Urological Diseases Research Center, Boston Children's Hospital, Boston, Massachusetts, United States of America.
PLoS One. 2013;8(1):e53430. doi: 10.1371/journal.pone.0053430. Epub 2013 Jan 4.
Smooth muscle contraction is a dynamic process driven by acto-myosin interactions that are controlled by multiple regulatory proteins. Our studies have shown that members of the AP-1 transcription factor family control discrete behaviors of smooth muscle cells (SMC) such as growth, migration and fibrosis. However, the role of AP-1 in regulation of smooth muscle contractility is incompletely understood. In this study we show that the AP-1 family member JunB regulates contractility in visceral SMC by altering actin polymerization and myosin light chain phosphorylation. JunB levels are robustly upregulated downstream of transforming growth factor beta-1 (TGFβ1), a known inducer of SMC contractility. RNAi-mediated silencing of JunB in primary human bladder SMC (pBSMC) inhibited cell contractility under both basal and TGFβ1-stimulated conditions, as determined using gel contraction and traction force microscopy assays. JunB knockdown did not alter expression of the contractile proteins α-SMA, calponin or SM22α. However, JunB silencing decreased levels of Rho kinase (ROCK) and myosin light chain (MLC20). Moreover, JunB silencing attenuated phosphorylation of the MLC20 regulatory phosphatase subunit MYPT1 and the actin severing protein cofilin. Consistent with these changes, cells in which JunB was knocked down showed a reduction in the F:G actin ratio in response to TGFβ1. Together these findings demonstrate a novel function for JunB in regulating visceral smooth muscle cell contractility through effects on both myosin and the actin cytoskeleton.
平滑肌收缩是一种由肌动球蛋白相互作用驱动的动态过程，受多种调节蛋白控制。我们的研究表明，AP-1 转录因子家族的成员控制平滑肌细胞（SMC）的离散行为，如生长、迁移和纤维化。然而，AP-1 在调节平滑肌收缩力方面的作用尚未完全了解。在这项研究中，我们表明，AP-1 家族成员 JunB 通过改变肌动蛋白聚合和肌球蛋白轻链磷酸化来调节内脏平滑肌的收缩性。转化生长因子-β1（TGFβ1）是一种已知的平滑肌收缩诱导剂，其下游的 JunB 水平被强烈上调。在基础和 TGFβ1 刺激条件下，通过凝胶收缩和牵引力显微镜测定，使用针对 JunB 的 RNAi 介导的沉默，显著抑制了原代人膀胱平滑肌细胞（pBSMC）的细胞收缩性。JunB 敲低不改变收缩蛋白α-SMA、钙调蛋白或 SM22α 的表达。然而，JunB 沉默降低了 Rho 激酶（ROCK）和肌球蛋白轻链（MLC20）的水平。此外，JunB 沉默减弱了 MLC20 调节磷酸酶亚基 MYPT1 和肌动蛋白断片蛋白 cofilin 的磷酸化。与这些变化一致，敲低 JunB 的细胞在 TGFβ1 刺激下显示肌动球蛋白 F:G 比值降低。这些发现共同证明了 JunB 通过对肌球蛋白和肌动蛋白细胞骨架的影响，在调节内脏平滑肌细胞收缩性方面具有新的功能。
