Phorbol ester stimulation of RasGRP1 regulates the sodium-chloride cotransporter by a PKC-independent pathway
Phorbol ester stimulation of RasGRP1 regulates the sodium-chloride cotransporter by a PKC-independent pathway
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DOI：
10.1073/pnas.0709506104
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发表时间：
2007-12-11
影响因子：
11.1
通讯作者：
Hoover, Robert S.
中科院分区：
文献类型：
--
作者：
Ko, Benjamin;Joshi, Leena M.;Hoover, Robert S.
氯化钠共转运体（NCC）是哺乳动物远曲小管（DCT）中主要的盐吸收途径，也是最有效的抗高血压药物之一噻嗪类利尿剂的作用部位。我们开发了一个细胞模型系统来评估天然表达NCC的哺乳动物细胞系——小鼠DCT （mDCT）细胞系的NCC功能。我们利用该体系研究了二酰基甘油（DAG）类似物酚酯（PE） 12- o -十四烷基酚-13-乙酸酯（TPA）对NCC的复杂调控。一般认为，pe通过激活PKC介导其对转运体的作用。然而，至少还有五个其他DAG/PE目标。在这里，我们描述了DAG/PE效应的一个替代靶点，Ras guanyl释放蛋白1 （RasGRP1）如何介导PE诱导的NCC功能和表面表达的抑制。通过对噻嗪类药物敏感的Na-22（+）摄取来评估NCC的功能，发现TPA完全抑制NCC功能。生物素化实验表明，这一结果主要是由于NCC的表面表达减少。虽然PKC抑制剂对这种抑制没有影响，但MAPK抑制剂完全阻止了TPA的作用。RasGRP1通过激活小G蛋白Ras激活MAPK通路。基因沉默RasGRP1可以阻止pe介导的NCC活性抑制、Ras的H-Ras亚型的激活和ERK1/2 MAPK的激活。这一发现证实了RasGRP1通过刺激MAPK通路介导pe诱导的NCC活性抑制的关键作用。
The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the mammalian distal convoluted tubule (DCT) and is the site of action of one of the most effective classes of anti hypertensive medications, thiazide diuretics. We developed a cell model system to assess NCC function in a mammalian cell line that natively expresses NCC, the mouse DCT (mDCT) cell line. We used this system to study the complex regulation of NCC by the phorbol ester (PE) 12-O-tetradecanoylphorbol-13-acetate (TPA), a diacylglycerol (DAG) analog. It has generally been thought that PEs mediate their effects on transporters through the activation of PKC. However, there are at least five other DAG/PE targets. Here we describe how one of those alternate targets of DAG/PE effects, Ras guanyl-releasing protein 1 (RasGRP1), mediates the PE-induced suppression of function and the surface expression of NCC. Functional assessment of NCC by using thiazide-sensitive Na-22(+) uptakes revealed that TPA completely suppresses NCC function. Biotinylation experiments demonstrated that this result was primarily because of decreased surface expression of NCC. Although inhibitors of PKC had no effect on this suppression, MAPK inhibitors completely prevented the TPA effect. RasGRP1 activates the MAPK pathway through activation of the small G protein Ras. Gene silencing of RasGRP1 prevented the PE-mediated suppression of NCC activity, the activation of the H-Ras isoform of Ras, and the activation of ERK1/2 MAPK. This finding confirmed the critical role of RasGRP1 in mediating the PE-induced suppression of NCC activity through the stimulation of the MAPK pathway.