轉錄因子Oct-2參與脂多醣刺激RAW264.7細胞resistin基因表現之研究

Oct-2 transcription factor is involved in the regulation of LPS-induced resistin gene expression in RAW264.7 cells

藍雅馨(Ya-Hsin Lan)

指導教授 ： 呂紹俊

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.01409

摘要

2001年，resistin因其作用可能與胰島素抗阻（insulin resistance）相關而得名，然而之後許多研究resistin與肥胖及第二型糖尿病的關聯性顯示不一致的實驗結果，到目前為止的研究成果推論出resistin可能參與調控血糖恆定、脂肪生成分化和發炎反應。
本實驗室以lipopolysaccharide（LPS）處理小鼠巨噬細胞RAW264.7發現resistin mRNA表現增加，探討其分子機制的實驗結果顯示resistin promoter-996至-969的28bp和-914至-907的octamer區域（element）皆為LPS促進resistin promoter轉錄活性的重要反應序列。接續已知的研究結果，利用resistin promoter-996~-969及-927~-896兩段DNA鹼基對分別作為探針（probe）進行凝膠電泳位移分析（EMSA），兩種探針都會與RAW264.7細胞核萃取液特異性結合形成DNA/protein複合物，其中-984至-979（CTATCT）互補股序列為GATA區域，EMSA及啟動子活性分析實驗結果顯示GATA區域並不參與LPS誘導resistin promoter活性增加，resistin promoter -996~-969中的LPS重要反應序列仍需要以不同方式去探討。而-914至-907（ATTTGCAT）為octamer區域，利用未標定的-927~-896 bp或consensus octamer oligonucleotides都可以競爭掉探針與核蛋白質的結合，但若將未標定的-927~-896 bp中ATTGCAT改變成GCCGCAT作為抑制物，則競爭能力明顯下降。目前已知有兩種octamer element binding proteins Oct-1及Oct-2，RT-PCR及西方墨點法的實驗結果顯示Oct-1和Oct-2 mRNA及蛋白質在RAW264.7細胞皆有表現，且處理過LPS的RAW264.7細胞中Oct-2 mRNA和蛋白質表現有顯著增加的現象，Oct-1則沒有明顯變化。利用biotin-labeled-octamer探針與streptavidin磁球結合，再與RAW264.7細胞核萃取液結合，得到與octamer結合的蛋白質進行西方墨點法分析，結果顯示Oct-2的確與octamer區域結合，且經過LPS處理的細胞核萃取液Oct-2結合至octamer區域有增加的情形。ChIP assay的結果也顯示在in vivo狀態，LPS會活化Oct-2結合於resistin promoter。Reporter assay中利用Oct-1或Oct-2或GATA-2表現質體與pResistin(-996/+61)-Luc一起轉染RAW264.7細胞，結果顯示Oct-2可以提高LPS促進resistin promoter的活性，相對的Oct-1卻有抑制性的作用，GATA-2則沒有影響。實驗室之前的結果指出LPS增加resistin表現可被MEK或PI3K或p38的抑制劑（U0126、Ly294002及SB203580）所抑制，Oct-2表現質體與pResistin(-996/+61)-Luc一起轉染的RAW264.7細胞，LPS引起的resistin promoter轉錄活性會被U0126及Ly294002抑制73 %及90 %，而轉染pResistin (-968/+61)-Luc的細胞，LPS所增加的luciferase也同樣被U0126及Ly294002抑制67 %及89 %。綜合以上結果，LPS調控Oct-2及另一未確認的因子來提高resistin基因的表現；LPS促進Oct-2 mRNA和蛋白質表現並經由PI3K和MEK途徑活化Oct-2蛋白質而增加resistin的轉錄作用。

關鍵字

脂多醣

並列摘要

Resistin is named for resistance to insulin. Studies so far suggest that resistin may involve glucose homeostasis, adipogenesis and inflammatory response.
From our previous studies, LPS increases resistin expression in vivo and in vitro. LPS also increases resistin mRNA expression in RAW264.7 cells, a mouse macrophage-like cell line. Our previous results showed that an octamer (ATTTGCAT) element, located at -914 to -907, and an unidentified element, within -996 to -969, are essential for maximal promoter activity in response to LPS stimulation. Two resistin promoter fragments, -996~-969 bp and -927∼-896 bp, were 32P-labeled and used as probe in EMSA assays, the results showed that both probes could form specific DNA/protein complexes with nuclear proteins from RAW264.7 cell. Cold -927∼-896 fragment and consensus octamer oligonucleotides completely competed for the formation of DNA/protein complex using the -927~-896 probe; however, octamer mutant probe (ATTTGCAT→GCCTGCAT) only partially competed the formation of the DNA/protein complex. Two nuclear proteins, Oct-1 and Oct-2, are known octamer-binding proteins. To identify which of them is involved in LPS induced resistin gene expression, we first checked whether Oct-1 and Oct-2 are expressed in RAW264.7. Both Oct-1 and Oct-2 mRNAs (detected by RT-PCR) and proteins (detected by Western blotting) are detectable in RAW264.7. However, only Oct-2 mRNA and protein levels were significant up-regulated by LPS in RAW264.7. Binding of Oct-2 in RAW264.7 nuclear extracts to the resistin promoter were demonstrated by using biotinylated -927~-896 bp DNA fragment captured by streptavidin magnetic beads and Chromatin immunoprecipitation (ChIP) assay. Co-transfection of RAW264.7 with pResistin(-996/+61)-Luc and pCG-Oct-1 and/or pCG-Oct-2 further demonstrated that Oct-2, but not Oct-1, is responsible for the activation of resistin promoter upon LPS treatment.
Taken together, our results showed that both -996~-969 bp and the octamer element located at -914 to -907, are essential for maximal promoter activity in response to LPS stimulation. In addition, we found that Oct-2, but not Oct-1, is responsible for LPS activation of resistin promoter. We also found, for the first time, that mRNA and protein levels of Oct-2 were significantly up-regulated by LPS in RAW264.7 cells, and activation of Oct-2 may involve PI3K and MEK pathways.

並列關鍵字

RAW264.7 ； LPS ； resistin ； Oct-2

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