Involvement of the MAPK-kinase pathway in the PTH-mediated regulation of the proximal tubule type IIa Na+/Pi cotransporter in mouse kidney
Involvement of the MAPK-kinase pathway in the PTH-mediated regulation of the proximal tubule type IIa Na+/Pi cotransporter in mouse kidney
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DOI：
10.1007/s00424-002-0969-8
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发表时间：
2003-04-01
影响因子：
4.5
通讯作者：
Wagner, CA
中科院分区：
文献类型：
--
作者：
Bacic, D;Schulz, N;Wagner, CA
磷酸盐在近端小管中的重吸收主要由IIa型Na+/P-i协同转运蛋白（NaPi-IIa）介导，并受多种因素（包括膳食磷酸盐摄入和甲状旁腺激素（PTH））的严格调节。PTH信号通过顶端和基底外侧PTH受体，并诱导NaPi-IIa的快速内化和随后的降解。至少有两个信号级联可以被PTH激活：PLC/PKC和cAMP/PKA通路。OK细胞培养的最新证据表明MAPK激酶参与PTH作用。在这里，我们使用新鲜分离的冠状小鼠肾切片和孵育他们在生理缓冲液中的存在和不存在的PTH与抑制剂和激活剂的各种信号级联反应，以进一步研究的事件，导致内化的NaPi-IIa。α-微管蛋白、肌动蛋白和几种刷状缘膜蛋白的免疫染色模式没有改变，证明了在实验期间切片的完整性。在孵育45分钟后，应用PTH（100 nM）诱导了NaPi-IIa刷状缘染色和内化的强烈减少。然而，Na+/硫酸盐协同转运蛋白（NaSi）的定位不受影响。PKC抑制剂白屈菜红碱（1 μ M）或MAPK激酶（ERK 1/2）抑制剂PD 098059（20 μ M）可完全阻止NaPi-IIa的内化。在没有PTH的情况下，单独使用两种抑制剂均无作用。PTH诱导ERK 1/2 MAPK-激酶磷酸化，PD 098059可阻止其磷酸化。PD 098059完全阻止8-Br-cAMP单独激活cAMP/PKA通路，而PKC激活剂1，2-二辛酰甘油（DOG）激活PLC/PKC通路和8-Br-cGMP激活PKG通路诱导NaPi-IIa内化，PD 098059仅部分阻断。抑制SB 203580或激活茴香霉素的p38激酶通路没有影响NaPi-IIa本地化控制条件下或PTH刺激后。此外，PD 098059可以减少PTH诱导的NaPi-IIa蛋白减少。这些结果表明，ERK 1/2 MAPK激酶通路在PTH信号传导中起着核心作用，导致近端小管中II型NaPi-IIa共转运蛋白的特异性内化和随后的降解。
Reabsorption of phosphate in the proximal tubule is mainly mediated by the type IIa Na+/P-i cotransporter (NaPi-IIa) and tightly regulated by a variety of factors including dietary phosphate intake and parathyroid hormone (PTH). PTH signals through both apical and basolateral PTH receptors and induces the rapid internalization and subsequent degradation of NaPi-IIa. At least two signalling cascades can be activated by PTH: the PLC/PKC and the cAMP/PKA pathways. Recent evidence from OK cell culture suggested the involvement of MAPK kinases in the PTH action. Here we used freshly isolated coronal mouse kidney slices and incubated them in a physiological buffer in the absence and presence of PTH with inhibitors and activators of the various signalling cascades to further study the events leading to internalization of NaPi-IIa. No alterations in the pattern of immunostaining for alpha-tubulin, actin and several brush border membrane proteins demonstrated intactness of the slices over the experimental period. Application of PTH (100 nM) induced a strong decrease of NaPi-IIa brush border staining and internalization after 45 min of incubation. The localization of the Na+/sulphate cotransporter (NaSi), however, was not affected. The internalization of NaPi-IIa could be completely prevented by the PKC inhibitor chelerythrine (1 muM) or the MAPK-kinase (ERK1/2) inhibitor PD098059 (20 muM). Without PTH both inhibitors alone had no effect. PTH induced phosphorylation of the ERK1/2 MAPK-kinases which was prevented by PD 098059. Separate activation of the cAMP/PKA pathway by 8-Br-cAMP was completely prevented by PD098059 whereas activation of the PLC/PKC pathway by the PKC activator 1,2-dioctanoyl-snglycerol (DOG) and the PKG pathway by 8-Br-cGMP induced internalization of NaPi-IIa which could be only partly blocked by PD 098059. Inhibition by SB203580 or activation by anisomycin of the p38 kinase pathway had no influence on NaPi-IIa localization under control conditions or after PTH stimulation. Furthermore, the PTH-induced decrease in NaPi-IIa protein could be reduced by PD 098059. These results suggest that the ERK1/2 MAPK kinase pathway plays a central role in the signalling of PTH leading to specific internalization and subsequent degradation of the type II NaPi-IIa cotransporter in the proximal tubule.