Nakagawa Taeko, Yamaguchi Masayoshi
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
J Cell Biochem. 2007 Apr 1;100(5):1178-90. doi: 10.1002/jcb.21105.
The regulatory role of regucalcin on cell responses for tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1) was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2-transfected cells (transfectant) were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture, cells were further cultured for 24-72 h in medium without BS containing either vehicle, TNF-alpha (0.1 or 1.0 ng/ml of medium), or TGF-beta1 (1.0 or 5.0 ng/ml). Culture with TNF-alpha or TGF-beta1 caused a significant decrease in the number of wild-type cells. This decrease was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with TNF-alpha (1.0 ng/ml) or TGF-beta1 (5.0 ng/ml). This DNA fragmentation was significantly suppressed in transfectants. TNF-alpha- or TGF-beta1-induced cell death was significantly prevented in culture with caspase-3 inhibitor (10(-8) M). Nitric oxide (NO) synthase activity in wild-type cells was significantly increased by addition of calcium chloride (10 microM) and calmodulin (5 microg/ml) into the enzyme reaction mixture. This increase was significantly suppressed in transfectants. Culture with TNF-alpha caused a significant increase in NO synthase activity in wild-type cells. The effect of TNF-alpha was not seen in transfectants. Culture with TGF-beta1 did not cause a significant increase in NO synthase activity in wild-type cells and transfectants. Culture with TNF-alpha or TGF-beta1 caused a remarkable increase in alpha-smooth muscle actin in wild-type cells. This increase was significantly prevented in transfectants. The expression of Smad 2 or NF-kappaB mRNAs was significantly increased in transfectants as compared with that of wild-type cells. Smad 3 or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expression was not significantly changed in transfectants. NF-kappaB mRNA expression in wild-type cells was significantly increased with culture of TNF-alpha. Smad 2 mRNA expression was significantly enhanced in wild-type cells cultured with TGF-beta1. These effects of TNF-alpha or TGF-beta1 were not significantly enhanced in transfectants. This study demonstrates that overexpression of regucalcin has suppressive effects on cell responses which are mediated through intracellular signaling pathways of TNF-alpha or TGF-beta1 in kidney NRK52E cells.
使用过表达调节钙素的克隆正常大鼠肾近端小管上皮NRK52E细胞，研究调节钙素对肿瘤坏死因子-α（TNF-α）或转化生长因子-β1（TGF-β1）细胞反应的调节作用。将NRK52E细胞（野生型）和稳定转染调节钙素（RC）/pCXN2的细胞（转染子）在含有5%牛血清（BS）的培养基中培养72小时，以获得亚汇合单层细胞。培养后，将细胞在不含BS的培养基中进一步培养24 - 72小时，培养基中含有溶剂、TNF-α（0.1或1.0 ng/ml培养基）或TGF-β1（1.0或5.0 ng/ml）。用TNF-α或TGF-β1培养导致野生型细胞数量显著减少。在过表达调节钙素的转染子中，这种减少被显著阻止。琼脂糖凝胶电泳显示，用TNF-α（1.0 ng/ml）或TGF-β1（5.0 ng/ml）培养的贴壁野生型细胞存在低分子量脱氧核糖核酸（DNA）片段。这种DNA片段化在转染子中被显著抑制。用半胱天冬酶-3抑制剂（10^(-8) M）培养可显著阻止TNF-α或TGF-β1诱导的细胞死亡。向酶反应混合物中添加氯化钙（10 μM）和钙调蛋白（5 μg/ml）可显著增加野生型细胞中的一氧化氮（NO）合酶活性。在转染子中，这种增加被显著抑制。用TNF-α培养可使野生型细胞中的NO合酶活性显著增加。在转染子中未观察到TNF-α的这种作用。用TGF-β1培养未导致野生型细胞和转染子中的NO合酶活性显著增加。用TNF-α或TGF-β1培养可使野生型细胞中的α-平滑肌肌动蛋白显著增加。在转染子中，这种增加被显著阻止。与野生型细胞相比，转染子中Smad 2或核因子-κB（NF-κB）mRNA的表达显著增加。转染子中Smad 3或甘油醛-3-磷酸脱氢酶（G3PDH）mRNA表达未显著变化。用TNF-α培养可使野生型细胞中的NF-κB mRNA表达显著增加。用TGF-β1培养的野生型细胞中Smad 2 mRNA表达显著增强。在转染子中，TNF-α或TGF-β1的这些作用未显著增强。本研究表明，调节钙素的过表达对通过肾NRK52E细胞中TNF-α或TGF-β1的细胞内信号通路介导的细胞反应具有抑制作用。
