山羊 caveolin-1 啟動子活性及其於山羊乳腺上皮細胞中受胰島素調控之探討

Abstract

Caveolin-1 (Cav-1) is a major integral membrane protein in caveolae of cell membrane. Cav-1 possesses versatile functions, such as cholesterol binding and transport, inhibition of signaling molecules, and suppression of oncogenic transformation. In mouse mammary glands, Cav-1 expression is dramatically down-regulated during late pregnancy and lactation. In addition, Cav-1 null mice showed accelerated mammary gland development and premature lactation during pregnancy. In preparation for lactation, the development of rodent mammary gland increases insulin sensitivity during late pregnancy due to an augmented kinase activity of the insulin receptor. Insulin is an essential lactogenic hormone, plays a central role during lactation via activation the PI3-kinase/Akt pathway. Insulin was also assumed to perform a role of maintenance of mammary epithelial cell survival. In previous study, the RNA and protein levels of Cav-1 were down-regulated by treatment with insulin and were increased by treatment with LY294002 in C2C12 mouse myoblasts and NIH 3T3 that were stably expressing the human insulin receptor. The molecular mechanism of regulating Cav-1 expression by insulin in mammary gland remains largely unknown. Because the caprine Cav-1 promoter sequence is unpublished, the Nested-PCR primers were designed to obtain the sequence based on ovis Cav-1 promoter and its partial 5’-UTR (2497 bp). The caprine Cav-1 promoter F2R3 (2739 bp) included partial 5’-UTR was cloned. To compare 975 bases of the upstream regulatory region of the Cav-1 promoters (Cav-1 promoter FR4) between caprine versus human and mouse, the sequence similarity is 74.2% and 64.3%, respectively. Furthermore, to analyze the correct transcription start site of caprine Cav-1 gene, the rapid amplification of cDNA ends (5’-RACE) has been done. As 5’-RACE products, by the sequence analysis, products containing 61 bp of 5’-UTR were obtained from caprine Cav-1 mRNA. In order to confirm whether the insulin regulated the activity of Cav-1 gene, the plasmids possessed caprine Cav-1 promoter with different lengths (Cav-1 F2R3 and Cav-1 FR4) drove firefly luciferase reporter gene were constructed, and the promoters activity were examined in immortalized caprine mammary epithelial cells (CMEC) by Dual-Glo Luciferase system. The Cav-1 promoter constructs and pGL4.83-TK were transiently co-transfected into CMECs. Cells were treated with insulin (5 μg/mL) for 24 hours after transfected, luciferase activity was measured. The results have shown that Cav-1 promoter F2R3 activity was decreased from 16.62 to 10.04 ( F Luc/R Luc ratio, p<0.05), and Cav-1 promoter FR4 activity has the similar result from 5.40 to 3.47 (F Luc/R Luc ratio, p<0.05). To investigate whether caprine Cav-1 promoter activity could be regulated by insulin through PI3-K/Akt pathway, CMECs were co-transfected Cav-1 promoter FR4 and constitutively active form of Akt for 24 hours, the promoter activity was remarkable decreased. In protein level, after treatment with insulin (5 μg/mL) for 24 and 48 hours, the endogenous Cav-1 expression was down-regulated, but increased when blocked the PI3-K/Akt pathway by PI3-kinase inhibitor, LY294002. Taken together, this study succeeded in cloning caprine Cav-1 promoter and indicated that transcription start site contained 61 bp of upstream sequence from start codon. Insulin signaling decreases of Cav-1 promoter activity and protein expression partially via PI3-K/Akt pathway. Therefore, the future work will focus on analyzing the insulin response elements in caprine Cav-1 promoter, and investigating the current transcription factor regulating the promoter activity.