Whiteside Catharine, Munk Snezana, Ispanovic Eric, Wang Hong, Goldberg Howard, Kapus Andras, Xia Ling
University Health Network, Toronto, Ont., Canada.
Nephron Exp Nephrol. 2008;109(2):e46-56. doi: 10.1159/000142099. Epub 2008 Jul 4.
BACKGROUND/AIMS: We postulated that alpha-smooth muscle actin expressed in primary cultured mesangial cells is down-regulated in 3-dimensional (D) culture and up-regulated by high glucose and growth factors.
Primary rat mesangial cells were growth-arrested in 5.6 mM (NG) or 30 mM (HG) glucose for 14 days in 3-D Matrigel. Alpha-SM actin expression was analyzed by immunoblotting, real-time PCR and by alpha-SM actin promoter activity in response to 24 h stimulation with endothelin-1 (ET-1), angiotensin II (Ang II) or HG.
Alpha-SM actin mRNA, protein and promoter activity were reduced to significantly lower levels in 3-D cells compared to cells in 2-D. Up-regulation of alpha-SM expression was stimulated by ET-1, Ang II and HG. Specific inhibitors of protein kinase C (PKC)-alpha, -beta or -zeta prevented alpha-SM upregulation in HG. In NG, PKC and ERK1/2 activation were required for alpha-SM actin accumulation in 3-D in response to ET-1 or Ang II. In HG, enhanced expression of alpha-SM actin in response to ET-1 or Ang II was unchanged during PKC or ERK1/2 inhibition.
Mesangial cells in 3-D express low levels of alpha-SM actin representing a more differentiated state. Regulation of alpha-SM actin expression is dependent on specific PKC isozyme and ERK1/2 signaling.
背景/目的：我们推测，原代培养的系膜细胞中表达的α-平滑肌肌动蛋白在三维（3D）培养中表达下调，而在高糖和生长因子作用下表达上调。
原代大鼠系膜细胞在3D基质胶中于5.6 mM（正常血糖，NG）或30 mM（高血糖，HG）葡萄糖中生长停滞14天。通过免疫印迹、实时PCR以及在给予内皮素-1（ET-1）、血管紧张素II（Ang II）或高糖刺激24小时后检测α-平滑肌肌动蛋白启动子活性来分析α-平滑肌肌动蛋白的表达。
与二维培养的细胞相比，三维培养的细胞中α-平滑肌肌动蛋白的mRNA、蛋白及启动子活性均显著降低。ET-1、Ang II和高糖可刺激α-平滑肌肌动蛋白表达上调。蛋白激酶C（PKC）-α、-β或-ζ的特异性抑制剂可阻止高糖条件下α-平滑肌肌动蛋白的上调。在正常血糖条件下，PKC和ERK1/2的激活是三维培养中ET-1或Ang II诱导α-平滑肌肌动蛋白积累所必需的。在高糖条件下，PKC或ERK1/2抑制期间，ET-1或Ang II诱导的α-平滑肌肌动蛋白表达增强未发生改变。
三维培养的系膜细胞表达低水平的α-平滑肌肌动蛋白，代表一种更分化的状态。α-平滑肌肌动蛋白表达的调节依赖于特定的PKC同工酶和ERK信号通路。
