Wang A, Pokhrel B, Hernandez G Perez, Jiang H
School of Animal Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
School of Animal Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
J Dairy Sci. 2024 Dec;107(12):11139-11148. doi: 10.3168/jds.2024-24905. Epub 2024 Aug 3.
Cow milk is rich in protein. Major cow milk proteins include α casein (CSN1S1), α casein (CSN1S2), β casein (CSN2), κ casein (CSN3), lactalbumin α (LALBA), and β-LG. These milk proteins are produced through gene expression in the mammary epithelial cells. Little is known about the molecular mechanism that mediates the expression of milk protein genes in cows. In this study, we tested the hypothesis that the expression of milk protein genes in cows is mediated by STAT5A, a transcription factor that is induced to bind and activate the transcription of target genes by extracellular signals such as prolactin. To circumvent the need for prolactin-responsive bovine mammary epithelial cells, we generated a plasmid that expresses a constitutively active bovine STAT5A variant, bSTAT5ACA. Transfection of the bovine mammary epithelial cell line MAC-T cells with the bSTAT5ACA expression plasmid caused a more than 100,000-fold and 600-fold increase in the expression of CSN1S1 and CSN1S2 mRNAs, respectively, compared with transfection of the wild-type bovine STAT5A (bSTAT5A) expression plasmid. Transfection of bSTAT5ACA, however, had no significant effect on the expression of CSN2, CSN3, LALBA, or LGB mRNA in MAC-T cells. Transfection of bSTAT5ACA caused a more than 260-fold and 120-fold increase in the expression of a luciferase reporter gene linked to the bovine CSN1S1 and CSN1S2 promoters in MAC-T cells, respectively, compared with that of bSTAT5A. The bovine CSN1S1 and CSN1S2 promoters each contain a putative STAT5 binding site, and gel-shift and super-shift assays confirmed bSTAT5ACA binding to both sites. These results together suggest that STAT5A plays a major role in regulating the expression of CSN1S1 and CSN1S2 genes in the bovine mammary epithelial cells and that STAT5A regulates the expression of these genes at least in part by binding to the STAT5 binding sites in their promoter regions. These results also suggest that STAT5A does not play a major role in regulating the expression of other major milk protein genes.
牛奶富含蛋白质。主要的牛奶蛋白包括α-酪蛋白（CSN1S1）、α-酪蛋白（CSN1S2）、β-酪蛋白（CSN2）、κ-酪蛋白（CSN3）、α-乳白蛋白（LALBA）和β-乳球蛋白（β-LG）。这些乳蛋白是通过乳腺上皮细胞中的基因表达产生的。关于介导奶牛乳蛋白基因表达的分子机制知之甚少。在本研究中，我们检验了这样一个假设：奶牛乳蛋白基因的表达是由STAT5A介导的，STAT5A是一种转录因子，可被催乳素等细胞外信号诱导结合并激活靶基因的转录。为了避免对催乳素反应性牛乳腺上皮细胞的需求，我们构建了一个表达组成型活性牛STAT5A变体bSTAT5ACA的质粒。与野生型牛STAT5A（bSTAT5A）表达质粒转染相比，用bSTAT5ACA表达质粒转染牛乳腺上皮细胞系MAC-T细胞，分别导致CSN1S1和CSN1S2 mRNA表达增加了100000倍以上和600倍。然而，bSTAT5ACA转染对MAC-T细胞中CSN2、CSN3、LALBA或LGB mRNA的表达没有显著影响。与bSTAT5A相比，bSTAT5ACA转染分别使与牛CSN1S1和CSN1S2启动子相连的荧光素酶报告基因在MAC-T细胞中的表达增加了260倍以上和120倍。牛CSN1S1和CSN1S2启动子各自包含一个假定的STAT5结合位点，凝胶迁移和超迁移分析证实bSTAT5ACA与这两个位点结合。这些结果共同表明，STAT5A在调节牛乳腺上皮细胞中CSN1S1和CSN1S2基因的表达中起主要作用，并且STAT5A至少部分地通过结合其启动子区域中的STAT5结合位点来调节这些基因的表达。这些结果还表明，STAT5A在调节其他主要乳蛋白基因的表达中不发挥主要作用。
