Morales Victoria, Gonzalez-Robayna I, Santana M Pino, Hernandez Inmaculada, Fanjul Luisa F
Department of Biochemistry and Physiology, Facultad de Medicina, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, 35016, Spain.
Endocrinology. 2006 Dec;147(12):5932-9. doi: 10.1210/en.2006-0635. Epub 2006 Aug 31.
The proinflammatory cytokine TNFalpha has important actions at the level of the ovary, including inhibition of P450 aromatase (P450AROM) activity and the secretion of inhibin, two proteins that are markers of the granulosa cell's differentiated status. Because the transcription of both P450AROM and inhibin alpha-subunit can be suppressed in the ovary by the inducible repressor isoform of cAMP-responsive element binding modulator (ICER), we have investigated whether TNFalpha and its intracellular messenger ceramide can induce ICER expression and the mechanisms whereby the induction is accomplished. ICER mRNA levels were assessed by RT-PCR in granulosa cells treated with TNFalpha, the ceramide-mobilizing enzyme sphingomyelinase (SMase), or C6-cer, a cell-permeant ceramide analog. Rapid (3 h) yet transient increases in the four isoforms of ICER were observed in response to all treatments. Likewise, ICER protein measured by immunoprecipitation with a specific antibody increases after TNFalpha, SMase, or C6-cer treatment. The mandatory phosphorylation of cAMP-responsive element binding was also observed in response to TNFalpha, SMase, or C6-cer and shown to be prevented by the p44/42 MAPK-specific inhibitor PD098059 but no other kinase blockers. Activation of p44/42 MAPK by the cytokine and its messenger was subsequently demonstrated as well as the inhibition of ICER expression by PD098059. Finally, the blocking of p44/42 MAPK activation prevented TNFalpha inhibition of FSH-dependent increases in P450AROM and inhibin alpha-subunit mRNA levels, thus indicating that p44/42 MAPK-mediated ICER expression may be accountable for the effects of TNFalpha on the expression of both proteins.
促炎细胞因子肿瘤坏死因子α（TNFα）在卵巢水平具有重要作用，包括抑制细胞色素P450芳香化酶（P450AROM）活性和抑制素分泌，这两种蛋白是颗粒细胞分化状态的标志物。由于cAMP反应元件结合调节因子的诱导型阻遏物亚型（ICER）可在卵巢中抑制P450AROM和抑制素α亚基的转录，我们研究了TNFα及其细胞内信使神经酰胺是否能诱导ICER表达以及诱导的机制。通过逆转录聚合酶链反应（RT-PCR）评估用TNFα、神经酰胺动员酶鞘磷脂酶（SMase）或C6-神经酰胺（一种可透过细胞的神经酰胺类似物）处理的颗粒细胞中ICER mRNA水平。对所有处理均观察到ICER的四种亚型迅速（3小时）但短暂增加。同样，用特异性抗体免疫沉淀法测定的ICER蛋白在TNFα、SMase或C6-神经酰胺处理后增加。对TNFα、SMase或C6-神经酰胺的反应中也观察到cAMP反应元件结合蛋白的必需磷酸化，并显示可被p44/42丝裂原活化蛋白激酶（MAPK）特异性抑制剂PD098059阻断，但不能被其他激酶阻滞剂阻断。随后证明了细胞因子及其信使对p44/42 MAPK的激活以及PD098059对ICER表达的抑制。最后，阻断p44/42 MAPK激活可防止TNFα对促卵泡激素（FSH）依赖性P450AROM和抑制素α亚基mRNA水平增加的抑制作用，因此表明p44/42 MAPK介导的ICER表达可能是TNFα对这两种蛋白表达影响的原因。
