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Smooth muscle (SM) development consists of several processes, including cell fate determination, differentiation, and maturation. The molecular mechanisms controlling SM early differentiation have been studied extensively. However, little is known about the mechanism underlying SM maturation. Cell division cycle 7 (Cdc7) has been shown to regulate cell fate determination in the initial phase of transforming growth factor-β (TGF-β)-induced SM differentiation. Our present study indicates that Cdc7 also regulates SM maturation. Knockdown of Cdc7 suppresses TGF-β-induced expression of SM myosin heavy chain, a late marker of SM differentiation. Cdc7 overexpression, on the other hand, enhances SM myosin heavy chain expression. Interestingly, Cdc7 activates the mRNA expression and promoter activity of myocardin (Myocd), a master regulator of SM differentiation, whose transcription is blocked in the initial phase of the differentiation because TGF-β does not induce Myocd mRNA until after the early SM markers are induced. These data suggest that Cdc7 mediates TGF-β-induced SM maturation via activation of Myocd transcription. Mechanistically, Cdc7 physically and functionally interacts with Nkx2.5 to regulate Myocd promoter activity. Cdc7 appears to enhance Nkx2.5 binding to Myocd promoter, leading to Myocd activation. Taken together, our studies demonstrate that Cdc7 regulates the initial and late phase of SM differentiation through distinct mechanisms.
Vascular smooth muscle (SM)2 differentiation is an important process during vasculogenesis and angiogenesis, and it is recognized that alterations in SM phenotype contribute to the progression of several cardiovascular diseases, including atherosclerosis, systematic and pulmonary hypertension, and restenosis (1–7). Although the term differentiation generally refers to the entire process by which committed but undifferentiated SMs or SM precursors acquire their cell-specific phenotypes, this process can be divided into three phases: cell fate determination (early or initial phase), differentiation (intermediate phase), and maturation (late phase) (8). The different phases can be tracked by the expression of different contractile proteins. Smooth muscle α-actin (α-SMA) and SM22α are early markers of SM differentiation whereas calponin and smooth muscle myosin heavy chain (SMMHC) represent the intermediate and late phase markers, respectively (8–12). Although we have gained a great deal of understanding on the cell fate determination and differentiation process, molecular mechanisms governing SM maturation remain largely unknown.
Transforming growth factor-β (TGF-β) plays critical roles in the SM differentiation (13–16). TGF-β also regulates cell proliferation in different physiological contexts (8, 17–19). Our previous studies have shown that cell division cycle 7 (Cdc7), a cell cycle regulator facilitating initiation of DNA replication, regulates the initiation program of TGF-β-induced SM differentiation via interaction with Smad3 while stimulating cell proliferation (20). It is unknown, however, whether Cdc7 plays a role in SM maturation.
Myocardin (Myocd), a serum response factor (SRF) coactivator, is considered as a master regulator for SM differentiation. Overexpression of Myocd activates multiple CArG-containing SM marker genes including α-SMA, SM22α, and calponin (21–24). Myocd was found to be involved in TGF-β-induced SM differentiation (25). Paradoxically, expression of early SM markers emerges prior to the detectable level of endogenous Myocd mRNA expression in the embryonic dorsal aorta, suggesting a minor role of Myocd in the initiation of SM differentiation (22, 26–28). Moreover, expression of some SM-associated genes appears to be independent of Myocd (29) (30). However, Myocd is able to induce contractile SM phenotype (31), suggesting that Myocd plays a major role in SM maturation. The molecular mechanism underlying Myocd induction during SM maturation, however, remains largely unknown. In this study, we found that Myocd expression in TGF-β-induced SM maturation is regulated by Cdc7. Cdc7 appears to induce Myocd gene transcription via interaction with Nkx2.5, a critical transcription factor for Myocd gene expression.
C3H10T1/2 (10T1/2) cells, a murine embryonic mesenchymal progenitor cell line that has been shown to differentiate into a mature SM lineage (13), were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 5% glutamine. For plasmid transfection, cells were cultured in 12- or 6-well plates or 6-cm dishes for 24 h with 80–90% of confluence followed by transfection using Lipofectamine LTX (Invitrogen) according to the manufacturer's instruction.
Total RNA from cultured cells was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription was performed using an iScript cDNA Synthesis kit (Bio-Rad). qPCR was performed in a Mx3005P qPCR machine using SYBR Green master mix (Agilent Technologies). Each sample was amplified in triplicate. Myocd primers were 5′-ACC CAT GGA CTC TGC CTA TG-3′ (forward) and 5′-AGG GGT ATT GCT CAG TGG TG-3′ (reverse). Nkx2.5 binding site and Cdc7 primers were reported previously (20).
