Patel Keyur, Harding Pamela, Haney Lisa B, Glass William F
Department of Pathology and Anatomy, Eastern Virginia Medical School, Norfolk 23501, USA.
J Cell Physiol. 2003 Jun;195(3):435-45. doi: 10.1002/jcp.10267.
Mesangial cells in diverse glomerular diseases become myofibroblast-like, characterized by activation of smooth muscle alpha-actin (alpha-SMA) expression. In cultured mesangial cells, serum-deprivation markedly increases alpha-SMA expression, cell size, and stress fiber formation. Since stress fibers are assembled from actin monomers, we investigated the hypothesis that alterations in stress fiber formation regulate alpha-SMA expression and hypertrophy. Human mesangial cells were treated with agents that disrupt or stabilize actin stress fibers. Depolymerization of actin stress fibers in serum-deprived cells with actin-depolymerizing agents, cytochalasin B (CytB) and latrunculin B (LatB), or with inhibitors of Rho-kinase, Y-27632 and HA-1077 decreased alpha-SMA mRNA as judged by Northern blot analysis. Western blot analysis showed that CytB also reduced alpha-SMA protein levels. In serum-fed cells, agents that stabilized actin stress fibers, jasplakinolide (Jas) and phalloidin, increased alpha-SMA mRNA and protein. Treatment of human or rat mesangial cells with CytB, LatB, or Y-27632 decreased alpha-SMA promoter activity. In contrast, Jas increased promoter activity 5.6-fold in rat mesangial cells. The presence of an RNA polymerase inhibitor blocked degradation of alpha-SMA mRNA in cells treated with CytB suggesting that destabilization of this message is dependent on a newly transcribed or rapidly degraded factor. Inhibition of actin polymerization by CytB, LatB, Y-27623, and HA-1077 inhibited incorporation of (3)[H]-leucine into newly synthesized protein. Additionally, CytB and LatB decreased cell volume as determined by flow cytometry. Collectively, these results indicate that the state of polymerization of the actin cytoskeleton regulates alpha-SMA expression, hypertrophy, and myofibroblast differentiation in mesangial cells.
在各种肾小球疾病中，系膜细胞会变成肌成纤维细胞样，其特征是平滑肌α-肌动蛋白（α-SMA）表达激活。在培养的系膜细胞中，血清剥夺会显著增加α-SMA表达、细胞大小和应力纤维形成。由于应力纤维由肌动蛋白单体组装而成，我们研究了以下假说：应力纤维形成的改变调节α-SMA表达和肥大。用人系膜细胞用破坏或稳定肌动蛋白应力纤维的试剂处理。用肌动蛋白解聚剂细胞松弛素B（CytB）和拉春库林B（LatB）或Rho激酶抑制剂Y-27632和HA-1077使血清剥夺细胞中的肌动蛋白应力纤维解聚，通过Northern印迹分析判断，α-SMA mRNA减少。蛋白质印迹分析表明，CytB也降低了α-SMA蛋白水平。在血清喂养的细胞中，稳定肌动蛋白应力纤维的试剂茉莉酮酸内酯（Jas）和鬼笔环肽增加了α-SMA mRNA和蛋白。用CytB、LatB或Y-27632处理人或大鼠系膜细胞会降低α-SMA启动子活性。相反，Jas使大鼠系膜细胞中的启动子活性增加5.6倍。RNA聚合酶抑制剂的存在阻断了用CytB处理的细胞中α-SMA mRNA的降解，表明该信息的不稳定依赖于新转录或快速降解的因子。CytB、LatB、Y-27623和HA-1077对肌动蛋白聚合的抑制抑制了（3）[H]-亮氨酸掺入新合成的蛋白质中。此外，通过流式细胞术测定，CytB和LatB降低了细胞体积。总体而言，这些结果表明肌动蛋白细胞骨架的聚合状态调节系膜细胞中α-SMA表达、肥大和肌成纤维细胞分化。
