Bernard Daniel J
Center for Biomedical Research, Population Council, 1230 York Avenue, New York, New York 10021, USA.
Mol Endocrinol. 2004 Mar;18(3):606-23. doi: 10.1210/me.2003-0264. Epub 2003 Dec 30.
The activins are pleiotropic members of the TGFbeta superfamily. Within the anterior pituitary gland, activins stimulate FSH synthesis in an autocrine/paracrine fashion by stimulating transcription of the FSHbeta subunit gene. Here, the mechanisms mediating this effect were investigated in the murine gonadotrope cell line, LbetaT2. Recombinant activin A and activin B dose- and time-dependently stimulated endogenous FSHbeta mRNA expression. FSHbeta primary transcript and mRNA levels were increased within 30-60 min, but these effects were blocked by preincubation with the transcription inhibitor actinomycin-D, suggesting that the FSHbeta gene is a direct target of the activin signal transduction cascade. In other systems, activin signals are transduced through a heteromeric serine/threonine receptor complex, which includes the signaling activin type IB receptor [activin receptor-like kinase 4 (ALK4)]. Transfection of a constitutively active form of the receptor, ALK4T206D, stimulated FSHbeta mRNA levels. Overexpression of the inhibitory SMAD7 blocked this effect, as well as activin-stimulated FSHbeta transcription. Because SMAD7 functions by preventing access of SMAD2 and SMAD3 to ALK4, these data suggested that both activins and ALK4 require SMAD2 and/or SMAD3 to affect FSHbeta transcription. Consistent with this idea, activin A stimulated SMAD2 and SMAD3 phosphorylation and nuclear translocation within 5-10 min in LbetaT2 cells. Transient transfection of SMAD3, but not SMADs 1, 2, 4, 5, or 8, stimulated endogenous FSHbeta mRNA levels. The results of SMAD2 transfection studies were inconclusive, however, because of a persistent failure to overexpress the full-length SMAD2 protein specifically in LbetaT2 cells. To assess more directly roles for both SMAD2 and SMAD3 in activin-stimulated FSHbeta expression, RNA interference was used to decrease endogenous SMAD protein levels in LbetaT2 cells. Activin A- and ALK4T206D-stimulated transcription of the FSHbeta gene were significantly attenuated by the depletion of either SMAD2 or SMAD3. Collectively, these data suggest that activins use both SMAD2- and SMAD3-dependent mechanisms to stimulate FSHbeta transcription in mouse gonadotrope cells.
激活素是转化生长因子β（TGFβ）超家族的多效性成员。在前脑垂体中，激活素通过刺激促卵泡激素β（FSHβ）亚基基因的转录，以自分泌/旁分泌方式刺激FSH的合成。在此，我们在小鼠促性腺激素细胞系LbetaT2中研究了介导这种效应的机制。重组激活素A和激活素B剂量和时间依赖性地刺激内源性FSHβ mRNA表达。FSHβ初级转录本和mRNA水平在30 - 60分钟内升高，但这些效应被转录抑制剂放线菌素-D预孵育所阻断，这表明FSHβ基因是激活素信号转导级联反应的直接靶点。在其他系统中，激活素信号通过异源二聚体丝氨酸/苏氨酸受体复合物转导，该复合物包括信号传导激活素IB型受体[激活素受体样激酶4（ALK4）]。转染组成型活性形式的受体ALK4T206D可刺激FSHβ mRNA水平。抑制性SMAD7的过表达阻断了这种效应以及激活素刺激的FSHβ转录。由于SMAD7通过阻止SMAD2和SMAD3接近ALK4发挥作用，这些数据表明激活素和ALK4都需要SMAD2和/或SMAD3来影响FSHβ转录。与此观点一致，激活素A在LbetaT2细胞中5 - 10分钟内刺激SMAD2和SMAD3磷酸化及核转位。瞬时转染SMAD3，但不是SMAD1、2、4、5或8，可刺激内源性FSHβ mRNA水平。然而，由于在LbetaT2细胞中持续无法特异性过表达全长SMAD2蛋白，SMAD2转染研究的结果尚无定论。为了更直接地评估SMAD2和SMAD3在激活素刺激的FSHβ表达中的作用，我们使用RNA干扰降低LbetaT2细胞中的内源性SMAD蛋白水平。SMAD2或SMAD3的缺失显著减弱了激活素A和ALK4T206D刺激的FSHβ基因转录。总体而言，这些数据表明激活素利用依赖SMAD2和SMAD3的机制来刺激小鼠促性腺激素细胞中的FSHβ转录。
