that specific inactivation of TGF- signaling in neural crest stem cells results in cardiovascular SMC defects. The TRII knock-out in neural crest cells abolished the development of smooth musculature at embryonic day 10.5. At later developmental stages, neural crest cells that failed to adopt a smooth muscle cell fate underwent apoptotic cell death. These results suggest that the smooth muscle -actin-positive cells generated from TGF--treated neural crest cells in culture indeed represent SMCs (31). A later report from a different group using the same strategy to mutate TRII in neural crest failed to identify SMC defects, possibly due to an in vivo compensatory mechanism or the use of a different TRII-floxed mouse line, as discussed by the authors (32). The function of TGF- signaling on VSMC differentiation from neural crest cells remains controversial. Our in vitro studies have shown that TGF- robustly induced expression of VSMC markers in neural crest Monc-1 cells (33). Further studies showed that Smad and RhoA play critical roles in mediating TGF--induced VSMC differentiation (34). Downstream targets of TGF- signaling important for VSMC differentiation from neural crest cells, however, remain to be determined.

In the present study, by performing Affymetrix cDNA array analysis and a small RNA interference (siRNA) assay, we have identified a protein, namely response gene to complement (RGC)-32, to be critical for TGF--induced VSMC differentiation. Further studies showed that RGC-32 controls VSMC differentiation by regulating the transcription of SMC-specific genes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—Monc-1 cells were grown as described (35). C3H10T1/2 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, glucose, and l-glutamine supplemented with penicillin.

Mouse RGC-32 Expression Vector Construction—Mouse RGC-32 cDNA was amplified from mRNA extracted from TGF--treated Monc-1 cells. The 5' primer included a BamHI restriction site for cloning, a Kozak sequence, and a T7 tag followed by a RGC-32 cDNA sequence. The 3' primer included RGC-32 cDNA sequence, a stop codon, and an XbaI restriction site. RGC-32 full-length cDNA was amplified with Vent DNA polymerase (New England Biolabs). For cloning, both pcDNA 3.0 vector and amplified RGC-32 cDNA were digested with BamHI and XbaI and then purified, followed by ligation using T4 DNA ligase (New England Biolabs). The cloned cDNA was verified by sequencing. RGC-32 overexpression in Monc-1 cells was confirmed by Western blot using T7 antibody (Novagen).

RNA Interference—To knock down RGC-32 expression, Monc-1 cells were transfected with RGC-32 siRNA using Lipofectamine 2000. 6 h later, the cells were treated with TGF- (5 ng/ml) for 24 h. Cells were then collected for analysis of RGC-32 and SMC marker expression. RGC-32 siRNAs (5'-CCUGGCCAUUCUUGGUUCACUAUAA-3') were purchased from Invitrogen.

Reverse Transcription-PCR (RT-PCR) and Quantitative PCR (qPCR)—RT-PCR and qPCR were performed as described previously. RGC-32 primer sequences were: 5'-AAGCCGCCCTCAGCGCAGAGCAG-3' (forward) and 5'-CATACTTGCTAAGGTCCTG-3' (reverse). Primers for SMC marker genes -SMA, SM22, calponin, and SMC-nonspecific gene c-fos were previously described (33).

Transfection and Luciferase Reporter Assay—-SMA wild type and CArG box mutants and myosin heavy chain (SM-MHC) promoter luciferase reporter constructs were generously provided by Dr. Gary Owens (36). SM22 promoter luciferase reporter construct was previously described (37). pcDNA or RGC-32 expression vector co-transfection with promoter construct into Monc-1 or C3H10T1/2 cells followed by luciferase assay was performed similarly as described previously (33), except that the cells were growth factor- (Monc-1 cells) or serum-(10T1/2 cells) starved and then TGF--treated for 48 h before luciferase assay.

Statistical Analysis—All values are expressed as mean ± S.E. Data were analyzed using analysis of variance with pairwise comparisons between groups. A level of p values < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have previously described that Smad and RhoA are the two pathways critical for TGF--induced SMC differentiation from Monc-1 cells. To identify the earliest downstream targets that may initiate the differentiation program, we treated Monc-1 cells with TGF- for 24 h and then performed microarray analyses using Affymetrix chips. RGC-32 was identified to be highly up-regulated (15-fold) by TGF- (data not shown). RGC-32 was first identified in oligodendrocytes in response to complement activation (38). It is a 14-kDa protein expressed in many adult human tissues including heart, brain, liver, skeletal muscle, placenta, kidney, and pancreas (39), and it is overexpressed in colon cancer and many tumors (40). Functionally, RGC-32 has been shown to play a role in cell cycle activation. It is a substrate and regulator of cyclin-dependent kinase p34^CDC2 (39, 41). Whether RGC-32 is involved in VSMC differentiation, however, remains to be determined.

To confirm the TGF- induction of RGC-32, we performed regular RT-PCR and qPCR to determine RGC-32 expression in Monc-1 cells. We found that TGF- significantly up-regulated RGC-32 expression in Monc-1 cells. qPCR showed that RGC-32 was significantly up-regulated as early as within 2 h of TGF- induction. The induction was increased 50-fold after 24 h of treatment and remained increased for up to 4 days, the longest time tested (supplemental Fig. S1).

To determine whether RGC-32 is functionally important for SMC differentiation, we used siRNA to knock down RGC-32 expression in Monc-1 cells and then treated the cells with TGF-. As shown in Fig. 1, RGC-32 siRNA effectively inhibited RGC-32 expression, and at the same time, blocked the expression of marker genes including -SMA, SM22, and calponin (Fig. 1A). Importantly, inhibition of RGC-32 expression correlated with the reduction of -SMA but did not affect SMC-nonspecific c-fos gene expression (Fig. 1B). Control siRNA did not inhibit the expression of any genes. These data suggest that RGC-32 is essential for TGF--induced SMC differentiation from Monc-1 cells.

