二十二碳六烯酸透過轉錄調控抑制結合四素的基因表達

Abstract

結合四素 (tetranectin) 會參與溶血作用 (fibrinolysis)，並與骨母細胞分化(osteoblast differentiation) 有關。其可藉由與組織型纖維蛋白溶酶原激活物 (tissue-type plasminogen activator) 結合，將無活性的纖溶酶原(plasminogen) 轉變成具活性的纖溶酶 (plasmin) 以裂解纖維蛋白(fibrin) (Clemmensen et al., 1986)。然而，近來本研究室與他人分別做出新奇發現而指出結合四素竟為人類初代脂肪細胞所分泌之脂泌素 (adipokine)，並與脂肪細胞生成 (adipogenesis) 有關 (丁博士研究室未發表資料)。 為瞭解結合四素在脂肪代謝上所扮演的角色及其基因調控機制，本試驗成功選殖豬隻結合四素的部分互補DNA序列 (463 bp)，以其設計引子並執行即時量化聚合脢連鎖反應(Real-time quantitative PCR) 以偵測先前收集之三十頭去勢保育豬組織樣本。三十頭保育豬隨機分配至三種飼糧處理組，每組十頭豬分別餵飼30天之2%大豆油 (soybean oil)、二十二碳六烯酸油 (DHA oil) 與牛油 (beef tallow) 之結合四素基因表現量。結果發現二十二碳六烯酸油飼糧處理降低結合四素在豬隻脂肪組織中之基因表現量 (P < 0.05)，在肝臟與肺臟中雖不具統計顯著性，但亦具相似之趨勢 (P = 0.08)。 再者，為確認二十二碳六烯酸油可降低結合四素的表現量，本試驗進一步分離豬隻初代脂肪細胞並分析100 μΜ二十二碳六烯酸及棕櫚油酸 (palmitic acid) 對結合四素表現量之影響。同樣地，結果顯示二十二碳六烯酸確實可降低其基因表現量，這些研究結果指出二十二碳六烯酸似可藉由轉錄調控的方式影響結合四素之基因表現量。 最後，為瞭解二十二碳六烯酸對結合四素轉錄調控的機制，本試驗成功選殖長度為1956 bp之豬隻結合四素假定啟動子 (putative promoter)，並藉由網路線上軟體TFsitescan預測此啟動子之轉錄因子調結結合位 (regulatory element) 並設計逐列截除啟動子片段所需之引子。並將此啟動子片段轉殖 (subclone) 至冷光酶報導載體 (luciferase reporter vector) 並轉染 (transfect) 至 HEK293T細胞以分析二十二碳六烯酸對結合四素啟動子轉錄活性之影響。結果顯示結合四素啟動子上有兩個區段會受二十二碳六烯酸所抑制，分別是 -1956 至 -1576 與 -559 至 -310處。同時網路線上軟體也預測另一膽固醇調節因子結合位 (Sterol regulatory element, SRE) 落於 -825 至 -559區間。若進一步將此膽固醇調節因子結合位突變則可削弱二十二碳六烯酸之抑制作用，此結果表示二十二碳六烯酸可藉由此調節因子抑制結合四素之基因表現。 綜合上述，本試驗研究結果顯示結合四素在脂肪細胞分化中扮演相關角色；而具抗肥胖與代謝症候群之二十二碳六烯酸則可藉由結合四素啟動子上之膽固醇調節因子結合位，抑制其基因表現量，但連結二十二碳六烯酸至膽固醇調節因子間之詳細訊息傳導機制則有待未來進一步之探討。

While tetranectin (TN) is a plasminogen-binding protein originally found to be involved in fibrinolysis and bone formation, it was recently identified by our group and others as an adipokine in human adipocyte culture media and implicated in adipogenesis and lipid metabolism. To elucidate its role and regulation in lipid metabolism, we firstly cloned the partial cDNA of porcine TN and used the sequence information to analyze the previously collected tissue samples of weaned piglets fed with 2% soybean oil, DHA oil or beef tallow (as-fed basis) by real-time qPCR and Western blotting analysis. Our results showed that the expression of TN was reduced in the adipose (P < 0.05), liver (P = 0.08) and lung (P = 0.08), from DHA-fed group (with lowered serum levels of triacylglycerol and cholesterol) compared to the beef tallow group, which was confirmed in primary porcine adipocyte culture treated with DHA. These results indicated that TN is involved in lipid metabolism and negatively regulated by DHA. To further investigate the transcriptional mechanism underlying DHA-suppressed TN expression, we cloned a 1956-bp putative porcine TN promoter by using the NCBI genomic sequences and “TFsitescan” software, transfected 293T cells with the luciferase gene constructs of serially-deleted TN promoter fragments and tested its transactivating activities by luciferase assay. Interestingly, two DHA-sensitive fragments from nucleotide position -1956 to -1576 and -559 to -310 were identified. In addition, a sterol regulatory element (SRE) was predicted within -825 to -559 by an on-line website program. Mutation on this SRE reduced its transactivating activity and rendered it resistant to DHA’s inhibitory effect, indicating its potential role in mediating DHA’s action. In conclusion, our current study suggests that TN plays a role in adipogenesis and lipid metabolism, and, for the first time, identified an SRE in TN’s promoter region, which is responsible for the negative effects of DHA on TN expression. The upstream events relaying DHA to SRE-mediated TN gene expression await future investigation.