HSD3B基因啟動子在H295R與JEG-3細胞中活性之探討

Abstract

3β-hydroxysteroid dehydrogenase (3β-HSD) 是HSD3B基因的產物，可將受質 分子△5的氫原子經由同素異構作用轉移至△4。類固醇荷爾蒙為膽固醇衍生物， 當P450scc將膽固醇轉換成孕烯醇酮，後續反應幾乎都要3β-HSD作用才能產生最 終產物。脊椎動物體內存在多種HSD3B基因，並具有組織表現專一性。已知人類 體中有HSD3B1、HSD3B2與五個偽基因 (pseudogene)，HSD3B1主要表現在胎盤 與周邊組織，而HSD3B2主要表現在腎上腺與性腺。 2009 年Doi 等人發現HSD3B1 mRNA 存在於腎上腺zona glomerulosa，而 HSD3B2 mRNA 大量表現於zona fasciculata，此現象與過去認為HSD3B1 只表現 在胎盤與周邊組織的認知不同。為探討HSD3B 啟動子的調控機制，從人類DNA 中建構出約2 kb 之HSD3B1 與HSD3B2 啟動子片段，檢測其於腎上腺皮質 (H295R) 與胎盤絨毛膜 (JEG-3) 細胞株的活性。結果HSD3B1 啟動子活性在 JEG-3 中特別明顯，而HSD3B2 啟動子在H295R 與JEG-3 都沒有顯現活性。此 外，給予forkolin、phorbol ester、血管張力素 (angiotensin II，Ang II) 與鉀離子 對HSD3B2 啟動子的轉錄活性皆無明顯的效應。但Ang II 會增加HSD3B1 啟動 子轉錄活性，且Ang II 下游轉錄因子Egr-1 具有同樣的效力，說明HSD3B1 啟動 子可能會受到Ang II-Egr-1 訊息路徑調控。 HSD3B1 啟動子在JEG-3 中有很顯著的活性。進一步建構不同長度的啟動 子於細胞中分析其活性，發現上游約238~500 bp 處可能有抑制性序列存在，而 在近端 50 ~ 100 bp 區域則找到兩個GATA 結合位。GATA 結合位發生突變會使 HSD3B1 啟動子活性顯著減少，代表這兩個結合位對於HSD3B1 啟動子活性是相 當重要的。此外，我們的實驗還顯示intron 1 的刪除會影響抑制性序列與GATA 序列的作用。這些結果說明活動於intron 1、抑制性序列與GATA 序列之分子， 可能會有交互作用進而調控HSD3B1 基因的表現。

3β-hydroxysteroid dehydrogenase (3β-HSD) is encoded by HSD3B gene. It catalyzes the conversion of △5-3β-hydroxysteroids into △4-ketosteroids. Steroids hormones are all derived from cholesterol, which is initially cleaved by the cytochrome P450scc to form prenenolone. 3β-HSD is involved in the subsequent formation of all classes of steroid hormones. There are multiple genes for HSD3B in vertebrate species and these genes showed tissue-specific expression. Human have two HSD3B genes, HSD3B1 and HSD3B2, and five pseudogene. HSD3B1 is the major type of placenta and peripheral tissues, and HSD3B2 is the major type of adrenal gland and gonads. In 2009, Doi et al. identified that HSD3B1 mRNA is present in the zona glomerulosa of human adrenal gland and HSD3B2 mRNA is dominantly present in the zona fasciculate. This result is different from previous notion. To study the regulation of HSD3B, I constructed about 2 kb promoter regions of HSD3B1 and HSD3B2 and examined the promoter activity in human adrenocortical (H295R) and placental chorion (JEG-3) cell. Activity of HSD3B1 promoter is strong in JEG-3, but HSD3B2 promoter has no significant activity in these cells. Treatment with forskolin, PMA, angiotensin II and potassium could not influence the promoter activity of HSD3B2 in H295R cells. However, angiotensin II and its downstream signal Egr-1 increased the activity of HSD3B1 promoter. It indicates that HSD3B1 promoter may be regulated by Ang II- Egr-1 pathway. Since HSD3B1 promoter shows a strong activity in JEG-3 cells, I further constructed deletion fragments of the promoter to examine the activity. A potential inhibitory sequences between -238 and -500 bp and two GATA binding sequences between -100 and -50 bp were identified. Mutation at either of the GATA core sequences remarkably decreased the activity of HSD3B1 promoter, indicating that both of GATA binding sites are important for the promoter function. Moreover, we found that intron 1 of HSD3B1 can influence the function of inhibitory sequences and GATA site. These results suggest that the factors involving in intron 1, inhibitory sequences, and GATA binding sites may interact to modulate the expression of HSD3B1 in JEG-3 cells.