Wei L, Wang L, Carson J A, Agan J E, Imanaka-Yoshida K, Schwartz R J
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA.
FASEB J. 2001 Mar;15(3):785-96. doi: 10.1096/fj.00-026com.
Activation of RhoA GTPase causes actin filament bundling into stress fibers, integrin clustering, and focal adhesion formation through its action on actin cytoskeleton organization. RhoA also regulates transcriptional activity of serum response factor (SRF). Recent studies in NIH 3T3 fibroblasts have shown that SRF activation by RhoA does not require an organized cytoskeleton and may be regulated by G-actin level. In cardiac myocytes, the organization of actin fibers into myofibrils is one of the primary characteristics of cardiac differentiation and hypertrophy. The primary purpose of this study was to examine if RhoA regulates SRF-dependent gene expression in neonatal cardiomyocytes in a manner different from that observed in fibroblasts. Our results show that RhoA-dependent skeletal alpha-actin promoter activation requires beta1 integrin and a functional cytoskeleton in cardiomyocytes but not in NIH 3T3 fibroblasts. Activation of the alpha-actin promoter by RhoA is greatly potentiated (up to 15-fold) by co-expression of the integrin beta1A or beta1D isoform but is significantly reduced by 70% with a co-expressed dominant negative mutant of beta1 integrin. Furthermore, clustering of beta1 integrin with anti-beta1 integrin antibodies potentiates synergistic RhoA and beta1 integrin activation of the alpha-actin promoter. Cytochalasin D and latrunculin B, inhibitors of actin polymerization, significantly reduced RhoA-induced activation of the alpha-actin promoter. Jasplakinolide, an actin polymerizing agent, mimics the synergistic effect of RhoA and beta1 integrin on the actin promoter. These observations support the concept that RhoA regulates SRF-dependent cardiac gene expression through cross-talk with beta1 integrin signal pathway via an organized actin cytoskeleton.
RhoA GTP酶的激活通过其对肌动蛋白细胞骨架组织的作用，导致肌动蛋白丝束集形成应力纤维、整合素聚集和粘着斑形成。RhoA还调节血清反应因子（SRF）的转录活性。最近在NIH 3T3成纤维细胞中的研究表明，RhoA对SRF的激活不需要有组织的细胞骨架，且可能受G-肌动蛋白水平的调节。在心肌细胞中，肌动蛋白纤维组织成肌原纤维是心脏分化和肥大的主要特征之一。本研究的主要目的是检验RhoA是否以不同于在成纤维细胞中观察到的方式调节新生心肌细胞中SRF依赖的基因表达。我们的结果表明，RhoA依赖的骨骼肌α-肌动蛋白启动子激活在心肌细胞中需要β1整合素和功能性细胞骨架，但在NIH 3T3成纤维细胞中则不需要。整合素β1A或β1D亚型的共表达可使RhoA对α-肌动蛋白启动子的激活大大增强（高达15倍），但与共表达的β1整合素显性负性突变体一起时则显著降低70%。此外，用抗β1整合素抗体使β1整合素聚集可增强RhoA和β1整合素对α-肌动蛋白启动子的协同激活。细胞松弛素D和Latrunculin B，肌动蛋白聚合抑制剂，显著降低RhoA诱导的α-肌动蛋白启动子激活。茉莉酸内酯，一种肌动蛋白聚合剂，模拟了RhoA和β1整合素对肌动蛋白启动子的协同作用。这些观察结果支持了这样一种概念，即RhoA通过有组织的肌动蛋白细胞骨架与β1整合素信号通路的相互作用来调节SRF依赖的心脏基因表达。
