Hsc70相互作用蛋白C端（CHIP）在心肌的保護及促進幹細胞特性機轉探討

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Title:	Hsc70相互作用蛋白C端（CHIP）在心肌的保護及促進幹細胞特性機轉探討 C-terminus of Hsc70-interacting protein (CHIP) provide beneficial effects: cardiac protection and stem cell properties
Authors:	馮致中;Chih-Chung Feng
Contributors:	臨床醫學研究所博士班
Keywords:	心血管疾病;心肌細胞肥大;幹細胞;Cardiovascular disease;stem cell
Date:	2018-08-06
Issue Date:	2018-12-25 10:37:45 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	心血管疾病在國人十大主要死因中位居前列。心血管疾病成因有許多種，除了心肌梗塞或肥胖，腎上腺素受體過度的激活也會誘導心肌細胞肥大導致心力衰竭。Human tumorous imaginal disc 1 (Tid1) 是為一佐監護蛋白且具有兩種異構體稱為短鏈的Tid1 (Tid1-S) 和長鏈的Tid1 (Tid1-L)。先前的研究表明Tid1在預防擴張型心肌病中也起著重要作用；然而，Tid1的異構體在心臟細胞凋亡和肥大中的作用尚不清楚。因此，本研究探討Tid1-S在異丙腎上腺素 (ISO) 誘導的心肌細胞肥大和細胞凋亡中的保護作用。我們使用H9c2細胞預先過表達Tid1-S，而後處理ISO來誘導肥大及凋亡，利用IHC、TUNEL assay、IFC、Co-IP和Western印跡評估其蛋白表現。從IHC結果中，我們發現人類心肌梗塞不同階段的組織中Tid1的表現增加。有趣的是，在本研究中，隨著ISO處理，Tid1表現下降且呈現時間依賴性。然而，Tid1-S的過表達抑制了NFATc3，BNP和calcineurin表現並抑制了NFATc3的易位作用。另一方面，Tid1-S過表達活化p-Aktser473並降低caspase-3和cytochrome c的表達。重要的是，我們發現Tid1的過表達增強了CHIP的表現，並且誘導了CHIP泛素化Gαs的活性進而導致Gαs降解程度的增加。本研究表明Gαs是為CHIP的一種新型底物，Tid1-CHIP- Gαs 之複合物在抑制ISO誘導的心肌細胞肥大和細胞凋亡方面起重要作用。間質幹細胞基於其多能性且取得方便，使其常用於目前研究幹細胞治療之細胞。先前的研究表明幾種 E3 連接?透過泛素化的途徑調節幹細胞自我更新、分化及重新編程，但是CHIP在調控幹細胞的角色還不清楚。本研究的目的為探討熱休克蛋白70羧基端作用蛋白（CHIP）E3連接?調節人類瓦頓氏膠間充質乾細胞（hWJMSC）的多能性及其他功能的因子。我們利用瞬時轉染將CHIP過表達於人類瓦頓氏膠間質幹細胞，而後分析間質幹細胞在存活、增殖、自我更新、遷移功能蛋白的表現。我們發現CHIP可以通過降低PTEN的表達來增加Akt的磷酸化，證實CHIP過度表達可以增強間質幹細胞的存活能力。CHIP也增強間質幹細胞自我更新蛋白Sox2和Nanog的表達。此外，CHIP通過E3連接?介導的泛素化與整聯蛋白β1和CD44以及其他表面受體相互作用調節間質幹細胞的遷移能力。有趣的是，我們還發現CHIP通過減少p21進而預防間質幹細胞受到氧化應激誘導的衰老。這些結果表明CHIP強化間質幹細胞中的多能性，在幹細胞治療中是為一新穎策略。 Cardiovascular disease has a high rank of the leading causes of mortality in the top ten leading causes of death in Taiwanese. Cardiovascular disease caused by various factors, including myocardial infarction or obesity. Moreover, adrenergic receptor over-activate also induces cardiac myocyte hypertrophy to lead to heart failure. Human tumorous imaginal disc 1 (Tid1), a chaperone protein and processes two isoforms called Tid1 short (Tid1-S) and Tid1 long (Tid1-L). The previous study suggests Tid1 also plays a major role in preventing dilated cardiomyopathy; however, the role of Tid1splicing variants in cardiac apoptosis and hypertrophy remains unclear. In this study, we aim to investigate the protective effect of Tid1-S in isoproterenol (ISO)-induced cardiomyoblast hypertrophy and apoptosis. H9c2 cells were pretreated Tid1 before ISO-induced hypertrophy and apoptosis and then evaluated by IHC, TUNEL assay, IFC, Co-IP, and Western blot. From the IHC experiment, we found that Tid1 proteins were increased in tissues from different stages of human myocardial infarction. Interestingly, Tid1 was decreased in time-dependent following ISO treatment in this study. However, over-expression of Tid1-S suppressed NFATc3, BNP, and calcineurin protein expression and inhibited NFATc3 nuclear translocation in ISO-induced H9c2. On the other hand, Tid1-S over-expression activated survival proteins p-AKTser473 and decreased caspase-3 and cytochrome c expression. Importantly, we found that overexpression of Tid1 enhanced CHIP expression, and induced CHIP to ubiquitinate Gαs, resulting in increased Gαs degradation. Our study showed that Gαs is a novel substrate of CHIP, and we also found that the Tid1-CHIP- Gαs complex plays an essential role in inhibiting ISO-induced cardiomyoblast hypertrophy and apoptosis. Mesenchymal stem cells are regarded as an attractive source of regeneration therapy based on their multipotent cell properties and are easy to obtain. Previous studies showed several E3 ligases modulating the stabilities and functions of various factors in different adult stem cell through the ubiquitylation pathway. The objective of this study is to characterize the C-terminus of Hsc70-interacting protein (CHIP) E3 ligase regulated the factors responsible for pluripotency maintenance or other functions of human Wharton’s jelly mesenchymal stem cells (hWJMSC). We found that CHIP can increase Akt phosphorylated through decreased expression of PTEN, suggesting survival pathway improvement by CHIP overexpression. CHIP overexpression also induced Sox2 and Nanog, which are the expression of stem cell self-renewal markers. In addition, CHIP alternated cell mobility via interact with Integrin β1 and CD44 and other surface receptors through E3 ligase-mediated ubiquitination. Interestingly, we also found CHIP prevented oxidative stress-induced senescence of hWJMSC via decreased p21. These results indicated that CHIP would be used to enhance multiple functions and adaptability in hWJMSC.
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