山羊 Stat5a 轉錄活化區域變異影響 β-酪蛋白啟動子活性之探討

Abstract

訊息傳導及轉錄活化因子 5a (signal transducers and activators of transcription 5a, Stat5a) 為 Stat 家族的一員，具有感知外在環境的變化以調控特定基因表現之能力。哺乳動物乳腺的發育及泌乳受到泌乳素 (prolactin, PRL) 所調控，在懷孕後期及泌乳時期，PRL 的分泌會刺激乳腺泡的生成以及乳蛋白基因的表現。在 PRL 的訊息傳遞路徑中，Stat5a 為主要的調控因子。PRL 經由乳腺上皮細胞上之 PRL 受器 (prolactin receptor, PRLR)，活化細胞質中的酪胺酸激酶 (janus kinase 2, Jak2)，活化後的 Jak2 會吸引 Stat5a 前來，並且磷酸化 Stat5a 上的酪胺酸殘基 (tyrosine residue, Y) Y694。Y694 磷酸化之 Stat5a 可形成 Stat5-Stat5 二聚體 (dimer)，並進入細胞核內，與乳蛋白基因啟動子 (promoter) 中的 GAS (gamma interferon activation site) 序列結合，活化乳蛋白的轉錄作用。由於 Stat5a 之於乳腺以及泌乳的角色非常重要，因此推測不同物種之間的 Stat5a 序列變異極有可能是造成各物種泌乳特性差異的主要原因。本研究比對山羊、牛、豬、人、大鼠、小鼠之 STAT5a cDNA 以及胺基酸序列，選取在 Stat5a 轉錄活化區域中物種間序列具特異性之胺基酸位置進行突變，探討此區域之不同位置突變對於 β-酪蛋白 (β-casein) 啟動子活性之影響。 。。自初代培養 (primary culture) 之山羊乳腺上皮細胞中選殖出山羊 STAT5a (caprine STAT5a, cSTAT5a) cDNA。將山羊之 Stat5a 胺基酸序列與牛、豬、人、大鼠、小鼠進行比對後發現 Stat5a 在物種間保留性很高，序列相似度高達 95% 以上，其中又以山羊及牛的序列相似程度最高，有 98.2%。比較 Stat5a 蛋白質上的 7 個功能性區域 (functional domain)，可發現位於羧基端的轉錄活化區域 (transactivation domain) 物種間差異性最大。值得注意的是，位在轉錄活化區域中的第 780 胺基酸位置，上述物種中只有山羊和牛為脯胺酸 (proline, P) (P780)，其餘物種皆為可被磷酸化之絲胺酸 (serine, S) (S780)。為了確認牛之 P780 並非個體差異，萃取 40 頭荷蘭牛之 genomic DNA 進行序列分析後發現序列結果皆為 P780。 。。以不朽化山羊乳腺上皮細胞株 (CMC) 作為研究平台，利用 Jak2 以及 MEK1 (Meiosis-specific serine/threonine protein kinase) 抑制劑與 PRL 共同處理 CMC，以確認 PRL 於 CMC 中的訊息傳導路徑。結果顯示當 Jak2 被抑制時會抑制 cStat5a 之磷酸化，並且使山羊 β-casein 啟動子活性下降；而處理 MEK1 抑制劑時，cStat5a 之磷酸化並不會受到影響，並且山羊 β-casein 啟動子活性顯著增加。為了解 cStat5a 對於山羊乳蛋白表現之影響，構築 5 種不同的 cStat5a 突變質體：Y694A、P780D、P780S、774/778/780A、774/778/780D。將其分別與 pGL4.18-F1B1 以及 pGL4.83-TK 共同轉染至 CMC 中，以 PRL 進行誘導後觀察不同的 cStat5a 突變對於 β-casein 啟動子活性之影響。結果發現，誘導後 12 小時 P780S 突變組的 β-casein 啟動子活性顯著高於控制組，而 Y694A 突變組則具有最低的 β-casein 啟動子活性。 。。綜上所述，CMC 中 PRL 的傳遞路徑以 Jak2/Stat5a 為主要，當抑制 MEK1 的訊息路徑時會增加山羊 β-casein 啟動子活性，推測 MEK1 路徑中具有能影響 β-casein 啟動子活性之因子存在。另外，山羊和牛皆具有與其他物種相異之 P780，將此位置及 Y694 分別突變後會影響 β-casein 啟動子活性，可知 P780 和 Y694 對於 Stat5a 之轉錄活化功能非常重要。已知 Y694 透過磷酸化來調控 Stat5a 的活性，而 P780 是如何去調控，亦或者是和其他胺基酸共同調控，則需要進一步的研究來釐清。

