130-kDa smooth muscle myosin light chain kinase is transcribed from a CArG-dependent, internal promoter within the mouse mylk gene

The 130-kDa smooth muscle myosin light chain kinase (smMLCK) is a Ca2+/CaM-regulated enzyme that plays a pivotal role in the initiation of smooth muscle contraction and regulation of cellular migration and division. Despite the critical importance of smMLCK in these processes, little is known about the mechanisms regulating its expression. In this study, we have identified the proximal promoter of smMLCK within an intron of the mouse mylk gene. The mylk gene encodes at least two isoforms of MLCK (130 and 220 kDa) and telokin. Luciferase reporter gene assays demonstrated that a 282-bp fragment (-167 to +115) of the smMLCK promoter was sufficient for maximum activity in A10 smooth muscle cells and 10T1/2 fibroblasts. Deletion of the 16 bp between -167 and -151, which included a CArG box, resulted in a nearly complete loss of promoter activity. Gel mobility shift assays and chromatin immunoprecipitation assays demonstrated that serum response factor (SRF) binds to this CArG box both in vitro and in vivo. SRF knockdown by short hairpin RNA decreased endogenous smMLCK expression in A10 cells. Although the SRF coactivator myocardin induced smMLCK expression in 10T1/2 cells, myocardin activated the promoter only two- to fourfold in reporter gene assays. Addition of either intron 1 or 6 kb of the 5' upstream sequence did not lead to any further activation of the promoter by myocardin. The proximal smMLCK promoter also contains a consensus GATA-binding site that bound GATA-6. GATA-6 binding to this site decreased endogenous smMLCK expression, inhibited promoter activity in smooth muscle cells, and blocked the ability of myocardin to induce smMLCK expression. Altogether, these data suggest that SRF and SRF-associated factors play a key role in regulating the expression of smMLCK.     

Smooth muscle-specific genes are differentially sensitive to inhibition by Elk-1

Understanding the mechanism of smooth muscle cell (SMC) differentiation will provide the foundation for elucidating SMC-related diseases, such as atherosclerosis, restenosis, and asthma. In the current study, overexpression of Elk-1 in SMCs down-regulated expression of several endogenous smooth muscle-restricted proteins, including telokin, SM22α, and smooth muscle α-actin. In contrast, down-regulation of endogenous Elk-1 in smooth muscle cells increased the expression of only telokin and SM22α, suggesting that smooth muscle-specific promoters are differentially sensitive to the inhibitory effects of Elk-1. Consistent with this, overexpression of the DNA binding domain of Elk-1, which acts as a dominant-negative protein by displacing endogenous Elk-1, enhanced the expression of telokin and SM22α without affecting expression of smooth muscle α-actin. Elk-1 suppressed the activity of smooth muscle-restricted promoters, including the telokin promoter that does not contain a consensus Elk-1 binding site, through its ability to block myocardin-induced activation of the promoters. Gel mobility shift and chromatin immunoprecipitation assays revealed that Elk-1 binds to a nonconsensus binding site in the telokin promoter and Elk-1 binding is dependent on serum response factor (SRF) binding to a nearby CArG box. Although overexpression of the SRF-binding B-box domain of Elk-1 is sufficient to repress the myocardin activation of the telokin promoter, this repression is not as complete as that seen with an Elk-1 fragment that includes the DNA binding domain. In addition, reporter gene assays demonstrate that an intact Elk-1 binding site in the telokin promoter is required for Elk-1 to maximally inhibit promoter activity. Together, these data suggest that the differential sensitivity of smooth muscle-specific genes to inhibition by Elk-1 may play a role in the complex changes in smooth muscle-specific protein expression that are observed under pathological conditions.     

Cooperation of myocardin and Smad2 in inducing differentiation of mesenchymal stem cells into smooth muscle cells

Several reports demonstrated that mesenchymal stem cells (MSCs) might differentiate into smooth muscle cells (SMCs) in vitro and in vivo. It has been shown that myocardin protein is a strong inducer of smooth muscle genes and MSCs can differentiate into SMCs in response to transforming growth factor-β (TGF-β). However, the relationship or link between myocardin and TGF-β3-induced MSC differentiation has not been fully elucidated. Here, we demonstrated that both myocardin and TGF-β3 were able to induce differentiation of rat bone marrow-derived MSCs toward smooth-muscle-like cell types, as evidenced by increasing expression of SMC-specific genes. Of note, myocardin cooperated with Smad2 to synergistically activate SM22α promoter and significantly enhance the expression of SM22α. Report assays with site-direct mutation analysis of SM22α promoter demonstrated that myocardin and Smad2 coactivated SM22α promoter mainly depending on CArG box and less on smad binding elements (SBE) sites as well. These findings reveal the cooperation of myocardin and Smad2 in process of MSC differentiation into SMCs.     

