Centre de Recherche en Reproduction Et Fertilité, Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, CRRF, Université de Montréal, Saint-Hyacinthe, Québec, Canada.
Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, 3200, Canada.
BMC Mol Cell Biol. 2023 Jun 20;24(1):21. doi: 10.1186/s12860-023-00482-5.
Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor-mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was previously shown as differentially expressed in granulosa cells (GC) of bovine pre-ovulatory follicles suggesting that JAK3 could modulate GC function and activation/inhibition of downstream targets. We used JANEX-1, a JAK3 inhibitor, and FSH treatments and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3, CDKN1B/p27 and MAPK8IP3/JIP3. Cultured GC were treated with or without FSH in the presence or not of JANEX-1. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was upregulated in GC treated with FSH and both were significantly decreased when JAK3 was inhibited. Proliferation markers CCND2 and PCNA were reduced in JANEX-1-treated GC and upregulated by FSH. Western blots analyses showed that JANEX-1 treatment reduced pSTAT3 amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX-1-treated GC. UHPLC-MS/MS analyses revealed phosphorylation of specific amino acid residues within JAK3 as well as CDKN1B and MAPK8IP3 suggesting possible activation or inhibition post-FSH or JANEX-1 treatments. We show that FSH activates JAK3 in GC, which could phosphorylate target proteins and likely modulate other signaling pathways involving CDKN1B and MAPK8IP3, therefore controlling GC proliferation and steroidogenic activity.
Janus 激酶 3（JAK3）是参与细胞因子受体介导的细胞内信号转导的 JAK 家族酪氨酸激酶蛋白之一，通过 JAK/STAT 信号通路。先前的研究表明，JAK3 在牛排卵前卵泡的颗粒细胞（GC）中差异表达，这表明 JAK3 可以调节 GC 功能和激活/抑制下游靶标。我们使用 JAK3 抑制剂 JANEX-1 和 FSH 处理，并分析增殖标志物、类固醇生成酶和包括 STAT3、CDKN1B/p27 和 MAPK8IP3/JIP3 在内的靶蛋白的磷酸化。在存在或不存在 JANEX-1 的情况下，用或不用 FSH 处理培养的 GC。CYP11A1 的表达，而不是 CYP19A1，在 FSH 处理的 GC 中上调，而当 JAK3 被抑制时，两者都显著降低。增殖标志物 CCND2 和 PCNA 在 JANEX-1 处理的 GC 中减少，并被 FSH 上调。Western blot 分析表明，JANEX-1 处理减少了 pSTAT3 的数量，而 JAK3 过表达增加了 pSTAT3。同样，即使在 JANEX-1 处理的 GC 中，FSH 处理也增加了 pSTAT3。UHPLC-MS/MS 分析显示 JAK3、CDKN1B 和 MAPK8IP3 内特定氨基酸残基的磷酸化表明，FSH 或 JANEX-1 处理后可能存在激活或抑制。我们表明，FSH 在 GC 中激活 JAK3，其可以磷酸化靶蛋白，并可能调节涉及 CDKN1B 和 MAPK8IP3 的其他信号通路，从而控制 GC 的增殖和类固醇生成活性。
