We have previously shown that several components of the RhoA signaling pathway control SMC phenotype by altering SRF-dependent gene expression. Because our genome wide analyses of chromatin structure and transcription factor binding suggested that the actin depolymerizing factor, DSTN, was regulated in a SMC-selective fashion, the goals of the current study were to identify the transcription mechanisms that control DSTN expression in SMC and to test whether it regulates SMC function. Immunohistochemical analyses revealed strong and at least partially SMC-selective expression of DSTN in many mouse tissues, a result consistent with human data from the GTEx consortium. We identified several regulatory regions that control DSTN expression including a SMC-selective enhancer that was activated by the MRTF/SRF, Notch/RBPJ, and SMAD transcription factors. Indeed, enhancer activity and endogenous DSTN expression were up-regulated by RhoA and TGF-β signaling and down-regulated by the Notch inhibitor, DAPT. We also showed that DSTN expression was decreased in vivo by carotid artery injury and in cultured SMC cells by PDGF-BB treatment. siRNA-mediated depletion of DSTN significantly enhanced MRTF-A nuclear localization and SMC differentiation marker gene expression; decreased SMC migration in scratch wound assays; and decreased SMC proliferation as measured by cell number and cyclin E expression. Taken together our data indicate that DSTN is a negative feedback inhibitor of RhoA/SRF-dependent gene expression in SMC that coordinately promotes SMC phenotypic modulation. Interventions that target DSTN expression or activity could serve as potential therapies for atherosclerosis and restenosis.

中文翻译：

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肌动蛋白解聚因子 Destrin 作为平滑肌细胞分化的负反馈抑制剂

我们之前已经表明，RhoA 信号通路的几个组成部分通过改变 SRF 依赖性基因表达来控制 SMC 表型。由于我们对染色质结构和转录因子结合的全基因组分析表明，肌动蛋白解聚因子 DSTN 以 SMC 选择性方式受到调节，因此本研究的目标是确定控制 SMC 中 DSTN 表达的转录机制并测试它是否调节SMC功能。免疫组织化学分析显示，在许多小鼠组织中，DSTN 强烈且至少是部分 SMC 选择性表达，这一结果与来自 GTEx 联盟的人类数据一致。我们确定了几个控制 DSTN 表达的调控区域，包括由 MRTF/SRF、Notch/RBPJ 和 SMAD 转录因子激活的 SMC 选择性增强子。事实上，增强子活性和内源性 DSTN 表达被 RhoA 和 TGF-β 信号传导上调，并被 Notch 抑制剂 DAPT 下调。我们还表明 DSTN 表达在体内因颈动脉损伤和在培养的 SMC 细胞中通过 PDGF-BB 处理而降低。siRNA 介导的 DSTN 消耗显着增强了 MRTF-A 核定位和 SMC 分化标记基因表达；在划伤试验中减少 SMC 迁移；通过细胞数量和细胞周期蛋白 E 表达测量的 SMC 增殖降低。综上所述，我们的数据表明 DSTN 是 SMC 中 RhoA/SRF 依赖性基因表达的负反馈抑制剂，可协同促进 SMC 表型调节。针对 DSTN 表达或活性的干预措施可以作为动脉粥样硬化和再狭窄的潜在疗法。增强子活性和内源性 DSTN 表达被 RhoA 和 TGF-β 信号上调，被 Notch 抑制剂 DAPT 下调。我们还表明 DSTN 表达在体内因颈动脉损伤和在培养的 SMC 细胞中通过 PDGF-BB 处理而降低。siRNA 介导的 DSTN 消耗显着增强了 MRTF-A 核定位和 SMC 分化标记基因表达；在划伤试验中减少 SMC 迁移；通过细胞数量和细胞周期蛋白 E 表达测量的 SMC 增殖降低。综上所述，我们的数据表明 DSTN 是 SMC 中 RhoA/SRF 依赖性基因表达的负反馈抑制剂，可协同促进 SMC 表型调节。针对 DSTN 表达或活性的干预措施可以作为动脉粥样硬化和再狭窄的潜在疗法。增强子活性和内源性 DSTN 表达被 RhoA 和 TGF-β 信号上调，被 Notch 抑制剂 DAPT 下调。我们还表明 DSTN 表达在体内因颈动脉损伤和在培养的 SMC 细胞中通过 PDGF-BB 处理而降低。siRNA 介导的 DSTN 消耗显着增强了 MRTF-A 核定位和 SMC 分化标记基因表达；在划伤试验中减少 SMC 迁移；通过细胞数量和细胞周期蛋白 E 表达测量的 SMC 增殖降低。综上所述，我们的数据表明 DSTN 是 SMC 中 RhoA/SRF 依赖性基因表达的负反馈抑制剂，可协同促进 SMC 表型调节。针对 DSTN 表达或活性的干预措施可以作为动脉粥样硬化和再狭窄的潜在疗法。