脂肪細胞分化過程中TTP、MKP-1與MAPK訊息傳遞路徑間之調控機制

The Regulatory Network between TTP, MKP-1 and MAPK Signaling in Adipogenesis

林念儀(Nien-Yi Lin)

指導教授 ： 林中天

共同指導教授 ： 張瀞仁

國立臺灣大學/獸醫專業學院/獸醫學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.02153

摘要

脂肪組織為身體的重要器官，掌管體內能量恆定，且可釋放多種激素以調節體內各種生理功能，脂肪細胞分化的異常可能引起肥胖以及相關的疾病。在以3T3-L1脂肪前驅細胞為模式的研究中發現，當細胞受到分化刺激時，多種極早期基因(immediate early gene)的表現會受到活化，包括RNA降解因子tristetraprolin (TTP)，TTP可藉由與mRNA 3’端非轉譯區(3’ untranslated region, 3’UTR)中特殊的多腺嘌呤-尿嘧啶序列(AU-rich element, ARE)結合而加速其降解，本論文的目標即在瞭解TTP在調節脂肪細胞分化過程中的角色。將與TTP結合的mRNA分離並鑑定後，發現TTP的表現可受到自我調控，TTP可藉由與自己的mRNA結合而降低本身的表現。本研究也分離到TTP的新標的─MKP-1 mRNA。研究發現當脂肪細胞受到分化刺激後，ERK pathway同時活化TTP與MKP-1的表現，MKP-1的表現可抑制ERK活性，進而造成此一訊息傳遞的負調控。而MKP-1的表現則受到TTP及另一個RNA結合因子HuR的調控，TTP與MKP-1 mRNA的結合造成RNA降解而抑制MKP-1表現，HuR與MKP-1的結合則可使MKP-1 mRNA更加穩定。MKP-1 3’UTR具有三段ARE，在RNA IP、RNA pull-down及REMSA等實驗中證實他們對TTP及HuR有不同的親和力，證據指出TTP與HuR皆傾向與ARE1和ARE2結合，對ARE3的結合力較低，有趣的是，即便如此，TTP仍可藉由其他不明方式調節ARE3。進一步研究發現，TTP對RNA的親和力可受到轉譯後修飾機制(posttranslational modification)的調節。p38 pathway的活化使TTP呈高度磷酸化的狀態，且對RNA的親和力降低；而ERK pathway造成的磷酸化雖然對TTP的分子量影響不大，但卻能有效增加其對RNA的親和力。另外，在進行RNA干擾(RNAi)實驗後發現，抑制TTP的表現同時也抑制脂肪細胞分化，但抑制MKP-1表現後，脂肪細胞的複製擴增(mitotic clonal expansion)增加且分化的程度更加提高，此結果顯示TTP及MKP-1在調節脂肪細胞分化有重要的功能。綜合上述，本研究顯示MAPK pathway可活化TTP基因的轉錄，又可藉由轉譯後修飾機制影響TTP的功能，而TTP可藉由調節MKP-1的表現而影響MAPK pathway的活性，彼此間形成精密的調控網絡，進而控制脂肪細胞分化。

關鍵字

脂肪細胞分化 ； 多腺嘌呤-尿嘧啶序列 ； TTP ； MKP-1 ； 轉錄後調節機制 ； 轉譯後修飾機制

並列摘要

Dysregulation of adipogenesis has been considered as a cause of the development of obesity and obesity-related diseases. Among the immediate early genes (IEGs) activated during the differentiation of 3T3-L1 preadipocytes, we identified tristetraprolin (TTP), a zinc finger-containing RNA-binding protein which was reported to bind AU-rich elements (AREs) of target mRNAs and induce their rapid degradation. To understand how TTP may be involved in regulating adipogenesis, the expression and mRNA targets of TTP were analyzed. Biochemical and functional assays showed that the expression of TTP per se was autoregulated. Sequence analysis of 3’UTR of IEG mRNAs differentially bound by TTP suggested a preferential binding sequence of UUAUUUAUU. In addition to TTP and the previously reported COX-2 mRNAs, we have also identified MKP-1 mRNA as a novel target of TTP. The expression of MKP-1 and TTP was simultaneously activated by ERK signaling, while MKP-1 protein functions as a feedback regulator of ERK. Moreover, the expression of MKP-1 was tightly controlled by ARE-binding proteins TTP and HuR. Binding of TTP to MKP-1 mRNA resulted in rapid decay and binding of HuR resulted in mRNA stabilization. The three AREs in MKP-1 3’UTR were differentially bound by TTP and HuR. More specifically, both of TTP and HuR preferentially bound to MKP-1 ARE1 and ARE2, but showed low binding affinity to MKP-1 ARE3. The RNA-binding affinity of TTP was posttranslationally modified by p38 and ERK signaling. TTP exhibited a decreased RNA binding while being phosphorylated by p38 signaling and showed an enhanced RNA binding while being phosphorylated by ERK signaling. The functional importance of TTP and MKP-1 during 3T3-L1 differentiation was further demonstrated by siRNA knockdown experiment. These results suggested that TTP can regulate the MKP-1 mRNA stability to subsequently control the activation of MAPK signaling pathways in adipocyte differentiation.

並列關鍵字

adipogenesis ； ARE ； TTP ； MKP-1 ； posttranscriptional regulation ； posttranslational modification

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