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(Circulation Research. 2004;95:340.)
© 2004 American Heart Association, Inc.

Editorials

Channeling to Myocardin

Joseph M. Miano

From the Center for Cardiovascular Research in the Aab Institute of Biomedical Sciences, University of Rochester School of Medicine, Rochester, NY.

Correspondence to Joseph M. Miano, Department of Medicine, University of Rochester School of Medicine, 601 Elmwood Ave, Rochester, NY 14642. E-mail j.m.miano{at}rochester.edu

Key Words: signal transduction • gene regulation • smooth muscle • serum response factor • myocardin

Myriad signal transduction pathways instruct genomes to transcribe genes. Insight into the signaling molecules converging on gene expression was accelerated in the 1980s with the discovery of numerous immediate early genes. The prototypic immediate early gene is c-fos, whose transcriptional regulation has been examined in intricate detail. Studies from the laboratories of Treisman,¹ Roeder,² and Weinberg³ defined an upstream c-fos enhancer that was responsive to several stimuli, including serum. The binding element was named serum response element and its core sequence (CCW₆GG) constitutes what we know as a CArG box. The laboratory of Treisman subsequently cloned the serum response factor (SRF) and 1 of the first signal>transcription factor>DNA binding element paradigms was established.⁴ To date, >60 SRF-dependent genes exist in mammalian genomes and, of these, nearly half are restricted to muscle.⁵ More than 100 hypothetical SRF-dependent genes await wet-laboratory validation (J.M.M., unpublished data, 2004).

Among signaling molecules, calcium stands as 1 with connections to virtually every biological process in nature, including gene transcription. Early studies showed an important role for calcium in the activation of c-fos transcription in neuronal cell types. Although several calcium channels could be linked to this process, it was the L-type voltage-sensitive calcium channel that was shown to be associated with c-fos induction.^6,7 Misra et al showed the c-fos CArG element was calcium responsive through enhanced SRF binding following phosphorylation of Ser103 on SRF.⁸ In vitro kinase assays verified SRF phosphorylation on Ser103 through calmodulin kinase IV, suggesting this calcium-dependent kinase was responsible for enhanced SRF binding to CArG and c-fos transcription.⁸ Later studies revealed that cAMP response element-binding protein (CREB) recruits one of its coactivators, CBP (CREB-binding protein), following calmodulin kinase-dependent phosphorylation of CREB, leading to c-fos activation in hippocampal neurons.⁹ Calcium-induced CBP recruitment to CREB likely lifts the repressive state of chromatin around the c-fos locus, permitting enhanced gene transcription.

SRF also requires 1 or more associated coactivators that either modify chromatin or connect the SRF complex to the RNA polymerase II holoenzyme. One such factor is myocardin (Mycd), a transcriptional coactivator cloned in an in silico screen for cardiac-restricted genes.¹⁰ Mycd lacks intrinsic DNA binding activity but associates with SRF and stimulates CArG-dependent promoter activity up to 3 orders of magnitude, making it perhaps the most potent mammalian coactivator known.¹⁰ In a series of complementary studies, Mycd was shown to be abundantly expressed in vascular smooth muscle cells (SMCs) and activate several CArG-dependent genes associated with the SMC promoterome.^11–14 Forced expression of Mycd in non-SMCs could impart a SMC-like phenotype with the activation of several endogenous SMC-restricted genes, including the smooth muscle isoforms of myosin heavy chain (MYH11), calponin (CNN1), -actin (ACTA2), and SM22 (TAGLN1), all of which are CArG-dependent.^11–14 Conversely, small interfering RNA knockdown of Mycd could inhibit the endogenous expression of SRF-dependent SMC marker genes,^12,13 and genetic ablation studies of Mycd in mice revealed an embryonic lethal phenotype with loss of differentiated vascular SMC.¹⁵ Thus, with a computer and a treasure trove of expressed sequence tag data at their fingertips, Olson and colleagues discovered what many investigators tried for more than a decade to find: a SMC-restricted transcription factor that could partially orchestrate a SMC differentiation program.¹⁰

Physiologists may ponder the excitement these findings have engendered in the molecular/developmental realm. One might ask, "Great, but do the cells over-expressing Mycd actually contract?" Moreover, are the upstream signaling molecules for contraction (eg, calcium influx) influencing the activity of Mycd and its effects on SMC gene expression? Although the former question remains an open one, the latter one is directly addressed in an article by Wamhoff et al, which appears in this issue of Circulation Research.¹⁶ These authors provide compelling data to support a role for calcium-induced Mycd effecting SRF-dependent gene transcription in vascular SMC. They show that cultured rat aortic SMCs display L-type voltage-sensitive calcium channel current, a finding that portends SMC contraction. The authors then go on to show that membrane depolarization elicits increases in the mRNA expression of c-fos, ACTA2, and MYH11, the latter gene representing a gold standard marker for SMC. Membrane depolarization also results in elevated CArG-dependent promoter activity, whereas the promoter activity of ACLP, which is CArG-independent,¹⁷ is unchanged. Interestingly, Mycd mRNA was induced with membrane depolarization. There are 3 conserved CArG elements in the first intron of Mycd, but they are located >6 kbp from the core promoter (J.M.M., unpublished data, 2004). None of the >60 SRF-dependent genes have CArG elements directing gene expression >3 to 4 kbp from the start site of transcription.⁵ Thus, it is unclear at this time whether distal CArG-like elements in Mycd are responsive to SRF and the effects of membrane depolarization.

