肝醣合成酶激酶-3b/乙型連環素訊息路徑與高度糖化終產物在LLC-PK1細胞所致效應之關係

Advanced glycation end-product-induced effects are dependent on glycogen synthase kinase-3b/b-catenin pathway in LLC-PK1 cells

林冠華(Kuan-Hua Lin)

指導教授 ： 莊麗月

高雄醫學大學/醫學院/醫學研究所/博士(2008年)

https://doi.org/10.6832/KMU.2008.00120

摘要

中文摘要

糖尿病腎病變是糖尿病病患嚴重的的併發症，是國人透析常見的原因，也是國內近年來十大死亡原因之一，尤其對病患的照護，以及所耗費的醫療資源非常龐大。而糖尿病腎病變的病理變化包括早期腎臟細胞增生、後期細胞肥大和細胞外間質的堆積，最後導致腎臟的纖維化。而高糖與高度糖化終產物與糖尿病腎病變的發生有著密切的關係。已有許多研究證明高度糖化終產物會在腎臟細胞誘發多個訊息傳遞路徑，如JAK2、MAPK和PKC等。最近有研究發現Wnt路徑與腎臟纖維化的關係，但其詳細的機轉仍未明暸。因此，我們利用豬類近端腎小管細胞 (LLC-PK1) 為對象，以高度糖化終產物作為模擬糖尿病的誘發因子，探討Wnt路徑與腎病變的關係。已知在高糖情況下，腎間質細胞會合成大量的肝醣合成酶激酶-3β (GSK-3β) 。因此，在探討Wnt路徑時，GSK-3β所扮演的角色更是我們研究的重點。實驗結果顯示高度糖化終產物與LLC-PK1細胞反應48小時後，乙型連環素的蛋白質隨著高度糖化終產物濃度的增加而大量減少，時間效應則在48小時後，乙型連環素的表現減少生成的效應最為顯著。以西方墨點法測量Wnt-4的蛋白表現，並沒有顯著的變化。由於GSK-3β為乙型連環素的上游調控蛋白，因此在4-48小時的反應時間中發現，GSK-3β的活化態蛋白 (GSK-3β-Tyr216) 表現增加，而其抑制性蛋白 (GSK-3β-Ser9)減少，且GSK-3β的活性也在4-48小時間有明顯的增加。我們以GSK-3β的抑制劑SB216763和GSK-3β shRNA進一步去確認GSK-3β在LLC-PK1細胞的角色，結果發現SB216763作用48小時，能緩解由高度糖化終產物誘發的乙型連環素的蛋白表現及細胞週期蛋白cyclin D1的減少；進一步也發現SB216763也能減緩高度糖化終產物誘發之膠原蛋白的生成及其蛋白表現量。此外，我們也以GSK-3β的shRNA抑制其表現，再次證明GSK-3b在上述反應的角色。因此，高度糖化終產物有可能透過其它途徑來活化GSK-3β。另外利用JAK2、PI3K、PKC、p38-MAPK及p42/44-MAPK路徑的抑制劑後發現皆能緩解高度糖化終產物所抑制的乙型連環素；但只有JAK2、PI3K、PKC及p38 MAPK的抑制劑能逆轉高度糖化終產物所致的cyclin D1的減少。動物實驗與糖尿病腎病變腎臟組織的免疫組織化學染色的結果，顯示罹患糖尿病三個月後的病鼠，其近端腎小管細胞的乙型連環素的蛋白量減少，與腎病變病患腎臟細胞生長抑制的病徵相符。綜合上述結果，我們認為高度糖化終產物能抑制近端腎小管細胞的增生及膠原蛋白的合成，且為GSK-3β依賴性，另外由高度糖化終產物所抑制的乙型連環素及cyclin D1的蛋白表現也與GSK-3β的調控有關。另外，PI3K、PKC、MAPK及JAK/STAT等訊息傳遞路徑也參與了高度糖化終產物所致的乙型連環素及cyclin D1蛋白表現的調節作用。

關鍵字

肝醣合成酶激酶-3b ； 乙型連環素

並列摘要

Diabetic nephropathy is the complication of diabetes mellitus. In Taiwan, diabetes mellitus and end-stage renal disease is the 5th and 8th leading cause of death, respectively. Diabetic nephropathy is a major cause of morbidity and mortality, which comprise of 20-40% of the diabetic patients. The pathogenesis of diabetic nephropathy is characterized by the proliferation of mesangial cells in the early stage but hypertrophy of renal cell at the late stage of diabetes mellitus. Moreover, the thickening of glomerular basement membrane and the accumulation of extracellular matrix leading to renal fibrosis is related of high glucose and the advanced glycation end-products (AGE) . However, the signal pathway associated with AGE in diabetic nephropathy is poorly understood. Recently, the evidence shows that Wnt pathway is associated with the formation of renal fibrosis. According to the previous studies, the glycogen synthase kinase-3β (GSK-3β) , one of the key mediator of Wnt pathway, plays a role in the regulation of glucose metabolism. Thus, we studied the role of β-catenin, and GSK-3β in AGE-induced effects in the proximal tubule-like LLC-Pk1 cells, and their relationship to the diabetic nephropathy.
We found that AGE (50, 75, 100 μg/ml) dose-dependently decreased β-catenin protein expression at 48 h incubation, but AGE did not decrease β-catenin protein expression until 48 h. Furthermore, we found that AGE (100 μg/ml) time-dependently (8-48 h) increased GSK-3β-Tyr216 (active GSK-3β) and time-dependently (4-24 h) decreased GSK-3β-Ser9 (inactive GSK-3β) protein expression. Meanwhile, AGE (100 μg/ml) activated GSK-3β kinase activity at 8-48 h. SB216763 (a GSK-3β inhibitor) attenuated AGE (100 μg/ml)-inhibited β-catenin protein expression at 48 h. SB216763 also attenuated AGE (100 μg/ml)-inhibited cell proliferation and attenuated AGE (100 μg/ml)-induced collagen production and type IV collagen protein expression at 48 h. We also found that JAk2, PI3K, MAPK, and PKC pathway inhibitor attenuated the AGE-inhibited β-catenin protein expression, but only JAk/STAT and MAPK pathway attenuated the AGE-inhibited cyclin-D1 protein expression. In immunohistochemistry experiments, we found that b-catenin level decreased in proximal tubules in 3-month STZ-induced diabetic rats and in patient with diabetic nephropathy. We concluded that AGE-inhibited cell proliferation and collagen production are dependent on GSK-3β in LLC-PK1 cells. Moreover, AGE-inhibted β-catenin and cyclin D1 protein expression are also dependent on GSK-3β.
It was also the first report that b-catenin levels decreased in renal tissue of patients with diabetic nephropathy.

並列關鍵字

GSK-3beta ； beta-catenin

參考文獻

參考文獻

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