探討轉錄因子LRH-1與GATA2/3的分子細胞機制

Molecular and cellular mechanisms of transcription factors LRH-1 and GATA2/3

賴財春(Tsai-Chun Lai)

指導教授 ： 胡孟君

國立臺灣大學/醫學院/生理學研究所/博士(2018年)

https://doi.org/10.6342/NTU201803646

摘要

轉錄因子作為轉錄作用起始的輔助因子，擔任生物體內參與調控基因表現的重責。本篇論文分別探討liver receptor homologue-1 (LRH-1) 以及GATA2/3於細胞中的分子機制。首先，LRH-1主要表現在肝腸組織以及卵巢，於肝臟代謝中扮演重要角色藉以維持生理恆定。現今有關調控LRH-1蛋白質穩定性的分子機制知之甚少。首先，我們發現LRH-1蛋白質水解路徑係經泛素蛋白酶體調控，其後說明LRH-1 蛋白質水解受CUL4-DDB1泛素連接酶調節。DNA damage-binding protein 2 (DDB2) 已知為一CUL4-DDB1之受質辨識組成單元，我們發現DDB2會影響LRH-1蛋白質生成量，並且證實DDB2直接與LRH-1的DNA-binding domain產生交互作用，並且促進LRH-1蛋白質泛素化使其送往蛋白酶體進行蛋白質水解。在人類肝癌細胞株HepG2中，我們發現胰島素會使內生性LRH-1蛋白質隨著處理劑量以及時間增加而增多，但LRH-1 mRNA表現量則不受影響。而胰島素處理降低LRH-1泛素化程度，顯示胰島素透過抑制泛素–蛋白酶體路徑調節LRH-1。此外，DDB2過量表現也降低LRH-1的轉錄活性，以及其下游基因之表現，而DDB2 knockdown卻發現glucokinase (GCK) 表現增加進而影響到HepG2對於葡萄糖吸收以及乳酸生成的能力，說明DDB2調控LRH-1之蛋白質穩定性，進而控制LRH-1在肝臟中的葡萄糖代謝功能。
LRH-1已知能夠調控許多類固醇生成酵素之表現，而HSD3B酵素參與三種固醇類賀爾蒙之合成，其中HSD3B1在胎盤的表現對於懷孕期間黃體素的產生十分重要。為了瞭解人類胎盤中HSD3B1基因調控的機制，我們分別將2.2 kb長度的啟動子片段以及個別片段刪除之啟動子選殖至具有螢光素酶 (luciferase) 報導基因的質體上，再進一步轉染入人類胎盤細胞株JEG-3進行分析。實驗結果發現近端-238/+337序列具有最高之轉錄活性，並發現此片段中具有兩個GATA位點，位點突變會大大地降低其在JEG-3細胞中的啟動子活性。進一步，我們發現GATA2以及GATA3能夠活化HSD3B1啟動子活性。EMSA實驗證實GATA2會結合到JEG-3細胞中的HSD3B1啟動子上的GATA片段。在JEG-3細胞中將GATA2 knockdown會顯著地降低HSD3B1表現，然而，GATA3 knockdown則是增加HSD3B1表現。最後，於西方墨點法分析發現GATA2在人類胎盤組織中具有高量表現，而非GATA3。本篇研究找出對於調控HSD3B1轉錄必要之GATA motif，並說明GATA2為一調控人類胎盤HSD3B1轉錄之重要分子。

