p38 MAPK mediates acid-induced transcription of PEPCK in LLC-PK1-FBPase+ cells
p38 MAPK mediates acid-induced transcription of PEPCK in LLC-PK1-FBPase+ cells
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DOI：
10.1152/ajprenal.00097.2002
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发表时间：
2002-10-01
影响因子：
4.2
通讯作者：
Gstraunthaler, G
中科院分区：
文献类型：
--
作者：
Feifel, E;Obexer, P;Gstraunthaler, G
LLC-PK1-FBPase(+) 细胞是一种糖异生和 pH 响应性肾近曲小管样细胞系。在酸性培养基 (pH 6.9) 中孵育时，LLC-PK1-FBPase(+) 细胞表现出氨生成率增加，以及谷氨酰胺酶和磷酸烯醇丙酮酸羧激酶 (PEPCK) mRNA 水平和酶活性增加。 PEPCK mRNA 的增加是由于响应细胞内酸中毒而启动的转录率增加。通过确定特定 MAPK 激活剂和抑制剂对基础和酸诱导的 PEPCK mRNA 水平的影响，检查已知 MAPK 活性（ERK1/2、SAPK/JNK、p38）在相关信号转导途径中的参与情况。将 LLC-PK1-FBPase(+) 培养物转移至酸性培养基中，分别导致 p38 和激活转录因子-2 (ATF-2) 的特异性磷酸化，从而激活。 Anisomycin (AI) 是一种强 p38 激活剂，可将 PEPCK mRNA 增加至与酸刺激观察到的水平相当。 AI还诱导p38和ATF-2的时间依赖性磷酸化。SB-203580，一种特异性p38抑制剂，阻断酸和AI诱导的PEPCK mRNA水平。蛋白质印迹分析表明，SB-203580 敏感的 p38α 亚型被强烈表达。 PEPCK启动子的cAMP-反应元件-1位点的八核苷酸序列与结合ATF-2的共有元件完美匹配。通过ELISA证明了ATF-2结合的特异性。我们得出结论，SB-203580敏感的p38α-ATF-2信号通路可能是肾LLC-PK1-FBPase(+)细胞中PEPCK mRNA水平的pH响应性诱导的介导物。
LLC-PK1-FBPase(+) cells are a gluconeogenic and pH-responsive renal proximal tubule- like cell line. On incubation with acidic medium (pH 6.9), LLC-PK1- FBPase(+) cells exhibit an increased rate of ammonia production as well as increases in glutaminase and phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels and enzyme activities. The increase in PEPCK mRNA is due to an enhanced rate of transcription that is initiated in response to intracellular acidosis. The involvement of known MAPK activities (ERK1/ 2, SAPK/ JNK, p38) in the associated signal transduction pathway was examined by determining the effects of specific MAPK activators and inhibitors on basal and acid- induced PEPCK mRNA levels. Transfer of LLC-PK1-FBPase(+) cultures to acidic medium resulted in specific phosphorylation, and thus activation, of p38 and of activating transcription factor- 2 (ATF- 2), respectively. Anisomycin (AI), a strong p38 activator, increased PEPCK mRNA to levels comparable to those observed with acid stimulation. AI also induced a time- dependent phosphorylation of p38 and ATF- 2. SB- 203580, a specific p38 inhibitor, blocked both acid- and AI- induced PEPCK mRNA levels. Western blot analyses revealed that the SB- 203580- sensitive p38alpha isoform is strongly expressed. The octanucleotide sequence of the cAMP- response element- 1 site of the PEPCK promotor is a perfect match to the consensus element for binding ATF- 2. The specificity of ATF- 2 binding was proven by ELISA. We conclude that the SB- 203580- sensitive p38alpha- ATF- 2 signaling pathway is a likely mediator of the pH- responsive induction of PEPCK mRNA levels in renal LLC-PK1- FBPase(+) cells.