ZFP36L1 Regulates the mRNA Expression of MAPK Phoshatase-1 and Other Immediate-Early Genes during the Early Stage of Adipogenesis

當3T3-L1脂肪細胞接收到分化訊號之刺激後，立即早期基因(Immediate early genes, IEGs)會立刻被大量的表現，但不久後其表現量又會快速的下降。此種特殊的基因表現方式已經知道是藉由調控其訊息RNA(messenger RNA, mRNA)的轉錄(transcription)和後轉錄(post-transcription)來達成。其中引起我們興趣的是，到底這類基因是經由何種機轉來達成如此劇烈的表現量變化。之前的研究指出，TTP蛋白質在3T3-L1脂肪細胞早期分化的時期會受到刺激而表現，並且會專一性的辨認一個立即早期基因MKP-1 mRNA上的多腺嘌呤-尿嘧啶序列(AU-rich elements, ARE)。TTP一旦與MKP-1 mRNA結合，就會導致此RNA的穩定性大幅下降，最後導致其表現量減少。然而在脂肪細胞的分化過程中，關於其他TTP家族成員的表現與功能至今還未被探討。因此，本篇研究主要的目的就是探討在脂肪細胞早期分化的過程中，另外一個TTP家族成員ZFP36L1所扮演的角色為何。藉由自製ZFP36L1專一性抗體的西方點漬法分析後發現，不同於TTP需要經由刺激才會被細胞表現的現象，ZFP36L1在脂肪細胞早期分化的過程中具有雙峰表現(biphasic expression)的特性。進一步研究發現，ZFP36L1是一個非常不穩定的蛋白質，但3T3-L1細胞經由刺激後導致ZFP36L1蛋白質量上的減少，卻主要是來自於轉譯層面(translational level)上的調控。同時也發現，除了在表現量上的差異外，ZFP36L1對於MKP-1 mRNA的結合與去穩定化的能力幾乎與TTP一樣，顯示細胞可能藉由同時調控ZFP36L1和TTP的表現，達到控制立即早期基因表現的目的。此外，在血清的刺激下，ZFP36L1會被 ERK/MAPK大量磷酸化，且一旦ZFP36L1被磷酸化後，其對於MKP-1 mRNA去穩定(destabilization)的能力會下降。最後，如果利用用病毒感染(lentiviral infection)的方式將小髮夾RNA(small-hairpin RNA, shRNA)送入3T3-L1中去抑制ZFP36L1的表現，MKP-1和立即早期基因的表現量都會大幅提高，而且會導致脂肪細胞無法順利分化。本篇研究提出了一個新的立即早期基因表現的調控模式，並且發現ZFP36L1在脂肪細胞分化過程中可能還扮演了重要的角色。Immediate-early genes (IEGs) are expressed instantly after the trigger of 3T3L1 preadipocyte differentiation and are under tight controls at transcriptional and post-transcriptional levels. The mechanisms underlying the rapid clearance of IEG mRNAs are of our interest. Our previous studies have revealed that Tristetraprolin (TTP), a RNA binding protein, recognized and destabilized MAP kinase phosphatase-1 (MKP-1) mRNAs through a mechanism called Adenyl/Uracil-rich (AU-rich) elements-mediated mRNA decay (AMD). However, the functions of other members in TTP protein family are not well-characterized in adipogenesis. In this study, we showed that ZFP36L1, another TTP family member, played roles in cell proliferation and post-transcriptional gene regulation in adipogenesis. In contrast to the inducible-property of TTP, ZFP36L1 protein was biphasically expressed within the first hour after induction and the expression of MKP-1 mRNA was reciprocal to the protein dynamics of ZFP36L1. This unusual protein dynamics was achieved by translational repression rather than by the change of ZFP36L1 protein stability. Importantly, the MKP-1 mRNA-binding and destabilizing abilities of ZFP36L1 were comparable to TTP and knockdown of ZFP36L1 in 3T3-L1 cells resulted in an upsurge of MKP-1 mRNA. Moreover, ZFP36L1 underwent a significant phosphorylation by ERK signaling immediately after induction and the phosphorylation status seemed to have some effects on its mRNA destabilizing ability. Finally, the effect of ZFP36L1 on the whole process of adipogenesis was investigated. This study elucidates a possible role of ZFP36L1 in adipogenesis and indicates that 3T3-L1 differentiating cells accomplish an elaborate regulation of MKP-1 mRNA expression by coordinating ZFP36L1 and TTP protein dynamics