關鍵字

轉錄因子 ； 泛素連接? ； 葡萄糖代謝 ； 類固醇生成

並列摘要

Transcription factors function as cofactor in gene transcription and account for regulation of gene expression. This study is aimed to figure out the molecular and cellular mechanisms of Liver receptor homologue-1 (LRH-1) and GATA2/3. Firstly, LRH-1 is predominantly expressed in enterohepatic tissues and ovary, and which is critical for hepatic metabolism to sustain homeostasis. Currently, the molecular mechanism underlying LRH-1 protein stability is poorly understood. First, we found that LRH-1 proteolysis is via ubiquitin-proteasome system, and we proved that CUL4-DDB1 ubiquitin ligase is involved in LRH-1 proteolysis. Furthermore, DNA damage-binding protein 2 (DDB2), functions as a well-known substrate recognition component of CUL4-DDB1, we observed that DDB2 affects LRH-1 protein levels, and indicated that DDB2 interacts with LRH-1 DNA-binding domain and targets LRH-1 for ubiquitin-proteasomal degradation. In human hepatoma (HepG2) cells, we observed that protein levels of endogenous LRH-1 are increased in a dosage- and time-dependent manner by insulin treatment, but without a change in mRNA levels of LRH-1. Then, insulin treatment decreased LRH-1 ubiquitination levels, indicating that insulin may stabilize LRH-1 protein via suppression of UPS. Moreover, overexpression of DDB2 diminished LRH-1 transcriptional activation and expression of target genes, whereas knockdown of DDB2 increased the expression of glucokinase (GCK) and influenced glucose uptake and lactate production in HepG2 cells, inferring that DDB2 regulates LRH-1 protein stability and modulates the hepatic glucose metabolism.
LRH-1 directly regulates expression of several steroidogenic enzymes, and HSD3B enzyme is essential for synthesis of three type steroid hormones. Among them, HSD3B1 expression is essential to produce progesterone for pregnancy maintenance. To understand the mechanisms of human HSD3B1 activation in the placenta, 2.2 kb of 5′-flanking sequence and 5′-deletions were fused to the luciferase reporter gene and transfected into human JEG-3 cells. The proximal -238/+337 sequence had the highest transcriptional activity, and we identified two GATA sites. Mutations of GATA sites greatly reduced promoter activity in JEG-3 cells, demonstrating the importance of GATA sites. Next, we observed that GATA2 and GATA3 both elevated HSD3B1 promoter activity. EMSA revealed the specific binding of GATA2 and GATA3 to the GATA sequences. GATA2 knockdown significantly reduced HSD3B1 expression in JEG-3 cells; however, GATA3 knockdown increased HSD3B1 expression. Western blot analysis revealed high levels of GATA2 but not GATA3 in human placental tissues. This study identified GATA motifs as essential control elements for HSD3B1 transcription and GATA2 as a novel transcriptional regulator of HSD3B1 expression in the human placenta.

並列關鍵字

Transcription factor ； ubiquitin ligase ； glucose metabolism ； steroidogenesis

參考文獻

Abbaszade, I. G., J. Arensburg, C. H. Park, J. Z. Kasa-Vubu, J. Orly et al., 1997 Isolation of a new mouse 3beta-hydroxysteroid dehydrogenase isoform, 3beta-HSD VI, expressed during early pregnancy. Endocrinology 138: 1392-1399.

Aberle, H., A. Bauer, J. Stappert, A. Kispert and R. Kemler, 1997 beta-catenin is a target for the ubiquitin-proteasome pathway. Embo j 16: 3797-3804.

Alkalay, I., A. Yaron, A. Hatzubai, A. Orian, A. Ciechanover et al., 1995 Stimulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B inhibitor for degradation via the ubiquitin-proteasome pathway. Proc Natl Acad Sci U S A 92: 10599-10603.

Annicotte, J. S., C. Chavey, N. Servant, J. Teyssier, A. Bardin et al., 2005 The nuclear receptor liver receptor homolog-1 is an estrogen receptor target gene. Oncogene 24: 8167-8175.

Ashida, H., M. Kim, M. Schmidt-Supprian, A. Ma, M. Ogawa et al., 2010 A bacterial E3 ubiquitin ligase IpaH9.8 targets NEMO/IKKgamma to dampen the host NF-kappaB-mediated inflammatory response. Nat Cell Biol 12: 66-73; sup pp 61-69.