Mark H. Hoofnagle

Myocardin Is a Key Regulator of CArG-Dependent Transcription of Multiple Smooth Muscle Marker Genes

Abstract— The interactions between serum response factor (SRF) and CArG elements are critical for smooth muscle cell (SMC) marker gene transcription. However, the mechanisms whereby SRF, which is expressed ubiquitously, contributes to SMC-specific transcription are unknown. Myocardin was recently cloned as a coactivator of SRF in the heart, but its role in regulating CArG-dependent expression of SMC differentiation marker genes has not been clearly elucidated. In this study, we examined the expression and the function of myocardin in SMCs. In adult mice, myocardin mRNA was expressed in multiple smooth muscle (SM) tissues including the aorta, bladder, stomach, intestine, and colon, as well as the heart. Myocardin was also expressed in cultured rat aortic SMCs and A404 SMC precursor cells. Of particular interest, expression of myocardin was induced during differentiation of A404 cells, although it was not expressed in parental P19 cells from which A404 cells were derived. Cotransfection studies in SMCs revealed that myocardin induced the activity of multiple SMC marker gene promoters including SM &agr;-actin, SM-myosin heavy chain, and SM22&agr; by 9- to 60-fold in a CArG-dependent manner, whereas myocardin short interfering RNA markedly decreased activity of these promoters. Moreover, adenovirus-mediated overexpression of a dominant-negative form of myocardin significantly suppressed expression of endogenous SMC marker genes, whereas adenovirus-mediated overexpression of wild-type myocardin increased expression. Taken together, results provide compelling evidence that myocardin plays a key role as a transcriptional coactivator of SMC marker genes through CArG-dependent mechanisms.

Control of SRF binding to CArG box chromatin regulates smooth muscle gene expression in vivo

Precise control of SMC transcription plays a major role in vascular development and pathophysiology. Serum response factor (SRF) controls SMC gene transcription via binding to CArG box DNA sequences found within genes that exhibit SMC-restricted expression. However, the mechanisms that regulate SRF association with CArG box DNA within native chromatin of these genes are unknown. Here we report that SMC-restricted binding of SRF to murine SMC gene CArG box chromatin is associated with patterns of posttranslational histone modifications within this chromatin that are specific to the SMC lineage in culture and in vivo, including methylation and acetylation to histone H3 and H4 residues. We found that the promyogenic SRF coactivator myocardin increased SRF association with methylated histones and CArG box chromatin during activation of SMC gene expression. In contrast, the myogenic repressor Kruppel-like factor 4 recruited histone H4 deacetylase activity to SMC genes and blocked SRF association with methylated histones and CArG box chromatin during repression of SMC gene expression. Finally, we observed deacetylation of histone H4 coupled with loss of SRF binding during suppression of SMC differentiation in response to vascular injury. Taken together, these findings provide novel evidence that SMC-selective epigenetic control of SRF binding to chromatin plays a key role in regulation of SMC gene expression in response to pathophysiological stimuli in vivo.

A G/C Element Mediates Repression of the SM22α Promoter Within Phenotypically Modulated Smooth Muscle Cells in Experimental Atherosclerosis

A hallmark of smooth muscle cell (SMC) phenotypic switching in atherosclerotic lesions is suppression of SMC differentiation marker gene expression. Yet little is known regarding the molecular mechanisms that control this process. Here we show that transcription of the SMC differentiation marker gene SM22&agr; is reduced in atherosclerotic lesions and identify a cis regulatory element in the SM22&agr; promoter required for this process. Transgenic mice carrying the SM22&agr; promoter–&bgr;-galactosidase (&bgr;-gal) reporter transgene were crossed to apolipoprotein E (ApoE)−/− mice. Cells of the fibrous cap, intima, and underlying media showed complete loss of &bgr;-gal activity in advanced atherosclerotic lesions. Of major significance, mutation of a G/C-rich cis element in the SM22&agr; promoter prevented the decrease in SM22&agr; promoter–&bgr;-gal reporter transgene expression, including in cells that compose the fibrous cap of the lesion and in medial cells in proximity to the lesion. To begin to assess mechanisms whereby the G/C repressor element mediates suppression of SM22&agr; in atherosclerosis, we tested the hypothesis that effects may be mediated by platelet-derived growth factor (PDGF)-BB–induced increases in the G/C binding transcription factor Sp1. Consistent with this hypothesis, results of studies in cultured SMCs showed that: (1) PDGF-BB increased expression of Sp1; (2) PDGF-BB and Sp1 profoundly suppressed SM22&agr; promoter activity as well as smooth muscle myosin heavy chain promoter activity through mechanisms that were at least partially dependent on the G/C cis element; and (3) a short interfering RNA to Sp1 increased basal expression and attenuated PDGF-BB induced suppression of SM22&agr;. Together, these results support a model whereby a G/C repressor element within the SM22&agr; promoter mediates transcriptional repression of this gene within phenotypically modulated SMCs in experimental atherosclerosis and provide indirect evidence implicating PDGF-BB and Sp1 as possible mediators of these effects.

