| 摘要: | 在我們實驗室過去的研究當中指出心肌細胞會在胞外訊息調控激?(Extracellular signal-regulated kinases, ERK)抑制劑的處理下降低血管緊張素II(Angiotensin II, Ang II)對於類胰島素生長因子II受體(Insulin-like growth factor II receptor, IGF-IIR)的活化效果。我們同時也觀察到Ang II會造成磷酸化的熱休克轉錄因子I(Heat Shock Factor I,HSF-I)蛋白於細胞質中累積並且與心臟細胞的肥大以及凋亡呈正相關性，然而ERK與HSF1之間的關係以及如何影響對於IGF-IIR調控肥大和凋亡路徑的機轉則仍然值得探討。
而本篇研究就是利用Ang II刺激心肌母細胞(H9c2)來模擬心臟在高血壓期間的狀態，證實Ang II可以透過血管緊張素II的第一型受體(Angiotensin II Type I Receptors, AT1R)激活ERK路徑，以至於IGF-IIR及其下游已知的肥大及凋亡蛋白的表現量上升。同時我們發現心肌母細胞在Ang II的刺激下ERK會針對HSF-1上的第三百零七個胺基酸──絲氨酸(Serine 307,S307)進行磷酸化的修飾，隨後引導糖原合成?激?III(Glycogen Synthase Kinase III, GSK3)針對HSF-1 S303進行磷酸化的修飾。然而Ang II經由ERK以及GSK3路徑提升IGF-IIR轉譯活化的比例卻並不顯著，大多則是直接抑制其蛋白質的降解作用。在心肌母細胞當中的IGF-IIR會因為Ang II的作用而降低其與泛素連結的能力，並且我們發現環指蛋白CXXVI (Ubiquitin ligase RING finger protein CXXVI, RNF126)在其中扮演著泛素連接?的角色。在HSF-1 S303磷酸化的情況下RNF126的表現量降低，使得IGF-IIR持續累積造成心臟傷害。最後則是依靠自發性高血壓老鼠作為模式生物，每天利用腹腔注射ERK抑制劑及GSK3抑制劑實際試驗透過這些路徑能夠治療心臟肥大的效果，並與現今已知被廣泛使用的治療藥物AT-1R拮抗劑比較，初步略見成效。
綜合以上觀測的結果，我們可以確信在心肌細胞中Ang II可以透過AT-1R的接收活化ERK/GSK3路徑並磷酸化修飾HSF-1 S303/307抑制RNF126加速IGF-IIR蛋白的累積，從而影響心肌細胞的肥大及凋亡。其中仍有許多不解之謎，包含AT-1R是直接或是間接活化ERK路徑、ERK/GSK3經過哪些因子影響HSF1磷酸化的修飾、磷酸化的HSF1是如何抑制蛋白?體與泛素的連接，種種謎團希望能在現有的研究資源中解密，而未來能否研製出比現今更具標靶性的抗心臟衰竭藥物，將會成為我們實驗室所研究的重要方向。
Hypertension-induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure (HF). Our previous finding showed that extracellular signal-regulated kinases (ERK) inhibitor effective suppressed cardiomyocyte hypertrophy and apoptosis via inhibiting Angiotensin II (Ang II)-induced insulin-like growth factor II receptor (IGF-IIR) expression. As well, we discovered Ang II-induced heat shock transcription factor I phosphorylation (pHSF1) accumulated in cytosol, which is positive correlated with cardiomyocyte hypertrophy and apoptosis. However, the detailed mechanism by which ERK affects HSF-I phosphorylation to regulate IGF-IIR in heart cell remains elusive.
In this study, we found that Ang II activated its downstream kinase ERK to increase IGF-IIR expression through its receptor, Angiotensin II type I receptor (AT1R). ERK activation subsequently phosphorylates HSF1 on serine 307 and leads to secondary phosphorylation by glycogen synthase kinase III (GSK3) on serine 303 in cardiac myoblast. Unexpectedly, we observed that Ang II-induced IGF-IIR expression was increased by ERK/GSK3 activation not though the DNA transcription, but via repressing IGF-IIR protein degradation. When the proteasome activity was inhibited, lots of ubiquitin-bound IGF-IIR complex was accumulated in cardiac myoblast, which is similar with the effect of Ang II. And we found that the E3 ubiquitin ligase of IGF-IIR, RING finger protein CXXVI (RNF126), was inhibited by p-HSF1 S303 to continually cause cardiomyocyte injury in hypertensive model. Afterwards, we confirmed direct effect of ERK/GSK3 inhibitor and compared with the widely used hypertension-Angiotensin II receptor blockers (ARBs) in vivo during 12 weeks Intraperitoneal(IP) injection into Wistar-Kyoto rats (WKY) and Spontaneously Hypertensive rat(SHR). Then we analyzed the cardiac function by echocardiography, observed the image of TUNEL, IHC, H&E staining on tissue sections, and isolated the left ventricular heart tissue to compare the target protein expression.
Finally, we concluded that ERK/GSK3-activated pHSF1 at serine 303 and 307 repress the protein degradation of IGF-IIR by RNF126 decrease was accelerating cardiomyocyte hypertrophy and apoptosis during hypertension-induced HF. These findings provided us HSF1-influenced associated protein have therapeutic value in developing treatments for cardiac disease. |
