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) 是 caveolae 細胞膜構造中主要的膜蛋白。Cav-1 蛋白質具有多種功能，參與膽固醇的結合與運輸、抑制訊息傳遞以及減緩細胞不正常轉型或癌化現象。小鼠於懷孕末期至泌乳時期，發現 Cav-1 在乳腺中的表現受到明顯抑制；除此之外，於 Cav-1 基因剔除小鼠中發現其乳腺有提早發育及提早泌乳的現象。齧齒類動物自懷孕末期開始，乳腺細胞會藉由提高胰島素受體的磷酸酶活性而增加對胰島素的敏感度。胰島素為一種泌乳內泌素，於哺乳動物的泌乳時期中扮演一重要角色，主要經由活化 PI3-kinase/Akt 訊息傳遞路徑進而調控乳腺細胞功能。胰島素被認為具有維持乳腺上皮細胞存活的能力。先前研究指出，在小鼠肌纖維母細胞 C2C12 及能夠穩定表現人類胰島素受體的 NIH 3T3 細胞中，胰島素能夠抑制 Cav-1 RNA 及蛋白質的表現量。若抑制細胞中 PI3-kinase/Akt 訊息傳遞路徑則使 Cav-1 表現量上升。然而在乳腺上皮細胞中，對於胰島素調控 Cav-1 表現的分子機制仍存在著許多未知。 目前山羊 Cav-1 啟動子的核苷酸序列尚未被發表，因此根據綿羊 Cav-1 啟動子包含部分 5’-UTR (2497 bp) 去設計引子，再透過 nested-PCR 以選殖出山羊 Cav-1 啟動子之序列 (Cav-1 F2R3, 2793 bp)。比較山羊與人類以及山羊與小鼠 Cav-1 基因序列 (自起始密碼至上游 975 個核苷酸序列，Cav-1 FR4)，發現相似度分別是 74.2% 及 64.3%。更進一步對山羊 Cav-1 進行分析，透過 5’-RACE 試驗，經序列比對分析後推測，山羊 Cav-1 mRNA 於 5’ 端含有 61 bp 之 5’-UTR。為了觀察胰島素於乳腺上皮細胞中是否具有調控 Cav-1 基因轉錄活性之能力，而構築不同長度的Cav-1啟動子片段 (Cav-1 F2R3 及 Cav-1 FR4) 於帶有螢火蟲冷光素酶報導基因的質體中，接著利用 Dual-Glo Luciferase 系統檢測 Cav-1 啟動子在不朽化山羊乳腺上皮細胞株 (CMEC) 中之活性。將帶有 Cav-1 啟動子之質體與 pGL4.83 TK 共同轉染入 CMEC 中，並於胰島素 (5.0 μg/ml) 處理細胞 24 小時後偵測其冷光之活性。結果顯示，Cav-1 F2R3 啟動子的活性從對照組的 16.62 下降至 10.04 (F Luc/R Luc ratio, p<0.05)，而 Cav-1 FR4 啟動子的活性也呈現出相似的結果，從 5.40 下降至 3.47 (F Luc/R Luc ratio, p<0.05)。為了探討胰島素抑制山羊 Cav-1 啟動子的活性是否經由 PI3-K/Akt 訊息傳導路徑所影響，於是將 Cav-1 FR4啟動子及穩定維持活化態的 Akt 共同轉染入 CMEC 之中，經 24 小時之後發現啟動子的活性明顯下降。反觀蛋白質層面，CMEC 受到胰島素 (5.0 μg/mL) 處理經 24 及 48 小時之後，內源性的 Cav-1 蛋白質表現量下降，若改以 PI3-K 訊息路徑抑制劑 LY294002 進行處理，則 Cav-1 蛋白質的表現量降低的情形則受到抑制。 綜上所述，本研究成功選殖山羊 Cav-1 啟動子，並推測轉錄起始點位於轉譯起始點上游第 61 個核苷酸序列中。胰島素可經由 PI3-K/Akt 訊息傳遞路徑以抑制 Cav-1 啟動子活性與蛋白質的表現。未來研究可朝向分析山羊 Cav-1 啟動子上受到胰島素調控的序列 (insulin response elements)，以及找出胰島素訊息傳遞路徑中，其下游具有調控 Cav-1 啟動子活性之轉錄因子。

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.