Because transcriptional regulation is an important process in SMC differentiation, we decided to test whether RGC-32 regulates SMC marker promoter activity. We cloned RGC-32 cDNA into pcDNA expression vector and then co-transfected the expression vector with -SMA, SM22, or SM-MHC promoter constructs into Monc-1 cells. Luciferase assays showed that RGC-32 significantly enhanced TGF--induced promoter activities of all the three marker genes (Fig. 2, A–C).

View larger version (35K): [in this window] [in a new window]   FIGURE 1. siRNA blockade of RGC-32 expression inhibits SMC marker expression. Monc-1 cells were transfected with RGC-32 (siRGC) or GFP (siGFP) siRNA followed by 1 day of TGF- treatment. RT-PCR (A) or qPCR (B) was performed to detect mRNA expression of the genes indicated. Data shown are the representative results from two separate experiments. *, p < 0.01 for comparison with TGF- and GFP siRNA-treated groups. Ctrl, control.

SRF and its cofactor myocardin play important roles in SMC differentiation (36, 46–55). TGF- has been shown to increase SRF binding to SM22 and SM -actin promoter in 10T1/2 cells (56, 57). To determine whether RGC-32 function is associated with SRF, we co-transfected RGC-32 expression vector with wild type -SMA promoter (from –2.6 to +2.8 kb) construct or the construct with mutation at CArG box in the promoter or first intron into Monc-1 cells. We found that CArG box mutation in the promoter region (CArGs A+B) completely blocked the RGC-32 function on -SMA promoter activity (Fig. 3). The intronic CArG mutation also significantly inhibited RGC-32 function but to a lesser extent than CArG A+B mutation (Fig. 3). These data suggest that RGC-32 controls SMC differentiation by regulating marker gene transcription in a CArG-dependent manner.

We reported previously that Smad and RhoA signaling are the two pathways critical for TGF--induced SMC differentiation from Monc-1 cells (33, 34). To determine whether Smad and RhoA signaling are important for RGC-32 activation, we used Smad7 to block Smad signaling, and C3 exotoxin to block RhoA signaling. We found that both Smad7 and C3 exotoxin significantly inhibited TGF--induced RGC-32 expression in Monc-1 cells (Fig. 4), suggesting that both Smad signaling and RhoA are important for RGC-32 activation during TGF--induced SMC differentiation from Monc-1 cells.

View larger version (44K): [in this window] [in a new window]   FIGURE 2. RGC-32 enhances promoter activity of SMC markers in Monc-1 and 10T1/2 cells. -SMA, SM22, or SM-MHC promoter construct was individually co-transfected with pcDNA 3.0 or RGC-32 expression vector into Monc-1 (A–C) or 10T1/2 (D–F) cells followed by TGF- (5 ng/ml) induction as indicated. Luciferase assays were then performed. Data shown were the representative results of three or four independent experiments. *, p < 0.01 for comparison with the TGF--treated pcDNA transfection groups. **, p < 0.05 for SM-MHC promoter comparison with pcDNA transfection and TGF--treated group (Monc-1 cells).

View larger version (12K): [in this window] [in a new window]   FIGURE 3. RGC-32 enhances -SMA promoter activity in a CArG-dependent manner. Wild type -SMA promoter (from –2.6 to +2.8 kb) construct or the construct with mutations at CArG box(es) in the promoter region (CArGm(A+B)) or in the first intron (CArGmIntr) was co-transfected with pcDNA or RGC-32 expression vector into Monc-1 cells as indicated. Luciferase assay was performed. Data shown were the representative results of three independent experiments. *, p < 0.01 for comparison with wild type -SMA promoter activity in the presence of RGC-32.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (33K): [in this window] [in a new window]   FIGURE 4. Smad and RhoA signaling are important for RGC-32 activation. Monc-1 cells were transfected with pcDNA or Smad7 expression plasmid (A) or treated with C3 exotoxin as described (34) (B) followed by TGF- (5 ng/ml) induction for 24 h as indicated. RT-PCR was performed to detect RGC-32 expression. Cyclophilin is an internal control.

TGF--induced SMC differentiation from Monc-1 cells requires both Smad and RhoA signaling (33, 34). Current studies demonstrate that both Smad and RhoA are important for RGC-32 activation. Therefore, we conclude that TGF--induced SMC differentiation from Monc-1 cells is regulated as follows. TGF- first activates Smad and RhoA signaling, which further activate RGC-32. RGC-32, together with other transcriptional factors including SRF and Smads, then turns on SMC maker gene transcription and initiates SMC differentiation.

	FOOTNOTES

 
^* This work was supported by an American Heart Association Scientist Development Grant (to S. C.) and National Institutes of Health Grants HL68686 (to C. S. W.) and DK52612 (to P. A. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains two supplemental figures.

¹ To whom correspondence should be addressed: Dept. of Pediatrics, Bldg. D, Rm. 363, Georgetown University Medical Center, 4000 Reservoir Rd., N. W., Washington, DC 20057. Tel.: 202-687-3401; Fax: 202-687-1503; E-mail: sc229{at}georgetown.edu

² The abbreviations used are: SMC, smooth muscle cell; VSMC, vascular SMC; TGF-, transforming growth factor-; TRII, TGF- type II receptor; RGC-32, response gene to complement 32; siRNA, small interfering RNA; -SMA, SM -actin; MHC, myosin heavy chain; SRF, serum-response factor; RT, reverse transcription; qPCR, quantitative PCR.

	ACKNOWLEDGMENTS

 
We thank Dr. Robert Lechleider for helpful advice and Dr. Gary Owens for generously providing -SMA and SM-MHC promoter constructs.

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