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

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.