葡萄糖激素受體對前列腺素還原酶-2基因表現調節之研究

The role of Glucocorticoid receptor(GR) in PTGR2 regulation

郭千榕(Chien-Jung Guo)

指導教授 ： 莊立民

國立臺灣大學/醫學院/分子醫學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201701785

摘要

脂肪細胞的分化是非常細膩、複雜的過程。受到眾多的訊息路徑的嚴密調控。本實驗室之前經由差異呈現法篩選出一個在脂肪細胞分化過程中高度表現的基因，即前列腺素還原酶 2 (Prostaglandin reductase 2，簡稱為 PTGR2)，PTGR2 可將 15-keto-PGE2 代謝成 13,14-dihydro-15- keto-PGE2。在先前的研究中發現，PTGR2 參與脂肪細胞的分化，此外，我們也發現在糖尿病模式鼠中的臟器脂肪細胞 PTGR2
有大量之表現。但 PTGR2 的調控之分子機制仍未明瞭，因此本篇論文藉由脂肪細胞分化過程來探討 PTGR2 基因之表現如何被調控。我們從啟動子中被不同藥物和不同訊息路徑抑制劑的研究中發現，PTGR2 可透過 Dexamethasone-glucocorticoidreceptor (GR) 的路徑而被調控。經由轉錄因子結合位的軟體預測中，我們發現在PTGR2 啟動子上可能的 GR 結合位置，再藉由點突變 GR 的結合位置，並測量啟動子的活性實驗，我們得知在-277/-283 的結合位置對 PTGR2 啟動子活性上升很重要。我們再以 chromatin immunoprecipitation (ChIP)的實驗，證明在脂肪細胞分化過程中的確有 GR 結合在 PTGR2 的啟動子上，證實了 GR 對 PTGR2 轉錄調節的分子機制。另外，我們也探討 PTGR2 在生物體內是否有受到 GR 調控的現象，我們發現在正常 B57CL/6 老鼠連續注射大量 dexamethasone 三天後，發現 PTGR2 在腹股溝的脂肪組織有顯著上升，透過老鼠和細胞的兩種不同的實驗模式，我們證明PTGR2 如何被 Dexamethasone-GR 的路徑調控的機制。

關鍵字

PTGR2 ； Dexamethasone ； GR ； 轉錄調節 ； 脂肪前驅細胞

並列摘要

Adipocyte differentiation is an elegant, complicated process involving sequentially activation of thousands of transcriptional factors. Our group previously discovered a novel enzyme, called prostaglandin reductase 2 (PTGR2) through mRNA differential display. PTGR2 is an oxidoreductase which catalyze 15-keto PGE2 to 13,14-dihydro-15-keto PGE2 as final product. In functional study, overexpression PTGR2 rather than its catalytic mutant inhibited adipocyte differentiation and relatively triacylglycerol content,which suggest a role of PTGR2 in adipogenesis. Additionally, PTGR2 is upregulated in omental fat in obese mouse model. However, the molecular mechanism of PTGR2 expression remains unknown. Results from promoter assay and inhibitors of different signaling pathways indicated that PTGR2 transcriptional regulation is possibly mediated via dexamethasone-GR pathway. Moreover, the predicted TF binding sites also indicated
presence of GR binding sites on PTGR2 promoter. With site-direct mutagenesis, we confirmed the proximal GR binding site at -277/-283 is responsible for promoter
activation by dexamethasone. We then performed chromatin immunoprecipitation (ChIP) and demonstrated a direct binding of GR on PTGR2 promoter site. To further investigate the regulation of PTGR2 in vivo, we discovered that dexamethasone pulse therapy significantly upregulate PTGR2 mRNA in in inguinal fat tissue. Our findings thus support the dexamethasone-GR pathway that leads to PTGR2 activation in both cell and animal models.

並列關鍵字

PTGR2 ； glucocorticoids ； GR ； transcriptional regulation ； 3T3-L1 cells.

參考文獻

31. Steven P. Weinstein, T.P., Alia Pritsker, and Richard S. Haber, Glucocorticoid-Induced Insulin Resistance: Dexamethasone Inhibits the Activation of Glucose

22. Hideyuki Sakoda, T.O., Motonobu Anai, Makoto Funaki, Kouichi Inukai, Hideki Katagiri,, Y.O. Yasushi Fukushima, Hiraku Ono, Midori Fujishiro, Masatoshi

1. Lau, D.C., et al., Adipokines: molecular links between obesity and atheroslcerosis.Am J Physiol Heart Circ Physiol, 2005. 288(5): p. H2031-41.

2. Seale, P. and M.A. Lazar, Brown fat in humans: turning up the heat on obesity.Diabetes, 2009. 58(7): p. 1482-4.

3. Farmer, S.R., Transcriptional control of adipocyte formation. Cell Metab, 2006.4(4): p. 263-273.