Gore A Jesse, Philips Daniel P, Miller William L, Bernard Daniel J
Department of Molecular and Structural Biochemistry, North Carolina State University, Box 7622, Raleigh, NC 27695-7622, USA.
Reprod Biol Endocrinol. 2005 Dec 29;3:73. doi: 10.1186/1477-7827-3-73.
Activins stimulate the synthesis of follicle stimulating hormone (FSH) in pituitary gonadotropes, at least in part, by inducing transcription of its beta subunit (Fshb). Evidence from several laboratories studying transformed murine LbetaT2 gonadotropes indicates that activins signal through Smad-dependent and/or Smad-independent pathways, similar to those used by transforming growth factor beta-1 (TGFB1) in other cell types. Therefore, given common intracellular signaling mechanisms of these two ligands, we examined whether TGFBs can also induce transcription of Fshb in LbetaT2 cells as well as in purified primary murine gonadotropes.
Murine Fshb promoter-reporter (-1990/+1 mFshb-luc) activity was measured in LbetaT2 cells treated with activin A or TGFB1, and in cells transfected with either activin or TGFB receptors. The ability of the ligands to stimulate phosphorylation of Smads 2 and 3 in LbetaT2 cells was measured by western blot analysis, and expression of TGFB type I and II receptors was assessed by reverse transcriptase polymerase chain reaction in both LbetaT2 cells and primary gonadotropes purified from male mice of different ages. Finally, regulation of endogenous murine Fshb mRNA levels by activin A and TGFB1 in purified gonadotropes and whole pituitary cultures was measured using quantitative RT-PCR.
Activin A dose-dependently stimulated -1990/+1 mFshb-luc activity in LbetaT2 cells, but TGFB1 had no effect at doses up to 5 nM. Similarly, activin A, but not TGFB1, stimulated Smad 2 and 3 phosphorylation in these cells. Constitutively active forms of the activin (Acvr1b-T206D) and TGFB (TGFBR1-T204D) type I receptors strongly stimulated -1990/+1 mFshb-luc activity, showing that mechanisms down stream of Tgfbr1 seem to be intact in LbetaT2 cells. RT-PCR analysis of LbetaT2 cells and whole adult murine pituitaries indicated that both expressed Tgfbr1 mRNA, but that Tgfbr2 was not detected in LbetaT2 cells. When cells were transfected with a human TGFBR2 expression construct, TGFB1 acquired the ability to significantly stimulate -1990/+1 mFshb-luc activity. In contrast to LbetaT2 cells, primary murine gonadotropes from young mice (8-10 weeks) contained low, but detectable levels of Tgfbr2 mRNA and these levels increased in older mice (1 yr). A second surprise was the finding that treatment of purified primary gonadotropes with TGFB1 decreased murine Fshb mRNA expression by 95% whereas activin A stimulated expression by 31-fold.
These data indicate that TGFB1-insensitivity in LbetaT2 cells results from a deficiency in Tgfbr2 expression. In primary gonadotropes, however, expression of Tgfbr2 does occur, and its presence permits TGFB1 to inhibit Fshb transcription, whereas activin A stimulates it. These divergent actions of activin A and TGFB1 were unexpected and show that the two ligands may act through distinct pathways to cause opposing biological effects in primary murine gonadotropes.
激活素至少部分地通过诱导促卵泡激素（FSH）β亚基（Fshb）的转录，刺激垂体促性腺细胞中促卵泡激素的合成。几个研究转化小鼠LβT2促性腺细胞的实验室提供的证据表明，激活素通过Smad依赖性和/或Smad非依赖性途径发出信号，类似于转化生长因子β-1（TGFB1）在其他细胞类型中所使用的途径。因此，鉴于这两种配体常见的细胞内信号传导机制，我们研究了TGFBs是否也能在LβT2细胞以及纯化的原代小鼠促性腺细胞中诱导Fshb的转录。
在用激活素A或TGFB1处理的LβT2细胞中，以及在用激活素或TGFB受体转染的细胞中，测量小鼠Fshb启动子-报告基因（-1990/+1 mFshb-luc）的活性。通过蛋白质免疫印迹分析测量配体刺激LβT2细胞中Smad 2和Smad 3磷酸化的能力，并通过逆转录聚合酶链反应评估LβT2细胞和从不同年龄雄性小鼠纯化的原代促性腺细胞中TGFB I型和II型受体的表达。最后，使用定量RT-PCR测量激活素A和TGFB1对纯化的促性腺细胞和全垂体培养物中内源性小鼠Fshb mRNA水平的调节。
激活素A在LβT2细胞中剂量依赖性地刺激-1990/+1 mFshb-luc活性，但TGFB1在高达5 nM 的剂量下没有作用。同样，激活素A而非TGFB1刺激这些细胞中Smad 2和Smad 3的磷酸化。激活素（Acvr1b-T206D）和TGFB（TGFBR1-T204D）I型受体的组成型活性形式强烈刺激-1990/+1 mFshb-luc活性，表明Tgfbr1下游的机制在LβT2细胞中似乎是完整的。对LβT2细胞和成年小鼠全垂体的RT-PCR分析表明，两者均表达Tgfbr1 mRNA，但在LβT2细胞中未检测到Tgfbr2。当用人类TGFBR2表达构建体转染细胞时，TGFB1获得了显著刺激-1990/+1 mFshb-luc活性的能力。与LβT2细胞相反，来自年轻小鼠（8-10周）的原代小鼠促性腺细胞含有低水平但可检测到的Tgfbr2 mRNA，并且这些水平在老年小鼠（1岁）中增加。另一个意外发现是，用TGFB1处理纯化的原代促性腺细胞可使小鼠Fshb mRNA表达降低95%，而激活素A则刺激其表达增加31倍。
这些数据表明，LβT2细胞中对TGFB1不敏感是由于Tgfbr2表达不足。然而，在原代促性腺细胞中确实存在Tgfbr2的表达，其存在使TGFB1能够抑制Fshb转录，而激活素A则刺激其转录。激活素A和TGFB1的这些不同作用是出乎意料的，表明这两种配体可能通过不同的途径在原代小鼠促性腺细胞中产生相反的生物学效应。
