Sandbo Nathan, Qin Yimin, Taurin Sebastien, Hogarth D Kyle, Kreutz Barry, Dulin Nickolai O
Section of Pulmonary and Critical Care Medicine, The University of Chicago Department of Medicine, 5841 S. Maryland Ave., MC 6076, Chicago, IL 60637, USA.
Mol Pharmacol. 2005 Mar;67(3):789-97. doi: 10.1124/mol.104.006874. Epub 2004 Nov 18.
Serum response factor (SRF) is activated by contractile and hypertrophic agonists, such as endothelin-1 (ET1) to stimulate expression of cytoskeletal proteins in vascular smooth muscle cells (VSMCs). While studying the regulation of smooth muscle alpha-actin (SMA) expression at the level of protein stability, we discovered that inhibition of proteasome-dependent protein degradation by N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) or lactacystin (LC) did not enhance the levels of SMA, but, unexpectedly, attenuated SMA expression in response to ET1, without affecting the viability of VSMCs. Down-regulation of SMA protein by MG132 or LC occurred at the level of SMA transcription and via the inhibition of SRF activity. By contrast, MG132 and LC potentiated the activity of activator protein-1 transcription factor. Regulation of SRF by MG132 was not related to inhibition of nuclear factor-kappaB, an established target of proteasome inhibitors, and was not mediated by protein kinase A, a powerful regulator of SRF activity. Signaling studies indicate that inhibition of ET1-induced SRF activity by MG132 occurs at the level downstream of heterotrimeric G proteins Gq/11 and G13, of small GTPase RhoA, and of actin dynamics but at the level of SRF-DNA binding. MG132 treatment did not result in ubiquitination or accumulation of SRF. By contrast, the levels of c-Jun were rapidly increased upon incubation of cells with MG132, and ectopic overexpression of c-Jun mimicked the effect of MG132 on SRF activity. Together, these data suggest that inhibition of proteasome results in down-regulation of SMA expression via up-regulation of c-Jun and repression of SRF activity at the level of DNA binding.
血清反应因子（SRF）可被收缩和肥大激动剂激活，如内皮素-1（ET1），以刺激血管平滑肌细胞（VSMC）中细胞骨架蛋白的表达。在研究平滑肌α-肌动蛋白（SMA）在蛋白质稳定性水平上的表达调控时，我们发现用N-苄氧羰基（Z）-亮氨酰-亮氨酰-亮氨酸（MG132）或乳胞素（LC）抑制蛋白酶体依赖性蛋白质降解并不能提高SMA的水平，但出乎意料的是，它会减弱VSMC对ET1的反应中SMA的表达，而不影响VSMC的活力。MG132或LC对SMA蛋白的下调发生在SMA转录水平，并通过抑制SRF活性实现。相比之下，MG132和LC增强了激活蛋白-1转录因子的活性。MG132对SRF的调节与抑制核因子-κB无关，核因子-κB是蛋白酶体抑制剂已确定的靶点，也不是由SRF活性的强大调节剂蛋白激酶A介导的。信号研究表明，MG132对ET1诱导的SRF活性的抑制发生在异三聚体G蛋白Gq/11和G13、小GTP酶RhoA以及肌动蛋白动力学的下游水平，但在SRF与DNA结合的水平。MG132处理并未导致SRF的泛素化或积累。相比之下，用MG132孵育细胞后，c-Jun的水平迅速升高，c-Jun的异位过表达模拟了MG132对SRF活性的影响。总之，这些数据表明，蛋白酶体的抑制通过上调c-Jun和在DNA结合水平抑制SRF活性导致SMA表达下调。