10T1/2 cells were treated with TGF-β or other factors as indicated. Cells were washed two times with cold PBS, followed by protein extraction using radioimmunoprecipitation assay buffer (50 mmol/liter Tris-HCI, pH 7.4, 1% Triton X-100, 0.25% w/v sodium deoxycholate, 150 mmol/liter NaCl, 1 mmol/liter EGTA, 0.1% SDS, protease inhibitors, phosphatase inhibitors). Protein concentration was measured using BCA Protein Assay reagent (Thermo Scientific). 5 or 10 μg of the lysates was resolved by SDS-PAGE and transferred to PVDF (Bio-Rad). Membranes were blocked with 5% nonfat dry milk in TBST and then incubated with primary antibodies in blocking buffer for 1–2 h followed by incubation with HRP-conjugated secondary antibody for 1 h (Sigma). Detection was performed with enhanced chemiluminescence (Millipore). Antibodies used were anti-Cdc7 (Santa Cruz Biotechnology), anti-Nkx2.5 (Santa Cruz Biotechnology), anti-α-SMA (Abcam), anti-SM22α (Abcam), anti-SMMHC (Biomedical Technologies, Inc.), and anti-α-tubulin (Cell Signaling).
Cells were washed with ice-cold lysis buffer containing protease inhibitor mix (Sigma). The lysates were incubated with Cdc7 or Nkx2.5 antibody for 1 h and then with protein A/G-agarose beads at 4 °C for 12 h. The immunoprecipitates were pelleted, washed, and subjected to immunoblotting using Nkx2.5 or Cdc7 antibody as described previously (32, 33).
Myocd promoter constructs were co-transfected into 10T1/2 cells with other plasmids as described previously (34). Cells were starved in serum-free medium for 24 h and then treated with 5 ng/ml TGF-β for 24 h. Luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega). Experiments were repeated at least three times, and the results from representative experiments are shown ± S.D.
ChIP assays were performed as described previously (35). Growth-arrested 10T1/2 cells were treated with TGF-β for 24 h. Chromatin complexes were immunoprecipitated with 1 μg of Nkx2.5 antibody or IgG (negative control). Semiquantitative PCR and qPCR were performed to amplify the Myocd promoter region containing Nkx2.5-binding element (NKE) using the following primer set: 5′-GTT CAG CAC TGC TTG TGG AA-3′ (forward) and 5′-TTT TCC CAT TCT CTG GGT TG-3′ (reverse).
All values are expressed as mean ± S.E. Data were evalutated using analysis of variance with pairwise comparisons between groups. A p value <0.05 was considered statistically significant.
Our previous study has shown that Cdc7 plays a role in initiating TGF-β-induced SM differentiation (20). To determine whether Cdc7 is important for SM maturation, we examined the expression of Cdc7 along with the mature SM marker SMMHC in cells treated with TGF-β for up to 48 h, a time past the initial stage of SM differentiation. As shown in Fig. 1, a very low level of SMMHC was expressed at 24 h after TGF-β treatment, but a significantly higher level of SMMHC expression was evident 48 h after TGF-β induction, indicating the formation of a mature SM phenotype. Cdc7 expression was significantly induced 24 h after TGF-β treatment (Fig. 1, A and B), consistent with its role in the initial phase of SM differentiation. Interestingly, Cdc7 expression was further increased by TGF-β during the SM maturation process (Fig. 1), suggesting that in addition to the initiation of SM differentiation, Cdc7 may also be involved in SM maturation.
To determine whether Cdc7 plays a role in SM maturation, we tested whether Cdc7 is required for the induction of SMMHC. As shown in Fig. 2, A and B, when Cdc7 expression was blocked by its shRNA, TGF-β-induced SMMHC expression was significantly attenuated, indicating that Cdc7 is essential for TGF-β-induced SM maturation. To further determine the role of Cdc7 in mature SM marker induction, we overexpressed Cdc7 in serum-starved cells without TGF-β addition and found that forced expression of Cdc7 in 10T1/2 cells induced a 2.5-fold increase in SMMHC expression (Fig. 2, C–E), indicating that Cdc7 alone is sufficient for promoting SM maturation.
Myocd is a master regulator for SM differentiation and is expressed mainly in SMs and cardiomyocytes. Myocd can induce a contractile SM phenotype (31). Indeed, Myocd alone can induce the mature SM marker SMMHC expression (Fig. 3, A and B), consistent with previous reports (24, 36). Because Cdc7 played a very important role in regulating mature SM marker gene expression, and Cdc7 was induced much earlier than Myocd by TGF-β (20), we sought to determine whether Cdc7 induces mature SM marker expression through Myocd. As shown in Fig. 3, C and D, Cdc7 induced SMMHC expression; however, dominant negative Myocd significantly inhibited Cdc7-induced SMMHC expression, indicating that Myocd is a downstream effector of Cdc7 during SM maturation. Because Myocd is a coactivator of SRF, we tested whether SRF is also important for Cdc7 function in SM maturation. As shown in Fig. 3, E and F, knockdown of SRF by shRNA blocked Cdc7-induced SMMHC expression. These data demonstrate that Cdc7 induces SM maturation in a Myocd/SRF-dependent manner.
