Abstract
Palladin, an actin associated protein, plays a significant role in regulating cell adhesion and cell motility. Palladin is important for development, as knockdown in mice is embryonic lethal, yet its role in the development of the vasculature is unknown. We have shown that palladin is essential for the expression of smooth muscle cells (SMC) marker genes and force development in response to agonist stimulation in palladin deficient SMCs. The goal of the study was to determine the molecular mechanisms underlying palladin's ability to regulate the expression of SMC marker genes. Results showed that palladin expression was rapidly induced in an A404 cell line upon retinoic acid (RA) induced differentiation. Suppression of palladin expression with siRNAs inhibited the expression of RA induced SMC differentiation genes, SM α-actin (SMA) and SM22, whereas over-expression of palladin induced SMC gene expression. Chromatin immunoprecipitation assays provided evidence that palladin bound to SMC genes, whereas co-immunoprecipitation assays also showed binding of palladin to myocardin related transcription factors (MRTFs). Endogenous palladin was imaged in the nucleus, increased with leptomycin treatment and the carboxyl-termini of palladin co-localized with MRTFs in the nucleus. Results support a model wherein palladin contributes to SMC differentiation through regulation of CArG-SRF-MRTF dependent transcription of SMC marker genes and as previously published, also through actin dynamics. Finally, in E11.5 palladin null mouse embryos, the expression of SMA and SM22 mRNA and protein is decreased in the vessel wall. Taken together, our findings suggest that palladin plays a key role in the differentiation of SMCs in the developing vasculature.
Highlights
Vascular smooth muscle cells (SMCs) are not terminally differentiated
Palladin was induced in SMC differentiation model The importance of palladin in SMC differentiation was examined in a clonal line of SMC progenitor cells designated A404 cells [36]
Results of the present studies showed a 70-fold increase in mRNA expression of the 92 kDa isoform of palladin 48 hrs after retinoic acid (RA) treatment (Fig. 1, p,0.05). These results are consistent with our previous findings showing induction of palladin expression within embryonic stem cells (ESC)-embryoid body (EB) treated with RA [26]
Summary
Vascular smooth muscle cells (SMCs) are not terminally differentiated. They have the ability to undergo phenotypic switching in association with pathological conditions such as vascular injury, post angioplasty stenosis, and atherosclerosis. The differentiation of SMC is characterized by the upregulation of SMC marker genes, which are associated with the contractile phenotype, such as SM alpha actin (SMA), SM myosin heavy chain (MHC) and SM22. The expression of SMC marker genes has been shown to be regulated by CArG-SRF complexes, by myocardin and by Myocardin Related Transcription Factors MRTF-A and MRTF-B that induce transcription of SMC marker genes in a CArG dependent manner [1,2,3,4,5]. The actin cytoskeleton is both an upstream regulator of MRTF activity, with monomeric (G) actin directly acting as a signal transducer, and a downstream effector of SRF resulting in activation of cluster genes encoding components of the actin cytoskeleton [8,9,10]
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