Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, USA.
PLoS One. 2010 Sep 22;5(9):e12823. doi: 10.1371/journal.pone.0012823.
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.
帕拉丁（Palladin）是一种与肌动蛋白相关的蛋白，在调节细胞黏附和细胞迁移方面发挥着重要作用。帕拉丁对于发育至关重要，因为在小鼠中敲低该基因会导致胚胎致死，但它在血管发育中的作用尚不清楚。我们已经表明，帕拉丁对于平滑肌细胞（SMC）标记基因的表达和对激动剂刺激的力发展是必需的，在缺乏帕拉丁的 SMC 中。本研究的目的是确定帕拉丁调节 SMC 标记基因表达的分子机制。结果表明，在 A404 细胞系中，视黄酸（RA）诱导分化后，帕拉丁的表达迅速诱导。用 siRNA 抑制帕拉丁的表达抑制了 RA 诱导的 SMC 分化基因 SM α-肌动蛋白（SMA）和 SM22 的表达，而过表达帕拉丁诱导了 SMC 基因的表达。染色质免疫沉淀试验提供了证据表明帕拉丁结合于 SMC 基因，而共免疫沉淀试验也表明帕拉丁与心肌相关转录因子（MRTFs）结合。内源性帕拉丁在核内成像，用莱普霉素处理后增加，并且帕拉丁的羧基末端与 MRTFs 在核内共定位。结果支持这样一种模型，即帕拉丁通过调节 CArG-SRF-MRTF 依赖的 SMC 标记基因的转录来促进 SMC 分化，并且如先前发表的那样，也通过肌动蛋白动力学。最后，在 E11.5 期帕拉丁缺失的小鼠胚胎中，血管壁中 SMA 和 SM22 mRNA 和蛋白的表达减少。总之，我们的发现表明，帕拉丁在发育中的血管中 SMC 的分化中起着关键作用。
