組蛋白甲基轉移酶Suv39h1在糖尿病腎病變的角色

Role of histone methyltransferase Suv39h1 in diabetic nephropathy

林聖軒

糖尿病腎病變 ； Suv39h1 ； 腎間質細胞 ； Suv93h1 ； H3K9me3 ； mesangial cell ； renal fibrosis

高雄醫學大學醫學研究所博士班學位論文

2016年

博士

莊麗月

繁體中文

糖尿病腎病變主要的病理特徵為高血糖、腎小管及腎絲球細胞肥大、腎絲球過濾率異常及細胞外間質堆積，最後造成腎臟纖維化。以表觀遺傳學(epigenetics)的觀點來探討基因調控(Gene regulation) 的研究中，組蛋白修飾作用(histone modification)是調控基因表現的一個重要機轉。而這樣調控基因的修飾作用在糖尿病腎病變領域的研究仍舊稀少且尚未釐清。Suppressor of Variegation 3-9 homolog 1 (Suv39h1)是一個組蛋白甲基轉移酶，將組蛋白histone 3(H3)尾段第九個離胺酸(lysine 9, K9)進行三甲基化(tri-methylations, me3) ，使得DNA與組蛋白纏繞緊密，進而抑制基因表現。先前的研究中指出，在糖尿病老鼠的血管平滑肌細胞中，Suv39h1及H3K9me3的表現量降低；而大量表現Suv39h1可以減緩缺血對心肌細胞造成的傷害。另外，高糖會降低Suv39h1及H3K9me3表現更在其他細胞被報導。因此，在糖尿病腎病變中Suv39h1扮演的角色和影響下游哪些分子為本研究所欲探討的議題。 以小鼠的腎臟間質細胞株 (MES13)作為實驗用細胞株，研究發現高糖、chaetocin (Suv39h1抑制劑)及Suv39h1 siRNA皆會降低Suv39h1並增加fibronectin(細胞外間質之組成蛋白)和p21WAF1(調控細胞週期因子)蛋白質表現量。而高糖及chaetocin也分別會增加fibronectin和p21WAF1的mRNA表現及其轉錄能力。高糖與chaetocin也皆會造成fibronectin和p21WAF1啟動子上H3K9me3降低。此外，過度表現Suv39h1能夠降低高糖誘發的fibronectin和p21WAF1蛋白質及mRNA表現量增加，同時亦能減緩高糖刺激造成的細胞肥大現象。另一方面，大量表現Suv39h1也能夠回復高糖使得fibronectin和p21WAF1啟動子上H3K9me3的減少。進一步的，發現LY294002或dominant-negative phosphoinositide 3-kinase (PI3K) mutant (Δp85)都能回復高糖所降低的Suv39h1以及高糖所增加的fibronectin和p21WAF1蛋白質的表現。在動物實驗方面，以鏈脲佐菌素(streptozotocin, STZ)誘發第一型糖尿病大鼠的動物實驗中，Suv39h1的mRNA與蛋白質表現量都比控制組低。另一方面，KMUP-1(7-{2-[4-(2-chlorobenzene)piperazinyl]ethyl-1,3-dime-thyl-xanthine)，為一個茶鹼衍生物的藥物，本實驗室最新研究發現能用來改善腎臟纖維化的現象。令人驚訝的是，高糖抑制Suv39h1的表現在加入KMUP-1後能回復此被減弱的現象，更凸顯了Suv39h1在治療糖尿病腎臟纖維化的角色。 總和以上結果，在腎臟間質細胞中，高糖經由活化PI3K pathway來抑制Suv39h1並且增加fibronectin和p21WAF1的表現。大量表現Suv39h1會透過回復fibronectin和p21WAF1啟動子上因為高糖所降低的H3K9me3，來抑制基因表現，進而減緩高糖刺激造成fibronectin和p21WAF1增加及細胞肥大的現象。因此，Suv39h1也許可當作一個治療糖尿病腎病變的新標靶分子。

Diabetic nephropathy (DN) is characterized by hyperglycemia, cell hypertrophy, accumulation of extracellular matrix (ECM), which result in renal fibrosis. The epigenetic histone modifications that regulate the genes associated with DN remain unknown. Suppressor of variegation 3-9 homolog 1 (Suv39h1) is a histone methyltransferase that trimethylates lysine 9 of histone H3 (H3K9me3), which results in gene silencing. A previous study found that H3K9me3 and Suv39h1 were decreased in diabetic mouse vascular smooth muscle cells whereas Suv39h1 overexpression attenuated ischemic myocardial injury. Moreover, high glucose (HG) decreased H3K9me3 and Suv39h1 levels in some cells. Thus, we studied the roles of Suv39h1 in HG-induced effects in MES13 (mouse mesangial) cells. We found that HG, chaetocin (a Suv39h1 inhibitor) and Suv39h1 siRNA decreased protein levels of Suv39h1 while increasing fibronectin and p21WAF1 protein levels. HG increased mRNA while chaetocin increased transcription of fibronectin and p21WAF1genes. Both HG and chaetocin decreased histone H3K9me3 levels at the promoters of fibronectin and p21WAF1 genes. Additionally, Suv39h1 overexpression attenuated HG-induced fibronectin and p21WAF1 mRNA and protein expressions while attenuating HG-induced cell hypertrophy. Suv39h1 overexpression also attenuated HG-suppressed histone H3K9me3 levels at the promoters of fibronectin and p21WAF1 genes. Moreover, LY294002 or the dominant-negative phosphoinositide 3-kinase (PI3K) mutant (Δp85) attenuated HG-decreased Suv39h1 and HG-induced fibronectin and p21WAF1 protein expressions. In the animal model, renal expression of Suv39h1 mRNA or protein was decreased in the streptozotocin-induced-8 weeks diabetic rats. On the other hand, KMUP-1(7-{2-[4-(2-chlorobenzene)piperazi-nyl]ethyl-1,3-dime-thyl-xanthine), a xanthine-based chemical compound, which ameliorated renal fibrosis effectively according our latest investigation reversed HG-decreased Suv39h1 expression. We concluded that HG decreased Suv39h1 via the PI3K pathway in mesangial cells. Inhibition of Suv39h1 increased fibronectin and p21WAF1 expressions. Moreover, Suv39h1 overexpression attenuated HG-induced fibronectin and p21WAF1 expressions and cell hypertrophy while attenuating HG-suppressed histone H3K9me3 levels at the promoters of fibronectin and p21WAF1 genes. Thus, Suv39h1 may provide a new renoprotective slight in diabetic nephropathy.