Department of Pharmacology and Therapeutics, McGill University, Montréal, QC, Canada.
Cell Signal. 2012 Aug;24(8):1632-40. doi: 10.1016/j.cellsig.2012.04.006. Epub 2012 Apr 20.
Activins stimulate follicle-stimulating hormone (FSH) β subunit (Fshb) gene transcription in pituitary gonadotrope cells. Previous studies suggest that activins signal via homolog of Drosophila mothers against decapentaplegic (SMAD) proteins to stimulate murine or porcine Fshb promoter activity in the gonadotrope-like cell line, LβT2. In contrast, activins were suggested to regulate the ovine Fshb promoter via a SMAD-independent pathway involving TGFβ associated kinase 1 (TAK1, MAP3K7) and p38 mitogen activated protein kinase (MAPK). Here, we examined roles for TAK1 and p38 in activin A-stimulated murine and ovine Fshb transcription. The TAK1 inhibitor 5Z-7-Oxozeanol (Oxo) significantly impaired fold activin A induction of murine and ovine Fshb promoter-reporters (Fshb-luc) in LβT2 cells, but only at concentrations 50-100 fold greater than its IC(50) for TAK1. Moreover, Oxo failed to inhibit activin A induction of endogenous Fshb mRNA levels or fold induction of Fshb-luc activity by a constitutively active form of the activin type I receptor (ALK4). Oxo, at a concentration 5-10 fold greater than its IC(50) for TAK1, attenuated TAK1/TAB2 stimulation of a p38-dependent reporter in the same cells. A Map3k7 siRNA impaired TAK1/TAB2-stimulated p38-dependent reporter activity, but failed to antagonize activin A-stimulated Fshb-luc. Though TAK1 was previously suggested to act via p38 to stimulate the ovine Fshb promoter, activin A failed to stimulate p38 phosphorylation in LβT2 cells. In apparent contrast, however, the p38 inhibitors SB203580 and SB202190 concentration-dependently attenuated activin A-induced Fshb-luc activity. Given the lack of p38 activation, we postulated that the inhibitors might non-selectively antagonize ALK4 activity. Indeed, both attenuated activin A-stimulated SMAD2 phosphorylation, consistent with direct antagonism of ALK4 kinase activity. Finally, we observed that RNA-mediated suppression of Smad4, and to a lesser extent Smad3, attenuated activin A induction of both murine and ovine Fshb promoter-reporters. Collectively, these data suggest that activin A signals via SMAD proteins, but not TAK1 or p38, to regulate murine and ovine Fshb transcription in gonadotrope-like cells.
激活素刺激垂体促性腺激素细胞中的卵泡刺激素 (FSH)β亚基 (Fshb) 基因转录。先前的研究表明，激活素通过果蝇母体对抗 decapentaplegic (SMAD) 蛋白的同源物 (SMAD) 蛋白信号传导，以刺激促性腺激素样细胞系 LβT2 中的鼠或猪 Fshb 启动子活性。相比之下，据报道激活素通过涉及 TGFβ 相关激酶 1 (TAK1，MAP3K7) 和 p38 有丝分裂原激活蛋白激酶 (MAPK) 的 SMAD 非依赖性途径来调节绵羊 Fshb 启动子。在这里，我们研究了 TAK1 和 p38 在激活素 A 刺激的鼠和绵羊 Fshb 转录中的作用。TAK1 抑制剂 5Z-7-Oxozeanol (Oxo) 显着削弱了激活素 A 对 LβT2 细胞中鼠和绵羊 Fshb 启动子报告基因 (Fshb-luc) 的诱导倍数，但仅在比其对 TAK1 的 IC50 高 50-100 倍的浓度下。此外，Oxo 未能抑制激活素 A 诱导的内源性 Fshb mRNA 水平或激活素 A 诱导的组成型激活的激活素 I 型受体 (ALK4) 活性的 Fshb-luc 诱导倍数。Oxo 在比其对 TAK1 的 IC50 高 5-10 倍的浓度下，减弱了 TAK1/TAB2 对同一细胞中 p38 依赖性报告基因的刺激。Map3k7 siRNA 削弱了 TAK1/TAB2 刺激的 p38 依赖性报告基因活性，但未能拮抗激活素 A 刺激的 Fshb-luc。尽管先前曾提出 TAK1 通过 p38 起作用以刺激绵羊 Fshb 启动子，但激活素 A 未能刺激 LβT2 细胞中的 p38 磷酸化。然而，明显相反的是，p38 抑制剂 SB203580 和 SB202190 浓度依赖性地减弱了激活素 A 诱导的 Fshb-luc 活性。鉴于缺乏 p38 激活，我们假设抑制剂可能非选择性地拮抗 ALK4 活性。事实上，两者均减弱了激活素 A 刺激的 SMAD2 磷酸化，这与 ALK4 激酶活性的直接拮抗一致。最后，我们观察到 RNA 介导的 Smad4 抑制，以及在较小程度上 Smad3 抑制，减弱了激活素 A 对鼠和绵羊 Fshb 启动子报告基因的诱导。总的来说，这些数据表明激活素 A 通过 SMAD 蛋白而不是 TAK1 或 p38 信号传导来调节促性腺激素样细胞中鼠和绵羊 Fshb 的转录。