Myocardin and Prx1 Contribute to Angiotensin II-Induced Expression of Smooth Muscle α-Actin

Abstract— Previous studies demonstrated that angiotensin II (Ang II)–induced hypertrophy of smooth muscle cells (SMCs) was associated with increased transcription of SM &agr;-actin gene. The aim of the present study was to determine whether myocardin, a SMC-selective cofactor of serum response factor (SRF), contributed to Ang II–induced increases in SM &agr;-actin transcription. Results showed that Ang II increased myocardin mRNA expression as well as SM &agr;-actin mRNA expression via the Ang II type 1 receptor in cultured rat aortic SMCs. Cotransfection studies revealed that CArG elements were required for Ang II–induced transcription of SM &agr;-actin gene, and a dominant-negative form of myocardin or a short interfering RNA (siRNA) specific for myocardin decreased Ang II–induced SM &agr;-actin transcription. Prx1, a homeodomain protein whose expression was increased by Ang II, also increased SM &agr;-actin gene transcription in part via CArG elements, and siRNA specific for Prx1 markedly decreased basal and Ang II–induced SM &agr;-actin transcription. Electrophoretic mobility shift assay showed that myocardin and Ang II, respectively, increased formation of a SMC-specific CArG-SRF-myocardin higher order complex. However, Ang II had no effect on binding between myocardin and SRF as determined by a mammalian two-hybrid assay, suggesting that Ang II–induced increases in formation of CArG-SRF-myocardin complex was the result of increased SRF binding to CArG elements and increased myocardin expression. Taken together, these results support a model in which Ang II–induced increases in expression of SM &agr;-actin are mediated through Prx1-dependent increases in SRF binding to CArG elements and subsequent recruitment of myocardin.

Assessment of Contractility of Purified Smooth Muscle Cells Derived from Embryonic Stem Cells

The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin‐resistance gene under the control of either a smooth muscle α‐actin (SMαA) or smooth muscle‐myosin heavy chain (SM‐MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription‐polymerase chain reaction, and flow cytometry and were designated APSCs (SMαA‐puromycin‐selected cells) or MPSCs (SM‐MHC‐puromycin‐selected cells), respectively. Both SMC lines displayed agonist‐induced Ca2+ transients, expressed functional Ca2+ channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as endothelin‐1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC‐derived cell lines, even after prolonged differentiation, highlighting the critical requirement for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.

PIAS1 Activates the Expression of Smooth Muscle Cell Differentiation Marker Genes by Interacting with Serum Response Factor and Class I Basic Helix-Loop-Helix Proteins

ABSTRACT Although a critical component of vascular disease is modulation of the differentiated state of vascular smooth muscle cells (SMC), the mechanisms governing SMC differentiation are relatively poorly understood. We have previously shown that E-boxes and the ubiquitously expressed class I basic helix-loop-helix (bHLH) proteins, including E2-2 and E12, are important in regulation of the SMC differentiation marker gene, the SM α-actin gene. The aim of the present study was to identify proteins that bind to class I bHLH proteins in SMC and modulate transcriptional regulation of SMC differentiation marker genes. Herein we report that members of the protein inhibitor of activated STAT (PIAS) family interact with class I bHLH factors as well as serum response factor (SRF). PIAS1 interacted with E2-2 and E12 based on yeast two-hybrid screens, mammalian two-hybrid assays, and/or coimmunoprecipitation assays. Overexpression of PIAS1 significantly activated the SM α-actin promoter and mRNA expression, as well as SM myosin heavy chain and SM22α, whereas a small interfering RNA for PIAS1 decreased activity of these promoters, as well as endogenous mRNA expression, and SRF binding to SM α-actin promoter within intact chromatin in cultured SMC. Of significance, PIAS1 bound to SRF and activated SM α-actin promoter expression in wild-type but not SRF−/− embryonic stem cells. These results provide novel evidence that PIAS1 modulates transcriptional activation of SMC marker genes through cooperative interactions with both SRF and class I bHLH proteins.

