- 學位論文
類胰島素生長因子-II接受體過度表現誘發心肌肥大、凋亡與纖維化之分子機轉探討：1．下游G蛋白訊息路徑之轉換活化；2．類胰島素生長因子-II接受體基因之調控機轉；3．全新類胰島素生長因子-II接受體之異構體發現
Molecular mechanisms of the overactivated IGF2R signaling pathway inducing cardiac hypertrophy, apoptosis and fibrosis：1. Role of the downstream G protein signaling switch；2. Transcriptional regulation of the IGF2R gene；3. Functional revealed of the novel IGF-IIR isoform
摘要
心肌肥大為心臟在面對各種刺激下的適應性反應，但隨著壓力不斷地增加且無法解除將造成心肌細胞凋亡導致心衰竭的發生。先前的研究發現在心臟病變的過程類胰島素生長因子-II(IGF-II)以及類胰島素生長因子第二型接受體(IGF2R)基因會大量上升可能是導致心肌肥大和凋亡以及心臟的纖維化的主因。但是心臟中詳細之IGF2R訊息路徑和其基因如何被調節以及其在心臟病變分子機轉中所扮演的角色仍不清楚；另外，我們將介紹一個全新的IGF2R基因異構體：2R-α的發現過程。首先，我們發現到在人類心肌梗塞和心結痂的檢體中較正常心組織有較高的IGF2R蛋白表現。為了專一性的活化IGF2R訊息路徑，我們採用兩種實驗策略：1．拮抗劑AG1024和IGF1R siRNA阻斷IGF1R訊息路徑；2．使用具專一性的Leu27IGF-II結合於IGF2R以避免IGF1R和InR的干擾。由實驗結果得知，IGF2R為一G protein-coupled receptor，在經IGF-II活化後改變其與small G-protein之結合能力。活化的IGF2R訊息路徑會經由Gαq、PKC-α/ CaMKII和calcineurin分別造成病理性心肌肥大和粒腺體依賴型的細胞凋亡，其完全不同於IGF1R訊息路徑所誘發的生理性心肌肥大和心肌的存活作用。我們同時發現IGF2R訊息路徑會透過活化MMP-9和Plasminogen Activators (PAs)影響著extra cellular matrix (ECM)的不正常的重組作用而導致心肌纖維化的發生。接著，我們進一步探討心臟病變過程中，調控igf2r基因大量表達的分子機轉。首先我們從兩個實驗方向進行探討：1．Epigenomic modification: 含DNA甲基化(DNA methylation)和組蛋白的乙醯化(Histone acetylation)；2．尋找可能參與啟動子活性(Promoter activity)的轉錄活化因子。實驗發現，於病態刺激下所造成之igf2r基因表現主要經由影響Histone acetylation造成，而與DNA methylation無關。利用建構含不同片段之igf2r promoter的報導基因載體，我們血管昇壓素(ANGII)會促使抑制性轉錄因子：熱刺激因子(HSF-1)從igf2r基因promoter上的HSF-Binding Element (HBE; -725~-704 bp)移除，進而開啟igf2r基因的表現。我們亦經由Rapid amplification of cDNA ends (RACE) PCR定義出一個具有4656bp長度的mRNA，起始於IGF2R基因intron 9的第645 bp，包含著exon 10-35 的序列，終止於intron 36的第455 bp位置。其可轉譯成具有1358個氨基酸的蛋白質，我們命名此IGF2R的異構體蛋白質為2R-α，並進一步探討其生理功能。綜合上述，我們認為透過抑制IGF2R訊息路徑和其基因之表達將提供治療心肌肥大和凋亡以及心臟纖維化進而減緩心衰竭進程的機會。同時相信，2R-α的發現與進一步功能的探討將對由IGF系統異常所造成的疾病帶來新的思考空間以及新的解決模式。
並列摘要
Cardiac hypertrophy is an adaptive response of heart under varied stresses. When stress has been accumulated, the transition from physiological hypertrophy to pathological hypertrophy results in promotion of heart failure. Our previous studies demonstrated upregulations of insulin-like growth factor II (IGF-II) and mannose 6-phosphate/IGF-II receptor (IGF2R) dose-dependently correlating with the progression of heart disease following complete abdominal aorta ligation, may play a critical role in angiotensin II (ANGII)-induced cardiomyocyte apoptosis. However, the detailed mechanisms of IGF2R in the promotion of heart failure in response to IGF-II remain unclear. Additionally, how igf2r gene up-regulation in response to pathological stresses is also poorly understood. Therefore, in the study I, we found a significant association of IGF2R overexpression with myocardial infarction and myocardial scars. Results of specifically activating IGF2R signaling through either inhibition of the IGF1R activity by IGF1R siRNA and AG1024 or using Leu27IGF-II analog, a ligand interact only with the IGF2R, revealed that IGF2R activated by IGF-II binding acted like a G protein-coupled receptor to activate PKC-α/CaMKII and calcineurin by association with Gαq, leading to pathological hypertrophy and mitochondria-dependent cell apoptosis in cardiomyocytes. Furthermore, we also found that IGF2R signaling activation disrupted the balance of MMP-9/TIMP-2 expressions and increased plasminogen activator (PAs) expression, resulting in the development of myocardial remodeling. In study II, we found the histone acetylation, but not the DNA methylation, is required for the induction of igf2r gene by ANGII. Moreover, when responding to ANGII, HSF-1, identified as a suppressor of igf2r gene under normal condition, slipped out of the HSF-binding element within the IGF2R promoter that contributes up-regulation of igf2r gene expression. Taken together, our study provides new insight into the gene regulation of IGF2R and the role of the IGF2R in the pathogenesis of cardiac disease. Suppression of IGF2R gene expression and its signaling pathways may be a good strategy to prevent the progression of heart failure. We also discovered a novel gene of IGF2R isoform, 2R-α, which transcripts 4656 nucleic acid mRNA examined by using the rapid amplification of cDNA ends (RACE) PCR and northern blots. After compared with IGF2R mRNA sequences, regardless of the same sequences from IGF2R’s exon 10 to exon 35, the 2R-α gene sequence unexpectedly comprise a partial fragment of intron 9 (645~806 bp) of IGF2R in its 5’ region and a partial fragment of intron 35 (1~455 bp) in its 3’ region. In addition, we found a new TATA box exists in the 5’ upstream region of 2R-α gene and its coding region starts at the 14 bp of exon 10, then stops in the 48bp of intron 35 that may encode a 1358 amino acids protein predicted by Open Reading Frame Finder. Furthermore, creating the antibody that recognized the 15 amino acidic sequence in N terminal：AYDESEDDTSDTTPC of predicted 2R-α protein sequences to confirm the translation of gene transcript into the 2R-α protein. Further to identify the role and function of this novel 2R-α gene in the future may provide a new concept of IGFs in regulating cell physiology and heart function.