Our previous studies have shown that TGF-β did not induce Myocd expression until 18 h of TGF-β treatment, preceding SM maturation (37), supporting the role of Myocd in regulating SM maturation. Interestingly, Cdc7 appeared to be essential for TGF-β-induced Myocd expression because knockdown of Cdc7 by shRNA blocked Myocd expression induced by TGF-β (Fig. 4, A and B). Conversely, overexpression of Cdc7 stimulated Myocd expression (Fig. 4, C and D). Moreover, knockdown of Cdc7 by shRNA significantly blocked TGF-β-induced Myocd promoter activity (Fig. 4E), suggesting that Cdc7 plays a role in Myocd gene transcription. Indeed, Cdc7 can activate Myocd promoter in a dose-dependent manner (Fig. 4F).
Nkx2.5 is a transcription factor regulating Myocd expression (38). It is also involved in TGF-β-induced Myocd promoter activity in 10T1/2 cells (37, 38). Because both Cdc7 and Nkx2.5 are essential for Myocd promoter activity, we sought to determine whether there is a functional interaction between Cdc7 and Nkx2.5 in Myocd gene transcription. As shown in Fig. 5A, although Cdc7 or Nkx2.5 alone significantly up-regulated Myocd promoter activity, the combination of Cdc7 and Nkx2.5 dramatically and synergistically increased the promoter activity by 3.7- and 10.2-fold compared with Cdc7 and Nkx2.5 alone group, respectively. To determine whether Cdc7 is required for Nkx2.5 activity, we knocked down Cdc7 by shRNA and found that blockade of Cdc7 expression suppressed Nkx2.5-induced Myocd activation in both vehicle- and TGF-β-treated cells (Fig. 5B). TGF-β did not induce a dramatic increase of Myocd promoter activity in Nkx2.5-overexpressed cells probably because TGF-β induced Myocd expression through the induction of Nkx2.5. Cdc7 did not affect Nkx2.5 expression because neither overexpression nor knockdown of Cdc7 had any effect on the Nkx2.5 protein level (Fig. 5C). These results led us to hypothesize that Cdc7 regulates Nkx2.5 function by modulating Nkx2.5 interaction with Myocd promoter. To test this hypothesis, we detected whether NKE mutation (38), which blocks Nkx2.5 binding to Myocd promoter, affects Cdc7 activity in activating the Myocd promoter. As shown in Fig. 5D, the NKE mutation markedly diminished Cdc7-induced Myocd activation by 62%, demonstrating that Cdc7-induced Myocd transcription depends on the interaction of Nkx2.5 with NKE in the Myocd promoter.
Because Cdc7 functionally interacted with Nkx2.5, and Cdc7 function was dependent on Nkx2.5 binding to Myocd promoter, we sought to examine whether Cdc7 physically interacts with Nkx2.5. A co-IP assay using endogenous proteins extracted from 10T1/2 cells treated with or without TGF-β showed that Nkx2.5 was co-immunoprecipitated with Cdc7 (Fig. 6A), and Cdc7 was co-immunoprecipitated with Nkx2.5 (Fig. 6B), indicating that Cdc7 physically interacted with Nkx2.5 in 10T1/2 cells. TGF-β appeared to enhance their interaction (Fig. 6, A and B).
Our previous studies have shown that the interaction of Nkx2.5 with Smad3 blocks Nkx2.5 binding to NKE in Myocd promoter in the early phase of SM differentiation (37). To determine whether the interaction of Nkx2.5 with Smad3 prevents Nkx2.5 from interacting with Cdc7, we performed co-IPs of Nkx2.5 with Smad3 and Cdc7 at early (4 h) and later (24 h) times following TGF-β treatment. As shown in Fig. 6, C and 6D, in the initial phase of TGF-β induction, 79% of Nkx2.5 interacted with Smad3. However, at the later phase, 77% of Nkx2.5 interacted with Cdc7. These data suggest that the interaction of Nkx2.5 with Smad3 and that with Cdc7 are mutually exclusive.
