前列腺素的分解代謝在脂肪細胞分化過程中所扮演的角色

The role of prostaglandin catabolism in adipocyte differentiation.

周玟伶(Wen-Ling Chou)

指導教授 ： 張智芬

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.01025

摘要

3T3-L1 細胞株是一個研究脂肪細胞分化機制最常用的模式系統，本論文中，前列腺素還原酉每(PGR-2)最初是一個利用mRNA差異呈現法(differential display)，被篩選出來的一個功能未知的基因；它是一個在3T3-L1 的脂肪細胞分化過程後期高度表現的基因，在組織的分布上，尤其在脂肪組織中具有特別高的表現。經過基因庫序列的比對分析，基因的選殖及其重組蛋白質的表現，酵素活性的測試，以及酵素反應產物的定性分析，從而證實此基因的表現具有催化15-keto prostaglandin E2 (15-keto-PGE2)還原成為13,14-dihydro-15-ketoprostaglandin E2的15-oxoprostaglandin- Δ13-reductase酵素活性，也因此將此基因定名為前列腺素還原酉每(PGR-2)。
前列腺素E2是一個半生期很短的活性小分子，其分解代謝先由
NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH)進行氧化反應產生15-keto-PGE2，再由NADPH/NADH-dependent 15-oxoprostaglandin-Δ13-reductase(PGR)進一步還原作用將15-keto-PGE2轉變為沒有活性的
13,14-dihydro-15-keto-PGE2。早期的研究發現脂肪組織具有高度的PGDH 與PGR之酵素活性，顯示前列腺素E2分解代謝在脂肪細胞中是旺盛的。
PPAR-γ 是一個在調控脂肪細胞分化上扮演重要角色的轉錄因子，在轉錄因子的分類上，它是屬於nuclear receptor的子家族成員，配體(ligand)對PPAR-γ的結合，使得轉錄輔抑制子從而脫離，並促使轉錄輔活化子返回與PPAR-γ結合，同時PPAR-γ藉由與retinoid X receptor (RXR)形成異二聚體才能進而活化其標的基因。目前臨床上治療糖尿病的藥物thiazolidinedione(TZD)，即是一類化學合成的PPAR-γ高親合性配體；過去一些天然的化學分子已被報導為潛在的內生性PPAR-γ的配體，但在生理上這些配體是否確實活化PPAR-γ則仍然沒有定論，有趣的是這些天然的化學分子往往伴隨不同的發炎刺激而產生，這與PPAR-γ日益明朗的抗發炎角色呈現正相關性。前列腺素E2的生成在許多發炎反應中皆會明顯增加，然而其代謝是否與調節PPAR-γ活性有關仍屬未知的議題。
本論文中，在3T3-L1 的細胞分化前期額外表現PGR-2 蛋白質時，脂肪細胞的分化明顯被抑制，但卻不影響分化前期PPAR-γ蛋白質的表現；然後，我進一步發現15-keto-PGE2是一個PPAR-γ的配體，藉由增進PPAR-γ與轉錄輔活化子的結合，而活化PPAR-γ的轉錄功能，使其能有效促進PPAR-γ標的基因的表現，與3T3-L1 的脂肪細胞分化；在293T細胞中，以轉染實驗表現PGR-2 蛋白質，的確能夠抑制15-keto-PGE2所引發的PPAR-γ的轉錄活化，反之，以轉染實驗表現前列腺素脫氫酉每15-hydroxyprostaglandin dehydrogenase (PGDH)蛋白質，使其氧化PGE2成為15-keto-PGE2，的確能夠促進PPAR-γ的轉錄活化。因此，根據以上的研究成果，我們認為細胞內PGE2的分解代謝，在調節PPAR-γ的轉錄活化的機制中，可能扮演一個重要的角色。

關鍵字

前列腺素 ； 脂肪細胞分化

並列摘要

3T3-L1 preadipocyte cell line has been used as a model for characterizing the events responsible for adipocyte differentiation. In this thesis, we used differential display to identify a novel gene, encoding prostaglandin reductase designated as PGR-2, in the beginning. Its expression pattern is abundant in adipose tissues and highly up-regulated in the late phase of 3T3-L1 adipocyte differentiation. By functional characterization, we found that PGR-2 is capable of converting 15-keto-PGE2 into 13,14-dihydro-15-keto-PGE2.
PGE2, a short lived mediator, is catabolized via an oxidation reaction catalyzed by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) for the generation of 15-keto-PGE2, which is further catabolized by a reduction reaction catalyzed by NADPH/NADH-dependent 15-oxoprostaglandin-Δ13-reductase (PGR). It has been shown that adipose tissue possesses high activity of both PGDH and PGR, indicating that PGE2 catabolism is highly active in adipocytes.
Peroxisome proliferator-activated receptor γ (PPAR-γ) plays important roles in adipogenesis. PPAR-γ is a ligand-dependent transcription factor that belongs to the nuclear receptor family, activating the transcription of its target genes as heterodimers with retinoid X receptor (RXR). Upon ligand binding, PPAR-γreleases bound corepressors and recruits coactivator for transcriptional activation. Thiazolidinediones (TZDs) are high-affinity synthetic agonists which have been widely used as insulin-sensitizing agents to treat type 2 diabetes. Several natural substances have been identified as potential endogenous PPAR-γ ligands. To date, their physiological significance and role as true endogenous activators for PPAR-γ have been uncertain. Interestingly, these identified ligands are usually associated with different inflammatory stimuli, which correlate with the anti-inflammatory role of PPAR-γ. The production of prostaglandin E2 (PGE2) is elevated in many inflammatory stimuli. However, little is known about whether the catabolism of PGE2 is associated with modulation of PPAR-γactivity.
In this thesis, enforced expression of PGR-2 represses transcriptional activity of PPAR-γand adipocyte differentiation of 3T3-L1 cells. Following these observations, we further found that 15-keto-PGE2, an intermediate metabolite within the PGE2 catabolic pathway, functions as a PPAR-γ ligand, by which it stimulates the differentiation of mouse fibroblasts into adipocytes. In accordance, overexpression of PGDH also increased PGE2-dependent activation of PPAR-γ. Our findings provide new insights into PGE2 catabolism in regulation of PPAR-γactivity.

並列關鍵字

prostaglandin ； PPAR-γ ； adipocyte differentiation

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