Lost in transdifferentiation

What are the true origins of the smooth muscle cells (SMCs) present in the intimal lesions of transplant arteriosclerosis? A new study in the JCI shows that Sca-1(+) cells purified from the mouse aortic root can migrate through an irradiated vein graft to the neointima of the vessel and transdifferentiate to express the early SMC differentiation marker gene SM22. Do Sca-1(+) cells transdifferentiate into SMC-like cells, or is activation of SMC marker genes a consequence of fusion of these cells with preexisting SMCs, a possibility raised by results of studies of adult stem cells in animal models of liver regeneration ? Or could this be bona fide transdifferentiation that recapitulates the pathologic processes in humans?

Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes

Myocardin is a serum response factor (SRF) coactivator exclusively expressed in cardiomyocytes and smooth muscle cells (SMCs). However, there is highly controversial evidence as to whether myocardin is essential for normal differentiation of these cell types, and there are no data showing whether cardiac or SMC subtypes exhibit differential myocardin requirements during development. Results of the present studies showed the virtual absence of myocardin(-/-) visceral SMCs or ventricular myocytes in chimeric myocardin knockout (KO) mice generated by injection of myocardin(-/-) embryonic stem cells (ESCs) into wild-type (WT; i.e., myocardin(+/+) ESC) blastocysts. In contrast, myocardin(-/-) ESCs readily formed vascular SMC, albeit at a reduced frequency compared with WT ESCs. In addition, myocardin(-/-) ESCs competed equally with WT ESCs in forming atrial myocytes. The ultrastructural features of myocardin(-/-) vascular SMCs and cardiomyocytes were unchanged from their WT counterparts as determined using a unique X-ray microprobe transmission electron microscopic method developed by our laboratory. Myocardin(-/-) ESC-derived SMCs also showed normal contractile properties in an in vitro embryoid body SMC differentiation model, other than impaired thromboxane A2 responsiveness. Together, these results provide novel evidence that myocardin is essential for development of visceral SMCs and ventricular myocytes but is dispensable for development of atrial myocytes and vascular SMCs in the setting of chimeric KO mice. In addition, results suggest that as yet undefined defects in development and/or maturation of ventricular cardiomyocytes may have contributed to early embryonic lethality observed in conventional myocardin KO mice and that observed deficiencies in development of vascular SMC may have been secondary to these defects.

Derivation of Contractile Smooth Muscle Cells from Embryonic Stem Cells

Smooth muscle cells (SMCs) play a key role in vascular physiology and pathology. An appreciation of normal SMCs developmental mechanisms will likely lead to a better understanding of disease processes and potentially to novel treatment strategies. We present a method for generating relatively pure populations of SMCs from embryonic stem cells (ESC) which display appropriate excitation and contractile responses to vasoactive agonists. We also present protocols for assessment of SMCs purity and identity by immunofluorescence, quantitative RT-PCR, and FACS. This ESC-based system has tremendous potential for studying developmental regulation of SMC lineage, as well as for possible SMC tissue engineering.

Assessment of contractility of purified smooth muscle cells derived from embryonic stem cells

The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin-resistance gene under the control of either a smooth muscle alpha-actin (SMalphaAlpha) or smooth muscle-myosin heavy chain (SM-MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription-polymerase chain reaction, and flow cytometry and were designated APSCs (SMalphaAlpha-puromycin-selected cells) or MPSCs (SM-MHC-puromycin-selected cells), respectively. Both SMC lines displayed agonist-induced Ca(2+) transients, expressed functional Ca(2+) channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as endothelin-1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC-derived cell lines, even after prolonged differentiation, highlighting the critical requirement for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.