Vascular Smooth Muscle Cells Spontaneously Adopt a Skeletal Muscle Phenotype: A Unique Myf5^–/MyoD⁺ Myogenic Program

Abstract

Smooth and skeletal muscle tissues are composed of distinct cell types that express related but distinct isoforms of the structural genes used for contraction. These two muscle cell types are also believed to have distinct embryological origins. Nevertheless, the phenomenon of a phenotypic switch from smooth to skeletal muscle has been demonstrated in several in vivo studies. This switch has been minimally analyzed at the cellular level, and the mechanism driving it is unknown. We used immunofluorescence and RT-PCR to demonstrate the expression of the skeletal muscle-specific regulatory genes MyoD and myogenin, and of several skeletal muscle-specific structural genes in cultures of the established rat smooth muscle cell lines PAC1, A10, and A7r5. The skeletal muscle regulatory gene Myf5 was not detected in these three cell lines. We further isolated clonal sublines from PAC1 cultures that homogeneously express smooth muscle characteristics at low density and undergo a coordinated increase in skeletal muscle-specific gene expression at high density. In some of these PAC1 sublines, this process culminates in the high-frequency formation of myotubes. As in the PAC1 parental line, Myf5 was not expressed in the PAC1 sublines. We show that the PAC1 sublines that undergo a more robust transition into the skeletal muscle phenotype also express significantly higher levels of the insulin-like growth factor (IGF1 and IGF2) genes and of FGF receptor 4 (FGFR4) gene. Our results suggest that MyoD expression in itself is not a sufficient condition to promote a coordinated program of skeletal myogenesis in the smooth muscle cells. Insulin administered at a high concentration to PAC1 cell populations with a poor capacity to undergo skeletal muscle differentiation enhances the number of cells displaying the skeletal muscle differentiated phenotype. The findings raise the possibility that the IGF signaling system is involved in the phenotypic switch from smooth to skeletal muscle. The gene expression program described here can now be used to investigate the mechanisms that may underlie the propensity of certain smooth muscle cells to adopt a skeletal muscle identity.(J Histochem Cytochem 48:1173-1193, 2000)

Myocardin is a bifunctional switch for smooth versus skeletal muscle differentiation

“…We hypothesize that a critical prerequisite for SMC-skeletal muscle transdifferentiation is the silencing of Myocd expression. In support of this premise, cultured SMCs with a propensity to transdifferentiate into skeletal muscle (14) display low-level expression of Myocd (20). It will be interesting to investigate whether the types of repressive mechanisms observed here also are operative in settings of SMC phenotypic modulation, as occurs during pathological vascular remodeling in vivo.…”

“…Indeed, overexpression of Myocd in PAC1 cells, which display features of both smooth and skeletal muscle (14), stimulated Myh11 and attenuated Myog expression (Fig. 3D), whereas siRNA knockdown of Myocd augmented expression of Myog mRNA (Fig.…”

SPARC-null mice exhibit increased adiposity without significant differences in overall body weight

“…Two micrograms of RNA were reverse-transcribed as described (15) in a 100-l reaction volume containing 400 units of Moloney murine leukemia virus reverse transcriptase and buffer (Promega)͞ 1.9 g of oligo dT15 (Invitrogen)͞1 mM each dNTP (Promega). The reactions were carried out for 1 h at 42°C and stored at Ϫ20°C.…”

“…PCR was carried out as described (15). Two microliters of the reverse-transcription reactions was added to the 20-l PCRs containing 1 unit of AmpliTAQ Gold (Applied Biosystems), 400 nM each primer, 200 M each dNTP, and 1.5 mM MgCl 2 in a buffer supplied with AmpliTAQ Gold.…”

Enhanced growth of tumors in SPARC null mice is associated with changes in the ECM

“…RNA was isolated with Tri Reagent (Molecular Research Center Inc., Cincinnati, Ohio, USA) according to the manufacturer's instructions. PCR was performed as described (18) with the following primer sequences: SPARC sense (5′-GTCCCACACTGAGCTGGC-3′) and anti-sense (5′-AAGCACAGAGTCTGGGTGAGTG-3′), collagen 1A1 sense (5′-GAACTTGGGGCAAGACAGT-CA-3′) and anti-sense (5′-GTCACGTTCAGTTGGTCAA-AGG-3′), LNα1 sense (5′-GATGCCATTGGCCTAGAGATTG-3′) and anti-sense (5′-GGATGGGAATGGGAGCTGA-3′), TGF-β1 sense (5′-ACCATCCATGACATGAACCG-3′) and antisense (5′-GGCTTGCGACCCACGTA-3′), VEGF sense (5′-AGGCTGCTGTAACGATGAAGC-3′) and antisense (5′-CCGCCTTGGCT-TGTCAC-3′), VEGFR1 sense (5′-GTGT-GCTTAGGTCGTGCACAC-3′) and anti-sense (5′-CTG-GATGAGAAACCTC-AGGCT-3′), VEGFR2 sense (5′-CG AGTCTGTCTACCTTGGAGGC-3′) and anti-sense (5′-CAGCCTGAGCCTTTACCGC-3′), neuropilin-1 (Npn-1) sense (5′-ACCACTGAT-AACTCGATTTGTCCG-3′) and antisense (5′-CGATCTTG-AACTTCCTCATGAACAC-3′), SC1 sense (5′-TGGTTCTTGCACGAACTTCC-3′) and anti-sense (5′-GAGAAGTTCAATGGGATGGTCTC-3′), and rpS6 sense (5′-AAGCTCCGCACCTTCTATGAGA-3′) and anti-sense (5′-TGACTGGACTCAGACTTAGAAGTAGAAGC-3′).…”