Luteinizing hormone signaling in Preovulatory follicles involves early activation of the epidermal growth factor receptor pathway
Luteinizing hormone signaling in Preovulatory follicles involves early activation of the epidermal growth factor receptor pathway
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DOI：
10.1210/me.2007-0246
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发表时间：
2008-04-01
影响因子：
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通讯作者：
Conti, Marco
中科院分区：
文献类型：
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作者：
Panigone, Sara;Hsieh, Minnie;Conti, Marco
促黄体生成素（LH）激活一系列信号传导事件，这些事件在卵巢排卵前卵泡中传播，以促进成熟卵子的排卵。对LH诱导排卵至关重要的是表皮生长因子（EGF）样生长因子的诱导以及EGF受体（EGFR）信号的转活化。由于这种转活化的时间尚未得到很好的表征，我们研究了培养卵泡中LH对EGF网络调节的动态变化。排卵前卵泡在有或没有重组LH和/或特异性抑制剂的情况下进行培养。通过免疫沉淀和蛋白质印迹分析检测EGFR和丝裂原活化蛋白激酶（MAPK）的磷酸化。通过半定量逆转录聚合酶链反应（RT - PCR），在重组LH刺激卵泡30分钟后检测到双调蛋白和上皮调节蛋白mRNA的增加，并在2小时后达到最大值。LH诱导的EGFR磷酸化在30分钟后也增加，并在2小时达到最大值。EGFR激活先于卵母细胞成熟且依赖环磷酸腺苷（cAMP），因为毛喉素同样激活EGFR。LH诱导的EGFR磷酸化对AG1478（一种EGFR激酶抑制剂）以及基质金属蛋白酶抑制剂GM6001和肿瘤坏死因子α蛋白酶抑制剂 - 1（TAPI - 1）敏感，这表明EGF样生长因子脱落参与其中。LH（而非双调蛋白）诱导的卵母细胞成熟和EGFR磷酸化对蛋白质合成抑制敏感。当用抗双调蛋白、上皮调节蛋白和β细胞素的中和抗体组合培养颗粒细胞时，EGFR磷酸化和MAPK激活受到抑制。在培养的卵泡中，LH诱导的MAPK激活被AG1478和GM6001部分抑制，表明该途径部分受EGF网络调节，但也涉及其他途径。因此，复杂的机制参与了排卵前卵泡内LH信号的快速放大和传播，包括EGF网络的早期激活。
LH activates a cascade of signaling events that are propagated throughout the ovarian preovulatory follicle to promote ovulation of a mature egg. Critical to LH-induced ovulation is the induction of epidermal growth factor (EGF)-like growth factors and transactivation of EGF receptor ( EGFR) signaling. Because the timing of this transactivation has not been well characterized, we investigated the dynamics of LH regulation of the EGF network in cultured follicles. Preovulatory follicles were cultured with or without recombinant LH and/or specific inhibitors. EGFR and MAPK phosphorylation were examined by immunoprecipitation and Western blot analyses. By semiquantitative RT-PCR, increases in amphiregulin and epiregulin mRNAs were detected 30 min after recombinant LH stimulation of follicles and were maximal after 2 h. LH-induced EGFR phosphorylation also increased after 30 min and reached a maximum at 2 h. EGFR activation precedes oocyte maturation and is cAMP dependent, because forskolin similarly activated EGFR. LH-induced EGFR phosphorylation was sensitive to AG1478, an EGFR kinase inhibitor, and to inhibitors of matrix metalloproteases GM6001 and TNF alpha protease inhibitor-1 (TAPI- 1), suggesting the involvement of EGF-like growth factor shedding. LH- but not amphiregulin-induced oocyte maturation and EGFR phosphorylation were sensitive to protein synthesis inhibition. When granulosa cells were cultured with a combination of neutralizing antibodies against amphiregulin, epiregulin, and betacellulin, EGFR phosphorylation and MAPK activation were inhibited. In cultured follicles, LH- induced MAPK activation was partially inhibited by AG1478 and GM6001, indicating that this pathway is regulated in part by the EGF network but also involves additional pathways. Thus, complex mechanisms are involved in the rapid amplification and propagation of the LH signal within preovulatory follicles and include the early activation of the EGF network.