Importantly, small interfering siMycd attenuated KCl-induced ACTA2 and MYH11 promoter activity, although endogenous gene expression was not measured. Consistent with the inability of Mycd to activate c-fos,¹⁰ siMycd did not alter membrane depolarization-induced c-fos promoter activity.¹⁶ Collectively, these data argue for calcium-mediated Mycd expression and downstream SMC CArG target gene expression.

One pathway of CArG-dependent gene expression in SMC is via the RhoA/Rho-associated kinase (ROK) pathway.^18,19 Wamhoff et al demonstrate nifedipine-sensitive depolarization-induced translocation of RhoA from cytosol to membrane where it is able to mediate downstream signaling. The authors discovered that inhibitors of ROK (Y-27632 and H1152) blocked KCl-induced SMC-CArG promoters but not the c-fos or ACLP promoters. On the other hand, the calmodulin kinase inhibitor KN93 blocked c-fos (but not SMC CArG) promoter activity. Thus, in SMC, membrane depolarization does not induce SMC CArG gene expression via calmodulin kinase-dependent phosphorylation of SRF on Ser103 (as observed with SRF over the c-fos promoter in neurons⁸). Rather, an unknown pathway of RhoA/ROK activation via membrane depolarization leads to selective CArG-dependent gene transcription, presumably through the induced expression of Mycd. These findings highlight the ability of SRF to toggle between disparate target gene sets based on unique signaling pathways emanating from an initial stimulus. An important area for future investigation will be to define the mechanisms of depolarization-induced Mycd gene expression, as well as posttranslational modifications of Mycd that increase its activity. For example, it will be of interest to determine whether Mycd is phosphorylated on membrane depolarization and, if so, how this translates into augmented activity.

As alluded to at the outset of this editorial, chromatin is a critical point of convergence for signaling and gene expression. Wamhoff et al used a quantitative chromatin immunoprecipitation assay to appraise the binding of SRF to SMC CArG elements in their native genomic context. Membrane depolarization evoked a nifedipine-sensitive 3-fold enrichment of SRF binding to CArGs in the ACTA2, MYH11, and c-fos promoters. The ROK inhibitor Y-27632 decreased both basal levels and KCl-induced binding of SRF to the ACTA2 and MYH11 promoters but not the c-fos promoter. How might elevated SRF binding to SMC CArGs be achieved following membrane depolarization? One possibility may be through a kinase that phosphorylates SRF on residues other than Ser103. Another possibility may be through the recruitment and/or modification of adjacent DNA binding proteins that interact with SRF and stabilize its binding to CArG.

The findings by Wamhoff et al raise the physiological question of SMC contractile activity. Rat aortic SMC rarely display contraction because of the dramatic loss in contractile elements on culturing. To circumvent this problem, the authors used an in vitro embryoid body model of SMC differentiation.²⁰ Here, Wamhoff et al show depolarization-induced SMC-like contractile activity concomitant with elevations in SMC CArG promoter activity. It will be of interest to assess the expression of Mycd in embryoid bodies and whether siMycd blocks both contractile activity and expression of SMC markers in this tractable model system.

The discovery of Mycd as a major component to a molecular switch for SMC differentiation represents a long-awaited advance and offers new directions for appraising the functionally differentiated SMC phenotype in development and disease. As molecular/developmental biologists, we often lose sight of the fact that a fully differentiated SMC is one that expresses a unique transcriptome and displays contractile activity. The report by Wamhoff et al provides key insight into a potential mechanism underlying a vascular SMC contractile phenotype. The elegant data in this report inspire several questions that are experimentally testable. (1) What is the mechanism for the observed downregulation of Mycd in vitro,¹¹ and how does membrane depolarization induce Mycd expression? (2) Does elevated Mycd expression elicit SMC contractile activity? (3) Are the two paralogs of Mycd (MRTF-A and MRTF-B²¹) similarly responsive to calcium transients and can they substitute for Mycd in membrane-depolarization induced gene expression? (4) What are the effects of SMC-matrix interactions on membrane depolarization, Mycd expression, and the acquisition of a contractile SMC phenotype? (5) What is the mechanism underlying membrane depolarization-induced SRF binding to CArG? Finally, how exactly does SRF-Mycd direct increases in SMC CArG-dependent gene expression? Does Mycd remodel chromatin as with CBP to increase DNA-binding factor accessibility? Does it act as a bridge between CArG-SRF and the RNA polymerase II holoenzyme. Or is Mycd, at 938 amino acids, yet another scaffold for the recruitment of additional coactivators of gene transcription? These and other questions are surely to be addressed in the years before us.

Acknowledgments

J.M.M. is supported by grants from the NIH (HL-62572 and HL-70077) and is an Established Investigator of the American Heart Association (0340075N). He dedicates his work to A.M.M. and E.A.M.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

References

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