Because Cdc7 functionally and physically interacted with Nkx2.5, we further explored the mechanism underlying Cdc7 function in SM maturation by testing whether Cdc7 modulates Nkx2.5 interaction with Myocd promoter. ChIP analysis showed that Nkx2.5 bound weakly to the NKE in Myocd promoter in the basal state (Fig. 7). TGF-β markedly increased the binding of Nkx2.5 to the NKE (Fig. 7). Blockade of Cdc7 expression by shRNA, however, significantly attenuated the TGF-β-enhanced Nkx2.5 binding to the promoter (Fig. 7), indicating that Cdc7 is essential for Nkx2.5 interaction with Myocd promoter. These data demonstrate that Cdc7 regulates SM maturation through enhancing Nkx2.5 binding to Myocd promoter, leading to increased expression of Myocd, which in turn activates the expression of mature SM marker SMMHC.
SM maturation is a process by which multipotential cells in the developing organism acquire contractile SM characteristics that distinguish them from other cell types (4, 8). At present, the two marker proteins that provide the best definition of a mature contractile SM phenotype are SMMHC and smoothelin. SMMHC expression has never been detected in non-SMs in vivo and is the only marker protein that is SM-specific during embryogenesis (39). Our results show that TGF-β induces SMMHC expression in 10T1/2 cells after 48 h of stimulation while enhancing Cdc7 expression (Fig. 1). Because TGF-β downstream intermediate signaling molecules Smad2/3 are translocated to cytoplasm 18 h after TGF-β stimulation when they have completed their role in inducing early stage of SM differentiation (32, 37), molecular mechanisms other than Smad2/3 signaling pathway are required for mediating TGF-β-induced SM maturation. Our data demonstrate that Cdc7/Nkx2.5-mediated Myocd expression is one of these mechanisms. First, Cdc7 is essential for TGF-β-induced SMMHC expression (Fig. 2). Second, Cdc7 activates Myocd expression (Fig. 4). Finally, Myocd/SRF mediates Cdc7 function in inducing SMMHC expression because dominant negative Myocd or SRF shRNA blocks Cdc7-induced SMMHC expression (Fig. 3). These data establish a novel mechanism by which Cdc7 induces SM maturation via the induction of Myocd.
Cdc7 is not a transcription factor. Therefore, Cdc7 may serve as a coactivator in regulating Myocd expression. Nkx2.5 is a downstream transcription factor of PI3K signaling and is essential for Myocd expression in cardiac myocytes (38). Cdc7 appears to be an Nkx2.5 coactivator because Cdc7 and Nkx2.5 synergistically up-regulate Myocd transcription (Fig. 5). Moreover, Cdc7 physically interacts with Nxk2.5 (Fig. 6). Their interaction is enhanced by TGF-β probably because the enhanced expression of both Cdc7 and Nkx2.5 is induced by TGF-β. Most importantly, Cdc7 is required for TGF-β-enriched Nkx2.5 binding to Myocd promoter because knockdown of Cdc7 blocks the enhanced Nkx2.5 interaction with Myocd promoter (Fig. 7). Cdc7 may recruit more Nkx2.5 for binding to the NKE in the Myocd promoter in TGF-β-treated cells or enhance the binding affinity of Nkx2.5.
The present study together with our recently published results (20, 37) provide a model for Cdc7 function in the initial and late phase of TGF-β-induced SM differentiation, i.e. upon TGF-β stimulation, Smad3 is activated and translocated to nucleus where it binds to Cdc7 and Nkx2.5. Smad3 interaction with Cdc7 promotes transcription of the early SM markers such as α-SMA and SM22α (within 18 h of TGF-β stimulation). Smad3 interaction with Nkx2.5 blocks Nkx2.5 binding to NKE in Myocd promoter. After 18 h of TGF-β stimulation, Smad3 dissociates with Nkx2.5 and shuttles back to the plasma possibly due to the dephosphorylation of Smad3 by protein phosphatase 1A (40), which makes Nkx2.5 available for Cdc7. Cdc7 then interacts with Nkx2.5 to enhance Nkx2.5 binding to NKE in Myocd promoter, leading to the activation of Myocd transcription, which in turn promotes SMMHC expression (Fig. 8).
The limitation of this study is that it is unclear why Cdc7 does not bind to Nkx2.5 in the early phase of SM differentiation. The possible explanation is that Smad3 has a stronger binding affinity to Nkx2.5. In addition, Smad3 was translocated into nuclei within 10 min after TGF-β induction. Therefore, Smad3 has the advantage to preoccupy any available Nkx2.5 in the nuclei. Cdc7 is not induced until 2 h after TGF-β induction (20) and requires a high level of expression in inducing SMMHC expression, suggesting that Cdc7 may be less competitive in binding with Nkx2.5 compared with Smad3, which can be further studied in the future.
*This work was supported, in whole or in part, by National Institutes of Health Grants HL093429 and HL107526 (to S.-Y. C.).
2The abbreviations used are: