探討馬兜鈴酸與BLCAP基因對於人類腎臟細胞株的SMAD訊息傳遞路徑之影響

Abstract

馬兜鈴酸 (Aristolochic acid, AA) 為馬兜鈴科植物的天然成分，主要由馬兜鈴酸 Ⅰ (AA Ⅰ) 和馬兜鈴酸 Ⅱ (AA Ⅱ) 所組成，腎臟纖維化則是馬兜鈴酸腎病變的典型特徵。Bladder cancer associated protein (BLCAP) 在許多癌症中被認為扮演抑癌基因 (tumor suppressor gene) 的角色，然而目前對於BLCAP基因的相關生理功能及調控機制仍不清楚。本研究的主要目的在於利用人類胚胎腎臟細胞株 (HEK293 cell lines) 作為實驗模式，探討馬兜鈴酸I是否經由BLCAP基因調控Gremlin的表現，並且進一步探討馬兜鈴酸I和BLCAP基因對Smad訊息傳遞路徑的影響。 首先利用馬兜鈴酸I處理HEK293細胞24小時，發現細胞內Gremlin mRNA表現量上升，而BMP-7 mRNA和下游p-Smad1/5/8蛋白質以及Id2 mRNA的表現量下降，同時馬兜鈴酸I也會抑制BLCAP mRNA的含量。將帶有pLKO.1-shBLCAP質體的慢病毒感染HEK293細胞株 (shBLCAP/HEK293) 後，則發現抑制BLCAP基因的表現與否並不會影響Gremlin和BMP-7 mRNA訊號強弱，推測馬兜鈴酸 I在誘導腎臟纖維化的過程中，並不是經由BLCAP調控Gremlin的表現。 在shBLCAP/HEK293細胞中Smad1/5/8的蛋白質磷酸化，以及其下游的Id2 mRNA表現量皆會減少，此外BMP type-Ⅱ receptor (BMPR2)的mRNA表現量與控制組相比也顯著下降，推測抑制BLCAP所導致的Smad1/5/8磷酸化作用受阻可能是源於BMPR2 mRNA表現量下降的關係。另一方面，shBLCAP/HEK293細胞的生長速度明顯增加，而抑制BLCAP基因表現會誘導TGF-β1 mRNA上升和Smad2/3蛋白質磷酸化，其下游目標基因PAI-1、CTGF mRNA和microRNA-21 (miR-21)的表現量也有所增加，顯示BLCAP基因會負向調控TGF-β1/Smad2/3訊息傳遞路徑。利用細胞轉染的方式將miR-21 inhibitor送入shBLCAP/HEK293細胞中，確認miR-21表現下降，而且會令減緩抑制BLCAP基因所誘導的細胞生長速度，同時也發現抑制細胞中miR-21增加能夠回復BMPR2 mRNA的含量。 總和以上實驗結果顯示在HEK293細胞株中，馬兜鈴酸I並不是藉由BLCAP誘導Gremlin表現來干擾其相關BMP-7/Smad1/5/8訊息傳遞路徑，BLCAP基因主要是透過抑制BMPR2的表現來調控Smad1/5/8訊息傳遞路徑，同時對TGF-β1/Smad2/3訊息傳遞路徑進行反向調控。

Aristolochic acid (AA), a group of natural compound widely found in Aritolochia family of plants, is composed of AAI and AAII. Aristolochic acid nephropathy (AAN) is a rapidly progressive tubulointerstitial fibrosis associated with the intake of AA. Bladder cancer associated protein (BLCAP) is considered to be a potential tumor suppressor gene in numerous cancers. However, the biological function and regulation of BLCAP gene remains unclear. The aims of this study are focused on whether AAI-induced Gremlin upregulation is mediated by BLCAP gene and on the effects of AAI and BLCAP gene on the Smad signaling pathway in human embryonic kidney 293 (HEK293) cell line. Exposure of HEK293 cells to AAI for 24 h significantly increased the expression of Gremlin mRNA. This increase was in parallel with a down-regulation in BMP-7 mRNA and phosphorylated Smad1/5/8 protein levels. Besides, AAI also reduced the level of BLCAP transcript. To examine whether AAI regulated the expression of Gremlin through BLCAP gene, HEK293 cells were infected with lentivirus harboring pLKO.1-shBLCAP (shBLCAP/HEK293) to decrease the level of BLCAP mRNA. Downregulation of BLCAP transcript in shBLCAP/HEK293 cells did not affect the levels of Gremlin and BMP-7 mRNA. We found that Smad1/5/8 phosphorylation and its downstream Id2 mRNA were inhibited in shBLCAP/HEK293 cells due to a lower level of BMP type-II receptor (BMPR2) mRNA. On the other hand, knocking down BLCAP promoted the cell growth of shBLCAP/HEK293 cells, and also enhanced TGF-β1 mRNA signals, Smad2/3 phosphorylation and the expression of downstream targets, including PAI-1, CTGF, and microRNA-21 (miR-21). Transfection of miR-21 inhibitor into shBLCAP/HEK293 cells could recover the cellular proliferation induced by BLCAP deficiency and also restore the decreased signal of BMPR2 mRNA. Taken together, AAI induced the gremlin expression and its downstream BMP-7/Smad1/5/8 signaling in HEK293 cells without the involvement of BLCAP. Furthermore, BLCAP gene not only regulated Smad1/5/8 pathway by modulating BMPR2 expression, but also negatively affect TGF-β1/Smad2/3 signaling.