Nowak Grazyna, Clifton Ginger L, Godwin Malinda L, Bakajsova Diana
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, 4301 West Markham St., Little Rock, AR 72205, USA.
Am J Physiol Renal Physiol. 2006 Oct;291(4):F840-55. doi: 10.1152/ajprenal.00219.2005. Epub 2006 May 16.
Previously, we showed that oxidant exposure in renal proximal tubular cells (RPTC) induces mitochondrial dysfunction mediated by PKC-epsilon. This study examined the role of ERK1/2 in mitochondrial dysfunction induced by oxidant injury and whether PKC-epsilon mediates its effects on mitochondrial function through the Raf-MEK1/2-ERK1/2 pathway. Sublethal injury produced by tert-butylhydroperoxide (TBHP) resulted in three- to fivefold increase in phosphorylation of ERK1/2 and p38 but not JNK. This was followed by decreases in basal and uncoupled respirations (41%), state 3 respiration and ATP production coupled to complex I (46%), and complex I activity (42%). Oxidant exposure decreased aconitase activity 30% but not pyruvate, alpha-ketoglutarate, and malate dehydrogenase activities. Inhibition of ERK1/2 restored basal and state 3 respirations, DeltaPsi(m), ATP production, and complex I activity but not aconitase activity. In contrast, activation of ERK1/2 by expression of constitutively active MEK1 suppressed basal, uncoupled, and state 3 respirations in noninjured RPTC to the levels observed in TBHP-injured RPTC. MEK1/2 inhibition did not change Akt or p38 phosphorylation, demonstrating that the protective effect of MEK1/2 inhibitor was not due to activation of Akt or inhibition of p38 pathway. Inhibition of PKC-epsilon did not block TBHP-induced ERK1/2 phosphorylation in whole RPTC or in mitochondria. We conclude that 1) oxidant-induced activation of ERK1/2 but not p38 or JNK reduces mitochondrial respiration and ATP production by decreasing complex I activity and substrate oxidation through complex I, 2) citric acid cycle dehydrogenases are not under control of the ERK1/2 pathway in oxidant-injured RPTC, 3) the protective effects of ERK1/2 inhibition are not due to activation of Akt, and 4) ERK1/2 and PKC-epsilon mediate oxidant-induced mitochondrial dysfunction through independent pathways.
此前，我们发现肾近端小管细胞（RPTC）暴露于氧化剂会诱导由蛋白激酶C-ε（PKC-ε）介导的线粒体功能障碍。本研究探讨了细胞外信号调节激酶1/2（ERK1/2）在氧化剂损伤诱导的线粒体功能障碍中的作用，以及PKC-ε是否通过Raf-丝裂原活化蛋白激酶/细胞外信号调节激酶激酶1/2（MEK1/2）-ERK1/2途径介导其对线粒体功能的影响。叔丁基过氧化氢（TBHP）造成的亚致死性损伤导致ERK1/2和p38的磷酸化增加了三到五倍，但c-Jun氨基末端激酶（JNK）未增加。随后，基础呼吸和非偶联呼吸（降低41%）、状态3呼吸以及与复合体I偶联的三磷酸腺苷（ATP）生成（降低46%）和复合体I活性（降低42%）均下降。氧化剂暴露使乌头酸酶活性降低30%，但丙酮酸、α-酮戊二酸和苹果酸脱氢酶活性未受影响。抑制ERK1/2可恢复基础呼吸和状态3呼吸、线粒体膜电位（ΔΨm）、ATP生成以及复合体I活性，但不能恢复乌头酸酶活性。相反，通过组成型活性MEK1的表达激活ERK1/2，可将未受损RPTC中的基础呼吸、非偶联呼吸和状态3呼吸抑制至TBHP损伤的RPTC中观察到的水平。抑制MEK1/2不会改变蛋白激酶B（Akt）或p38的磷酸化，表明MEK1/2抑制剂的保护作用并非由于Akt的激活或p38途径的抑制。抑制PKC-ε不会阻断TBHP在整个RPTC或线粒体中诱导的ERK1/2磷酸化。我们得出以下结论：1）氧化剂诱导的ERK1/2而非p38或JNK的激活通过降低复合体I活性以及通过复合体I的底物氧化，减少线粒体呼吸和ATP生成；2）在氧化剂损伤的RPTC中，柠檬酸循环脱氢酶不受ERK1/2途径的调控；3）抑制ERK1/2的保护作用并非由于Akt的激活；4）ERK1/2和PKC-ε通过独立途径介导氧化剂诱导的线粒体功能障碍。