Signal transducers and activators of transcription 5a (Stat5a) is a member of Stat family which has the ability to sense and transmit environmental cues to regulate specific gene expression in the nucleus. During late gestation to lactation, the secretion of prolactin (PRL) stimulates the formation of alveolus and milk protein translation. Stat5a is the critical mediator in the PRL signaling pathway and regulates mammary gland development and lactation. PRL binds to prolactin receptor (PRLR) on mammary epithelial cell, leading dimerization of PRLRs and activates Janus kinase 2 (Jak2), then recruits cytoplasmic Stat5a to the Jak2. Stat5a was phosphorylated on tyrosine residue (Y694) by Jak2. Activated Stat5a forms Stat5-Stat5 dimer immediately and translocated into nucleus binds to specific DNA motif called GAS (gamma interferon activation site) element on milk protein promoter and initiate gene transcription. Since the pivotal role of Stat5a on lactation of mammary gland, the milk protein production difference between species may cause by the diversity of Stat5a sequence. In order to find the unique sites of STAT5a sequence among species, this study compared STAT5a cDNAs of caprine, bovine, pig, human, mouse, and rat and pick the variational sites to do mutation. Then study on the effects of different Stat5a mutation on β-casein promoter activity. The caprine STAT5a (cSTAT5a) cDNA was cloned from primary caprine mammary gland. The similarity of amino acid sequences among caprine, bovine, pig, human, mouse and rat is about 95%. Among species mentioned, caprine and bovine have most similar amino acid sequences, 98.2%. Stat5a protein possesses 7 functional domains, each has different structures and functions. Major variation was found in transactivation domain. It is interesting that both caprine and bovine have proline residue at 780 amino acid (P780) while other species are serine (S780) and were confirmed would be phosphorylated by previous study. In order to confirm proline residue in bovine is not due to individual difference, genomic DNA sequences from 40 Holstein cows were analyzed and found out all of them contained the same P780 amino acid sequences like caprine. In order to investigate the effects of cStat5a on milk protein transcription, using the immortalized caprine mammary epithelial cells (CMC) as experimental model. Treated CMC with Jak2 or MEK1 inhibitor to see the PRL signaling pathway on CMC. The results showed that β-casein promoter activity decreased under Jak2 inhibitor treatment and increased when treated with MEK1 inhibitor. Phosphorylation of Y694 also decreased under Jak2 inhibitor treatment. Then CMC was co-transfected β-casein promoter drove luciferase report gene and exogenous cStat5a. Five different mutants of cStat5a: Y694A, P780D, P780S, 774/778/780A and 774/778/780D were constructed. The different effects of mutated cStat5a on β-casein promoter activity were determined by exogenous expression of different mutated cStat5a in CMC and luminescence system was applied to quantitate the β-casein promoter activity. The result showed that after insulin-hydrocortisone-PRL (IHP) induction for 12 hours, P780S cStat5a had the highest induction ratio than wild type (WT) cStat5a, and Y694A cStat5a had the lowest β-casein promoter activity. Other mutations of cStat5a kept the same β-casein promoter activity with WT. Taken together, this study indicated that Jak2/Stat5a is the major downstream singling pathway of PRL. And there were some factors in MEK1 singling pathway which can regulate β-casein promoter activity. On the other hand, both of the amino acid P780 and Y694 in caprine and bovine are important for transcription activation of Stat5a in CMC. Future work is to focus on investigating the role of P780 in controlling the activity of caprine β-casein promoter and understanding why caprine and bovine which should have more activation state of Stat5a did not go the same way of evolution.