Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells.
Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells.
复制标题
小鼠 IMCD 细胞中多种丝裂原激活蛋白激酶受高渗透压调节。
DOI：
10.1152/ajprenal.1997.272.3.f305
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Heasley,LE
中科院分区：
文献类型：
--
作者：
Berl,T;Siriwardana,G;Ao,L;Butterfield,LM;Heasley,LE
髓内集管（IMCD）细胞通过合成转运体来适应高渗环境，转运体允许有机渗透物的积累。为了检测其他有丝分裂原活化蛋白（MAP）激酶的激活，将IMCD-3细胞提取物在高渗培养基（600 mosmol/kgH2O）中处理15分钟，用Mono Q快速高效液相色谱分离，并以表皮生长因子受体[EGFR-(662-681)]肽为底物进行检测。确定了三个活动峰。Western blotting显示，这些峰与Jun nh2末端激酶（JNK）、细胞外信号调节蛋白激酶、ERK1和ERK2以及p38 MAP激酶一致。为了评估ERK2激活在IMCD-3细胞中的功能意义，我们评估了上游调节蛋白激酶MAP/ERK激酶（MEK）抑制剂PD-098059的作用。PD-098059通过高渗性抑制ERK活化。然而，肌醇摄取的刺激（适应性的标志）在16小时后没有改变。直接测量JNK活性[GST-cJun-(1-79)的磷酸化]显示，随着NaCl或甘露醇的介质渗透压从300 mosmol/kgH2O增加到900 mosmol/kgH2O， JNK活性显著（20- 40倍）增加。尿素则引起了更温和的活性增加。反应迅速，早在暴露后2分钟就能检测到，在10-15分钟达到最大激活。细胞蛋白激酶C （PKC）因长期暴露于磷酯而下调，仅能最低限度地减弱JNK对高渗透压的反应，表明PKC缺乏参与。我们得出结论，在IMCD-3细胞中，通过高渗透压抑制ERK活化并不会阻止肌醇运输的渗透调节增加。这与erk在响应中的作用不一致。JNK和p38的作用尚未被排除，这些途径可能代表了有机渗透转运体基因随后转录和适应细胞外高渗性的启动事件。
Inner medullary collecting duct (IMCD) cells adapt to a hypertonic environment by synthesizing transporters that allow for accumulation of organic osmolytes. To examine for activation of additional mitogen-activated protein (MAP) kinases, extracts of IMCD-3 cells subjected to a hypertonic medium (600 mosmol/kgH2O) for 15 min were fractionated by Mono Q fast-performance liquid chromatography and assayed with the epidermal growth factor receptor [EGFR-(662-681)] peptide as substrate. Three peaks of activity were identified. Western blotting revealed that these peaks coincided with Jun NH2-terminal kinase (JNK), extracellular signal-regulated protein kinases, ERK1 and ERK2, and p38 MAP kinase. To assess the functional significance of ERK2 activation in IMCD-3 cells, the effect of PD-098059, an inhibitor of the upstream regulatory protein kinase MAP/ERK kinase (MEK) was assessed. PD-098059 inhibited ERK activation by hypertonicity. Yet, the stimulation of inositol uptake, a marker of adaptation, after 16 h was unaltered. Direct measurements of JNK activity [phosphorylation of GST-cJun-(1-79)] revealed a marked (20- to 40-fold) increase in activity as medium osmolality was increased from 300 to 900 mosmol/kgH2O with either NaCl or mannitol. Urea induced a more modest increase in activity. The response is prompt and detected as early as 2 min after exposure, reaching a maximum activation at 10-15 min. Downregulation of cellular protein kinase C (PKC) by chronic exposure to phorbol esters only minimally attenuated the JNK response to hyperosmolality, indicating a lack of involvement of PKC. We conclude that, in IMCD-3 cells, inhibition of ERK activation by hyperosmolality does not prevent osmoregulatory increase in inositol transport. This is not consistent with a role for ERKs in the response. The roles for JNK and p38 have not been ruled out, and these pathways may represent the initiating event in the subsequent transcription of organic osmolyte transporter genes and adaptation to extracellular hypertonicity.