Platelet-derived growth factor-BB represses smooth muscle cell marker genes via changes in binding of MKL factors and histone deacetylases to their promoters

A hallmark of smooth muscle cell (SMC) phenotypic switching is suppression of SMC marker gene expression. Although myocardin has been shown to be a key regulator of this process, the role of its related factors, MKL1 and MKL2, in SMC phenotypic switching remains unknown. The present studies were aimed at determining if: 1) MKL factors contribute to the expression of SMC marker genes in cultured SMCs; and 2) platelet-derived growth factor-BB (PDGF-BB)-induced repression of SMC marker genes is mediated by suppression of MKL factors. Results of gain- and loss-of-function experiments showed that MKL factors regulated the expression of single and multiple CArG [CC(AT-rich)(6)GG]-containing SMC marker genes, such as smooth muscle (SM) alpha-actin and telokin, but not CArG-independent SMC marker genes such as smoothelin-B. Treatment with PDGF-BB reduced the expression of CArG-containing SMC marker genes, as well as myocardin expression in cultured SMCs, while it had no effect on expression of MKL1 and MKL2. However, of interest, PDGF-BB induced the dissociation of MKL factors from the CArG-containing region of SMC marker genes, as determined by chromatin immunoprecipitation assays. This dissociation was caused by the competition between MKL factors and phosphorylated Elk-1 at early time points, but subsequently by the reduction in acetylated histone H4 levels at these promoter regions mediated by histone deacetylases, HDAC2, HDAC4, and HDAC5. Results provide novel evidence that PDGF-BB-induced repression of SMC marker genes is mediated through combinatorial mechanisms, including downregulation of myocardin expression and inhibition of the association of myocardin/MKL factors with CArG-containing SMC marker gene promoters.     

Sequences at the 3' side of the c-fos SRE mediate gene expression via an Sob1-dependent, TCF-independent pathway.

We previously described a 110-kDa tyrosine phosphoprotein, Sob 1, that regulates formation of the DNA binding complex Band A at the c-fos serum response element (SRE) during T cell activation. Using competition and mutant oligonucleotide analysis, we have determined that both the core CArG box of the c-fos SRE and the 3' sequences flanking the CArG box are necessary for stable Band A complex formation. Moreover, using transient transfection and reporter assays, we show that mutations affecting Band A complex formation in vitro also impaired serum induction of c-fos gene expression in vivo. Since mutation at this site has no effect on SRF binding, our results suggest that in combination with SRE/SRF, Sob 1-regulated factor(s) bind at the 3' side of SRE to form Band A, and this confers maximal serum induction of c-fos gene expression via the SRE.     

Distinct enhancers regulate skeletal and cardiac muscle-specific expression programs of the cardiac alpha-actin gene in Xenopus embryos

During vertebrate embryonic development, cardiac and skeletal muscle originates from distinct precursor populations. Despite the profound structural and functional differences in the striated muscle tissue they eventually form, such progenitors share many features such as components of contractile apparatus. In vertebrate embryos, the alpha-cardiac actin gene encodes a major component of the myofibril in both skeletal and cardiac muscle. Here, we show that expression of Xenopus cardiac alpha-actin in the myotomes and developing heart tube of the tadpole requires distinct enhancers within its proximal promoter. Using transgenic embryos, we find that mutations in the promoter-proximal CArG box and 5 bp downstream of it specifically eliminate expression of a GFP transgene within the developing heart, while high levels of expression in somitic muscle are maintained. This sequence is insufficient on its own to limit expression solely to the myocardium, such restriction requiring multiple elements within the proximal promoter. Two additional enhancers are active in skeletal muscle of the embryo, either one of which has to interact with the proximal CArG box for correct expression to be established. Transgenic reporters containing multimerised copies of CArG box 1 faithfully detect most sites of SRF expression in the developing embryo as do equivalent reporters containing the SRF binding site from the c-fos promoter. Significantly, while these motifs possess a different A/T core within the CC(A/T)(6)GG consensus and show no similarity in flanking sequence, each can interact with a myotome-specific distal enhancer of cardiac alpha-actin promoter, to confer appropriate cardiac alpha-actin-specific regulation of transgene expression. Together, these results suggest that the role of CArG box 1 in the cardiac alpha-actin gene promoter is to act solely as a high-affinity SRF binding site.     

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity

Mouse telokin and SM22 promoters have previously been shown to direct smooth muscle cell-specific expression of transgenes in vivo in adult mice. However, the activity of these promoters is highly dependent on the integration site of the transgene. In the current study, we found that the ectopic expression of telokin promoter transgenes could be abolished by flanking the transgene with insulator elements from the H19 gene. However, the insulator elements did not increase the proportion of mouse lines that exhibited consistent, detectable levels of transgene expression. In contrast, when transgenes were targeted to the hprt locus, both telokin and SM22 promoters resulted in reproducible patterns and levels of transgene expression in all lines of mice examined. Telokin promoter transgene expression was restricted to smooth muscle tissues in adult and embryonic mice. As reported previously, SM22 transgenes were expressed at high levels specifically in arterial smooth muscle cells; however, in contrast to randomly integrated transgenes, the hprt-targeted SM22 transgenes were also expressed at high levels in smooth muscle cells in veins, bladder, and gallbladder. Using hprt-targeted transgenes, we further analyzed elements within the telokin promoter required for tissue specific activity in vivo. Analysis of these transgenes revealed that the CArG element in the telokin promoter is required for promoter activity in all tissues and that the CArG element and adjacent AT-rich region are sufficient to drive transgene expression in bladder but not intestinal smooth muscle cells. visceral smooth muscle; development; myosin light chain kinase; embryos; CArG element