Regulation of β-Casein Gene Expression by Octamer Transcription Factors and Utilization of β-Casein Gene Promoter to Produce Recombinant Human Proinsulin in the Transgenic Milk

摘要

β-Casein is a major milk protein, which is synthesized in mammary alveolar secretory epithelial cells (MECs) upon the stimulation of lactogenic hormones, mainly prolactin and glucocorticoids (HP). Previous studies revealed that the proximal promoter (-258 bp to +7 bp) of the β-casein gene is sufficient for induction of the promoter activity by HP. This proximal region contains the binding sites for the signal transducer and activator of transcription 5 (STAT5), glucocorticoid receptor (GR), and octamer transcription factors (Oct). STAT5 and GR are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. This study investigated the functions of Oct-1 and Oct-2 in HP induction of β-casein gene expression. By transiently transfection experiment, we showed that individual overexpression of Oct-1 and Oct-2 further enhanced HP-induced β-casein promoter activity, respectively, while Oct-1 and Oct-2 knockdown significantly inhibited the HP-induced β-casein promoter activity, respectively. HP rapidly induced the binding of both Oct-1 and Oct-2 to the β-casein promoter, and this induction was not mediated by either increasing their expression or inducing their translocation to the nucleus. In MECs, Oct-2 was found to physically interact with Oct-1 regardless of HP treatment. However, HP induced physical interactions of Oct-1 or Oct-2 with both STAT5 and GR. Although the interaction between Oct-1 and Oct-2 did not synergistically stimulate HP-induced β-casein gene promoter activity, the synergistic effect was observed for the interactions of Oct-1 or Oct-2 with STAT5 and GR. The interactions of Oct-1 with STAT5 and GR enhanced or stabilized the binding of STAT5 and GR to the promoter. Abolishing the interaction between Oct-1 and STAT5 significantly reduced the hormonal induction of β-casein gene transcription. Thus, our study indicates that HP activate β-casein gene expression by inducing the physical interactions of Oct-1 and Oct-2 with STAT5 and GR in mouse MECs.There is a high and increasing demand for insulin because of the rapid increase in diabetes incidence worldwide. However, the current manufacturing capacities can barely meet the increasing global demand for insulin, and the cost of insulin production keeps rising. The mammary glands of dairy animals have been regarded as ideal bioreactors for mass production of therapeutically important human proteins. We tested the feasibility of producing human proinsulin in the milk of transgenic mice. In this study, four lines of transgenic mice were generated to harbor the human insulin gene driven by the goat β-casein gene promoter. The recombinant human proinsulin was detected in the milk by Western blotting and enzyme-linked immunosorbent assay. The highest expression level of human proinsulin was as high as 8.1 μg/µl in milk of transgenic mice at mid-lactation. The expression of the transgene was only detected in the mammary gland during lactation. The transgene expression profile throughout lactation resembled the milk yield curve, with higher expression level at middle lactation and lower expression level at early and late lactation. The blood glucose and insulin levels and major milk compositions of transgenic mice were not changed. The mature insulin derived from the milk proinsulin retained biological activity. Thus, our study indicates that it is practical to produce high levels of human proinsulin in the milk of dairy animals, such as dairy cattle and goat.