Overexpression of regucalcin enhances its nuclear localization and suppresses L-type Ca2+ channel and calcium-sensing receptor mRNA expressions in cloned normal rat kidney proximal tubular epithelial NRK52E cells
Overexpression of regucalcin enhances its nuclear localization and suppresses L-type Ca2+ channel and calcium-sensing receptor mRNA expressions in cloned normal rat kidney proximal tubular epithelial NRK52E cells
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DOI：
10.1002/jcb.20863
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发表时间：
2006-11-01
影响因子：
4
通讯作者：
Yamaguchi, Masayoshi
中科院分区：
文献类型：
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作者：
Nakagawa, Taeko;Yamaguchi, Masayoshi
利用过表达regucalcin（RC）的正常大鼠肾近端肾小管上皮细胞株NRK52E，研究RC对多种矿物质离子转运相关蛋白基因表达的影响。将NRK52E细胞（野生型）和稳定的RC/pCXN2转染子在含有5%牛血清（BS）的培养基中培养72小时以获得亚融合单层。培养72小时后，将细胞在含有载体、醛固酮（10（-8）或10（-7）M）或甲状旁腺激素（PTH）（1 - 34）（10（-8）或10（-7）M）且不含13 S的培养基中进一步培养24 - 72小时。RC明显定位于细胞核。RC的过表达导致大鼠外髓K+通道（ROMK）mRNA表达显著增加，而L型钙通道和钙敏感受体（CaR）mRNA表达显著降低。RC过表达对上皮钠通道（ENaC）、Na，K-ATP酶（α-亚基）、II型Na-Pi协同转运蛋白（NaPi-IIa）、血管紧张素原、Na +-Ca 2+交换蛋白和甘油醛-3-磷酸脱氢酶（G3PDH）mRNA表达无影响。此外，还研究了激素对基因表达的影响。用醛固酮（10（-8）或10（-7）M）培养导致野生型细胞中ENaC、Na、K-ATP酶和ROMK mRNA表达显著增加。这些增加在转染子中减弱。用PTH（10（-8）或10（-7）M）培养可显著降低野生型细胞中NaPi-IIa mRNA的表达。这种效应在转染子中没有改变。PTH显着降低血管紧张素原mRNA表达的野生型细胞和转染，而醛固酮没有影响。用PTH（10（-8）或10（-7）M）培养导致野生型细胞L型Ca~（2+）通道和CaR mRNA表达显著降低，而激素显著增加Na~+-Ca~（2+）交换器mRNA表达。PTH对L型Ca~（2+）通道、CaR和Na~+-Ca~（2+）交换体mRNA表达也有影响。本研究表明，RC的过表达导致其核定位的显着增加，并且在NRK52E细胞中的各种矿物离子调节蛋白中，它对调节细胞内Ca 2+信号的L型Ca 2+通道或CaR的基因表达具有抑制作用。
The effect of regucalcin (RC), a regulatory protein in intracellular signaling pathway, on the gene expression of various mineral ion transport-related proteins was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing RC. NRK52E cells (wild-type) and stable RC/pCXN2 transfectant were cultured for 72 h in medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured 24-72 h in a medium containing either vehicle, aldosterone (10(-8) or 10(-7) M), or parathyroid hormone (PTH) (1-34) (10(-8) or 10-7 M) without 13S. RC was markedly localized in the nucleus of transfectants. Overexpression of RC caused a significant increase in rat outer medullary K+ channel (ROMK) mRNA expression, while it caused a remarkable decrease in L-type Ca2+ channel and calcium-sensing receptor (CaR) mRNA expressions. Overexpression of RC did not have an effect on epithelial sodium channel (ENaC), Na, K-ATPase (alpha-subunit), Type II Na-Pi cotransporter (NaPi-IIa), angiotensinogen, Na+-Ca2+ exchanger, and glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions. Hormonal effect on gene expression, moreover, was examined. Culture with aldosterone (10(-8) or 10(-7) M) caused a significant increase in ENaC, Na, K-ATPase, and ROMK mRNA expressions in the wild-type cells. Those increases were weakened in the transfectants. Culture with PTH (10(-8) or 10(-7) M) significantly decreased NaPi-IIa mRNA expression in the wild-type cells. This effect was not altered in the transfectants. PTH significantly decreased angiotensinogen mRNA expression in the wild-type cells and the transfectants, while aldosterone had no effect. Culture with PTH (10(-8) or 10(-7) M) Caused a significant decrease in L-type Ca2+ channel and CaR mRNA expressions in the wild-type cells, while the hormone significantly increased Na+-Ca2+ exchanger mRNA expression. The effects of PTH on L-type Ca2+ channel, CaR, and Na+-Ca2+ exchanger mRNA expressions were also seen in the transfectants. This study demonstrates that overexpression of RC caused a remarkable increase in its nuclear localization, and that it has suppressive effects on the gene expression of L-type Ca2+ channel or CaR, which regulates intracellular Ca2+ signaling, among various regulator proteins for mineral ions in NRK52E cells.