Department of Pharmacology and Physiology, Faculty of Medicine, Université de Montréal, Montreal, QC H3C 3J7, Canada.
Department of Pharmacology and Physiology, Faculty of Medicine, Université de Montréal, Montreal, QC H3C 3J7, Canada.
Cell Signal. 2018 Jun;46:64-75. doi: 10.1016/j.cellsig.2018.02.012. Epub 2018 Feb 27.
Vascular smooth muscle cells (VSMC) can exhibit a contractile or a synthetic phenotype depending on the extracellular stimuli present and the composition of the extracellular matrix. Uncontrolled activation of the synthetic VSMC phenotype is however associated with the development of cardiovascular diseases. Here, we aimed to elucidate the role of the ARF GTPases in the regulation of VSMC dedifferentiation. First, we observed that the inhibition of the activation of ARF proteins with SecinH3, a blocker of the cytohesin ARF GEF family, reduced the ability of the cells to migrate and proliferate. In addition, this inhibitor also blocked expression of sm22α and αSMA, two contractile markers, at the transcription level impairing cell contractility. Specific knockdown of ARF1 and ARF6 showed that both isoforms were required for migration and proliferation, but ARF1 only regulated contractility through sm22α and αSMA expression. Expression of these VSMC markers was correlated with the degree of actin polymerization. VSMC treatment with SecinH3 as well as ARF1 depletion was both able to block the formation of stress fibres and focal adhesions, demonstrating the role of this GTPase in actin filament formation. Consequently, we observed that both treatments increased the ratio of G-actin to F-actin in these cells. The elevated amounts of cytoplasmic G-actin, acting as a signaling intermediate, blocked the recruitment of the Mkl1 (MRTF-A) transcription factor in the nucleus, demonstrating its involvement in the regulation of contractile protein expression. Altogether, these findings show for the first time that ARF GTPases are actively involved in VSMC phenotypic switching through the regulation of actin function in migration and proliferation, and the control of actin dependent gene regulation.
血管平滑肌细胞（VSMC）可以根据存在的细胞外刺激和细胞外基质的组成表现出收缩或合成表型。然而，合成 VSMC 表型的不受控制的激活与心血管疾病的发展有关。在这里，我们旨在阐明 ARF GTPases 在调节 VSMC 去分化中的作用。首先，我们观察到，用 SecinH3（细胞松弛素 ARF GEF 家族的阻断剂）抑制 ARF 蛋白的激活，降低了细胞迁移和增殖的能力。此外，这种抑制剂还在转录水平上阻断了 sm22α 和 αSMA 的表达，这两种收缩标志物，从而损害了细胞的收缩性。ARF1 和 ARF6 的特异性敲低表明，这两种同工型都需要迁移和增殖，但 ARF1 仅通过 sm22α 和 αSMA 的表达来调节收缩性。这些 VSMC 标志物的表达与肌动蛋白聚合的程度相关。VSMC 用 SecinH3 处理以及 ARF1 耗竭都能够阻止应力纤维和焦点黏附的形成，证明了这种 GTPase 在肌动蛋白丝形成中的作用。因此，我们观察到这两种处理都增加了这些细胞中 G 肌动蛋白与 F 肌动蛋白的比例。细胞质中大量的 G 肌动蛋白作为信号中间物，阻止了 Mkl1（MRTF-A）转录因子在核内的募集，表明其参与了收缩蛋白表达的调节。总之，这些发现首次表明，ARF GTPases 通过调节迁移和增殖中的肌动蛋白功能以及控制肌动蛋白依赖的基因调控，积极参与 VSMC 表型转换。
