Epidermal growth factor activates Na(+/)H(+) exchanger in podocytes through a mechanism that involves Janus kinase and calmodulin.
Epidermal growth factor activates Na(+/)H(+) exchanger in podocytes through a mechanism that involves Janus kinase and calmodulin.
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DOI：
10.1016/j.bbamcr.2009.03.006
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发表时间：
2009-07
期刊：
影响因子：
--
通讯作者：
Raymond JR
中科院分区：
文献类型：
--
作者：
Coaxum SD;Garnovskaya MN;Gooz M;Baldys A;Raymond JR
钠质子交换因子1(NHE-1)广泛表达，被多种生长因子激活，在调节细胞内pH、细胞体积、细胞增殖和细胞骨架等方面发挥重要作用。尽管它很重要，但人们对它在肾小球足细胞中的调控知之甚少。在本工作中，我们研究了培养的足细胞中表皮生长因子受体(EGFR)对NHE-1活性的调节。RT-PCR显示分化的足细胞表达NHE-1和NHE-2，以及EGFR亚单位EGFR/ErbB1、Erb3和ErbB4。用细胞传感器微生理仪测定，EGF引起的肾小管上皮细胞质子外流的浓度依赖性增加在5-(N-甲基-N-异丁基)阿米洛利或无钠溶液中被减弱。此外，Janus激酶(JAK2)和钙调蛋白(CaM)的药理抑制剂可减弱EGF诱导的NHE-1活性。免疫共沉淀研究表明，EGF诱导了JAK2和CaM之间以及CaM和NHE-1之间形成复合体。此外，EGF还增加了JAK2和CaM的酪氨酸磷酸化水平。EGFR激酶抑制剂AG1478可阻断NHE-1的激活，但不能阻断EGF诱导的JAK2或CaM的磷酸化。这些结果表明，表皮生长因子通过两条途径诱导足细胞NHE-1活性：(1)EGF→EGFR→JAK2激活(不依赖于EGFR酪氨酸激酶活性)→酪氨酸磷酸化CaM→CaM结合NHE-1→构象变化NHE-1→激活；以及(2)EGF→EGFR→EGFR激酶激活→与NHE-1结合(不依赖JAK2)→构象变化NHE-1→激活。
Sodium-proton exchanger type 1 (NHE-1) is ubiquitously expressed, is activated by numerous growth factors, and plays significant roles in regulating intracellular pH and cellular volume, proliferation and cytoskeleton. Despite its importance, little is known about its regulation in renal glomerular podocytes. In the current work, we studied the regulation of NHE-1 activity by the epidermal growth factor receptor (EGFR) in cultured podocytes. RT-PCR demonstrated mRNAs for NHE-1 and NHE-2 in differentiated podocytes, as well as for EGFR subunits EGFR/ErbB1, Erb3, and ErbB4. EGF induced concentration-dependent increases in proton efflux in renal podocytes as assessed using a Cytosensor microphysiometer, were diminished in the presence of 5-(N-methyl-N-isobutyl) amiloride or in a sodium-free solution. Furthermore, pharmacological inhibitors of janus kinase (Jak2) and calmodulin (CaM) attenuated EGF-induced NHE-1 activity. Co-immunoprecipitation studies determined that EGF induced formation of complexes between Jak2 and CaM, as well as between CaM and NHE-1. In addition, EGF increased levels of tyrosine phosphorylation of Jak2 and CaM. The EGFR kinase inhibitor, AG1478, blocked activation of NHE-1, but did not block EGF-induced phosphorylation of Jak2 or CaM. These results suggest that EGF induces NHE-1 activity in podocytes through two pathways: (1) EGF → EGFR → Jak2 activation (independent of EGFR tyrosine kinase activity) → tyrosine phosphorylation of CaM → CaM binding to NHE-1 → conformational change of NHE-1 → activation of NHE-1; and (2) EGF →EGFR → EGFR kinase activation → association of CaM with NHE-1 (independent of Jak2) → conformational change of NHE-1 → activation of NHE-1.
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