中間絲蛋白vimentin為PPARγ的結合蛋白之鑑定與研究

Abstract

過氧化體增殖劑活化受體γ (PPARgamma)是nuclear receptor家族中受配體(ligand)調控而活化的轉錄因子，它調控了參與分化、新陳代謝和免疫反應的基因表現。配體對PPARγ的結合使得轉錄輔抑制子脫離，並促使轉錄輔活化子返回與PPARγ結合進而活化基因轉錄的進行。PPARγ是信息傳遞的中樞因子，它的活性在不同的胞內運作機制下受到很完善的調控。致裂原(mitogen)的刺激會對PPARγ進行負向調控進而抑制其基因調控的活性，這樣的負調控主要是由胞外調節激酶（extracellular signal-regulated kinase ; ERK）/致裂原活化蛋白激酶(mitogen-activated protein kinase; MAPK)下游的信息梯瀑所主導。在致裂原與配體的刺激之下，MEKs和ERKs會快速轉移至細胞核中，ERKs進而在PPARγ上絲胺酸(Serine) 82/112的位置將其磷酸化，再由MEKs藉其上的NES(nuclear export signal)調控PPARγ被磷酸化後的核輸出。這樣大量的核輸出會降低PPARγ活化核目標基因的能力，進一步抑制其基因調控的功能。然而，PPARγ核輸出的實際運作機制及其送至細胞質後際而的命運仍有待後續研究。 運用LC/MS/MS的分析技術，我們從誘導分化的3T3-L1脂肪細胞萃取物中鑑定出一蛋白質vimentin會與PPARγ進行結合。隨著脂肪細胞的分化過程，vimentin與PPARγ的蛋白質表現量會相對應增加。在3T3-L1脂肪細胞中，我們運用免疫沉澱-西方墨點法及免疫細胞化學染色分析進一步驗證了vimentin與PPARγ會在細胞質間進行結合。而在PPARγ的配體誘導之下，vimentin會擇優地與磷酸化的PPARγ結合。 我們的實驗同時指出，在配體誘導之下PPARγ會被磷酸化，進而被送至細胞質與vimentin進行結合，這樣的一個運輸路徑是依賴exportin 1/CRM1來執行並受到leptomycin B所抑制。進一步詳細的實驗也說明了，vimentin和pPPARγ的結合除了如預期在細胞萃取物中不溶性的細胞骨架可以見到，同時也會在粒線體和內質網進行。

Peroxisome proliferators activated receptor gamma (PPARγ) is a ligand activated transcription factor of the nuclear receptor family that regulates genes involved in differentiation, metabolism and immunity. Upon ligand binding, PPARγ releases bound corepressors and recruits coactivators for transcriptional activation. As a central signaling component, the activity of PPARγ is well regulated under various cellular processes. Mitogenic stimulation exerts negative regulation that suppresses PPARγ’s genomic activity. This downregulation is mediated largely by the extracellular signal regulated kinase 1/2 (ERKs)/mitogen activated protein kinases (MAPKs) signaling cascade. Upon mitogen and ligand stimulation, MEKs and ERKs rapidly translocate into the nucleus followed by ERK-mediated PPARγ phosphorylation on Serine 82/112. Upon binding onto the phosphorylated PPARγ, the NES in the MEKs then mediated the export of phosphorylated PPARγ out of the nucleus. This massive nuclear export reduces the ability of PPARγ to transactivate nuclear target genes and thereby inhibits its genomic function. However, the exact mechanism of nuclear export of PPARγ and the subsequent fate of cytoplasmic PPARγ remain further elucidated. With advent of LC/MS/MS technique, we have identified a protein vimentin which was associated with PPARγ from the cell extracts of 3T3-L1 adipocytes upon induction of differentiation. During adipocyte differentiation, the expression of vimentin was increased in parallel of the increases of PPARγ. We confirmed the association of vimentin and PPARγ in the cytoplasmic compartment of 3T3-L1 adipocytes with immunoprecipitation-western blot and immunocytochemistry studies. Interestingly, vimentin was preferentially associated with the phospho-PPARγ especially after treatment of PPARγ ligand. Our data also suggest that phosphorylation of PPARγ appears after ligand treatment which leads to subsequent export of phosphorylated PPARγ to cytoplasm, at a leptomycin B-sensitive exportin 1/CRM1 dependent pathway, where interaction with vimentin occurs. Further detailed studies showed that the interaction of vimentin and pPPARγ may take place in mitochondria and ER in addition to the expected cytoskeleton in the insoluble portion of cell extracts.