Department of Biological Sciences, University of Arkansas, SCEN 601, Fayetteville, AR, 72701, USA.
Department of Biological Sciences, University of Arkansas, SCEN 601, Fayetteville, AR, 72701, USA; Lyon College, Math and Science Department, 2300 Highland Rd, Batesville, AR, 72501, USA.
Mol Cell Endocrinol. 2018 Dec 5;477:163-171. doi: 10.1016/j.mce.2018.06.014. Epub 2018 Jun 28.
In some freshwater fish species, the control of gill Na, Cl cotransporter (Ncc2b) by prolactin appears to be instrumental to ionic homeostasis. This study was carried out to examine the signaling pathways involved in prolactin-mediated salt retention using gill explants from Japanese medaka (Oryzias latipes). Ovine prolactin induced a concentration-dependent stimulation of ncc2b with significant effects of 10, 100 and 1000 ng of hormone per mL media (2-6 fold). To understand the molecular mechanisms mediating prolactin control of gill function, we analyzed effects on signaling pathways known to be involved in the hormones action in other systems, namely Stat5, Akt and Erk1/2. Their activation was examined in a time course and concentration response experiment. Prolactin (1 μg mL) induced a rapid phosphorylation (stimulation) of Stat5 (10 min) that reached a plateau after 30 min and was maintained for at least 120 min. The effect of prolactin on Stat5 phosphorylation was concentration-dependent (4-12 fold). No activation of Akt or Erk1/2 was observed in either experiment. The Stat5 activation was further investigated in localization studies that demonstrated strong nuclear expression of phosphorylated Stat5 in prolactin-treated gill ionocytes. Using specific inhibitors, we analyzed the signalling pathways mediating prolactin induction of gill ncc2b. Co-incubation experiments showed that Stat5 inhibition blocked prolactin's stimulation of ncc2b expression, while PI3K-Akt and Mek1/2-Erk1/2 pathway inhibitors had no effect. These findings show that ncc2b expression is dependent on prolactin's downstream activation of Stat5 and its subsequent nuclear translocation within branchial ionocytes.
在一些淡水鱼类物种中，催乳素对鳃 Na+、Cl-共转运蛋白（Ncc2b）的控制似乎对离子稳态很重要。本研究使用来自日本青鳉（Oryzias latipes）的鳃外植体，研究了催乳素介导的盐潴留中涉及的信号通路。绵羊催乳素诱导 ncc2b 的浓度依赖性刺激，激素浓度为 10、100 和 1000ng/mL 时具有显著效果（2-6 倍）。为了了解催乳素调节鳃功能的分子机制，我们分析了与其他系统中激素作用有关的信号通路的影响，即 Stat5、Akt 和 Erk1/2。在时间过程和浓度反应实验中检查了它们的激活。催乳素（1μg/mL）诱导 Stat5 的快速磷酸化（刺激）（10 分钟），在 30 分钟后达到平台期，并至少维持 120 分钟。催乳素对 Stat5 磷酸化的影响呈浓度依赖性（4-12 倍）。在这两个实验中都没有观察到 Akt 或 Erk1/2 的激活。进一步的定位研究表明，Stat5 激活在催乳素处理的鳃离子细胞中观察到强烈的磷酸化 Stat5 核表达。使用特异性抑制剂，我们分析了介导催乳素诱导鳃 Ncc2b 的信号通路。共孵育实验表明，Stat5 抑制阻断了催乳素对 Ncc2b 表达的刺激，而 PI3K-Akt 和 Mek1/2-Erk1/2 途径抑制剂没有影响。这些发现表明，Ncc2b 的表达依赖于催乳素对 Stat5 的下游激活及其随后在鳃离子细胞内的核转位。
