高糖誘導近端腎小管LLC-PK1細胞肥大的分子機轉： p21waf1/cip1蛋白質與PI3K訊息傳遞路徑的角色

The role of p21waf1/cip1and PI3K signaling pathway in HG induce cellular hypertrophy in proximal tubular cell line LLC-PK1

莊才德(Tsai-Der Chuang)

指導教授 ： 莊麗月

高雄醫學大學/醫學院/醫學研究所/博士(2007年)

https://doi.org/10.6832/KMU.2007.00124

摘要

我們以高糖培養近端腎小管細胞株LLC-PK1模擬糖尿病病患血糖控制不佳，高血糖情況下病患腎臟近端腎小管的病理可能機制。我們觀察到高糖培養LLC-PK1細胞會導致生長抑制與細胞肥大的現象，此與動物實驗及臨床觀察到的早期腎臟病變相似。並且由西方點墨法發現p21 waf1/cip1蛋白質會受到高糖影響呈現時間與劑量效應的增加，而且以shRNA技術也證明p21 waf1/cip1蛋白質與高糖抑制細胞生長和促進細胞肥大有關。我們更發現高糖增加p21 waf1/cip1蛋白質透過轉錄與轉譯作用的增加，進一步分析高糖促進轉錄作用的機制，發現高糖增加Sp1轉錄因子結合到p21 waf1/cip1啟動子-74~-86的位置。此與前人研究TGF-β1促進p21 waf1/cip1啟動子藉由Smad2/3與Sp1轉錄因子交互作用並結合到p21 waf1/cip1啟動子-74~-86的位置一樣。又由於高糖會增加TGF-β1 mRNA的增加，因此我們推測在近端腎小管高糖增加p21 waf1/cip1啟動子的機制有TGF-β1訊息傳遞的下游Smad2/3的參與。結果顯示高糖的確增加了Smad2/3的磷酸化作用與增加了Smads的轉錄活性(EMSA實驗)，並且也證明了高糖促進了Smad2/3與Sp1轉錄因子的交互作用，此結果釐清了高糖增加p21 waf1/cip1蛋白質的分子機制。除此之外，我們使用dominant negative Smad3質體Smad3△c轉染入細胞，也得到了能逆轉高糖增加p21 waf1/cip1啟動子與蛋白質的現象，並且也能逆轉高糖抑制細胞生長與促進肥大的作用。
由於乙型轉型生長因子(TGF-β)可誘導膠原蛋白、fibronectin、laminin的合成，更被認為是造成糖尿病腎臟肥大及腎臟硬化的最重要因素。但因為乙型轉型生長因子(TGF-β)是多功能的細胞激素，若全面抑制可能會有不良影響，因此若用dominant negative Smad來轉染或利用antisense或decoy oligonucleotides來抑制Smad，或許能只抑制其促進細胞外間質或促進肥大的作用而不致影響某些須TGF-β的正常功能，如此將會是更理想的方法。我們因此嚐試使用滲透式微幫浦(Osmotic minipumps)植入小鼠背部的方式，藉由持續緩慢釋出Smad3 antisense或對照組scrambled oligonucleotides 10天，觀察在STZ誘導的糖尿病小鼠中，Smad3 蛋白質的角色。由結果可得到Smad3 antisense能夠部分逆轉腎臟肥大的現象，且也減少了糖尿病高血糖引起的p21 waf1/cip1與TGF-β1 receptor II蛋白質的增加。此結果證明了在動物模式下亦可觀察到糖尿病高血糖會增加近端腎小管p21 waf1/cip1蛋白質的增加，而且與細胞實驗相同的，Smad3蛋白質參與了p21 waf1/cip1蛋白質的表現，也與腎臟的肥大有關。
我們並指出高糖透過氧化壓力、PKC訊息傳遞、p38 蛋白激酶及PI3-K訊息傳遞路徑促進p21waf1/cip1啟動子與蛋白質的增加。由於高糖透過活化PKC與p38 蛋白激酶與氧化壓力而調控下游基因表現不論在近端腎小管或其他細胞株皆已有前人研究探討過，但高糖對於PI3-K的影響則較少有人研究，基於此我們試著探討PI3-K訊息傳遞路徑在高糖增加p21waf1/cip1蛋白質中的角色。我們發現高糖促進PI3-K活性，也增加了下游Akt激酶的活性，使用PI3-K活性抑制劑LY294002能逆轉高糖抑制細胞生長與促進肥大的作用而dominant negative p85質體也能逆轉高糖促進肥大的作用，顯示PI3-K訊息傳遞路徑的確參與高糖促進肥大的作用。
除了PI3-K活性抑制劑LY294002能逆轉高糖增加p21waf1/cip1蛋白質外，△p85的大量表現也能減弱p21waf1/cip1蛋白質的量，即使外加高糖也能明顯抑制p21waf1/cip1蛋白質的增加。PI3-K活性抑制劑LY294002與大量表現△p85能明顯抑制高糖36小時所活化p21waf1/cip1蛋白質啟動子。以上結果顯示PI3K訊息傳遞的確參與了高糖對於p21waf1/cip1蛋白質的增加。另外使用PI3-K活性抑制劑LY294002也明顯的減少了p21waf1/cip1蛋白質與cyclinE /cdk 2複合物的結合，如此可說明為何PI3-K抑制劑LY294002能逆轉高糖抑制細胞生長與促進肥大的作用。
除此之外，我們也證明了高糖透過活化PI3-K訊息傳遞進而影響 Smad2/3轉錄因子的活化、與Sp1轉錄因子的交互作用以及Sp1轉錄因子的轉錄活性，因而活化了p21waf1/cip1的啟動子與蛋白質的增加，最後影響了細胞的生長與導致肥大。另外，由於PI3-K/mTOR訊息傳遞的途徑近來已被許多研究者証明參與了蛋白質的轉譯作用，所以我們藉由使用mTOR蛋白質專一性抑制劑Rapamycin 探討PI3-K/mTOR訊息傳遞途徑是否也參與了高糖的影響。結果顯示PI3-K/mTOR訊息傳遞途徑在高糖抑制細胞生長與促進肥大方面的確扮演了重要的角色，而且PI3-K/mTOR訊息傳遞也參與高糖活化p21waf1/cip1的啟動子增加了p21waf1/cip1的mRNA與蛋白質。
綜合上述的研究結果，我們得到高糖活化PI3K訊息傳遞而導致生長抑制與細胞肥大。PI3K訊息傳遞除了透過影響Smad3的活化之外也影響了mTOR蛋白質而調控了p21waf1/cip1啟動子的活化、mRNA與蛋白質的表現，使p21waf1/cip1蛋白質總量增加而使細胞週期停滯於G1晚期，並由於mTOR蛋白質影響的轉譯作用而促使細胞走向肥大。除了PI3K訊息傳遞，我們亦發現高糖活化PKC訊息傳遞、p38 蛋白激酶及增加氧化壓力而調控了p21waf1/cip1蛋白質的表現。

關鍵字

高糖 ； 近端腎小管 ； 細胞肥大

並列摘要

The present studies investigated the role of high glucose inhibit cell proliferation in kidney tubular epithelial cells. In LLC-PK1 cells, p21 protein (not p27 or p15) increased after 24-to -48-h exposure to high glucose. The key role of p21 protein on cell cycle arrest was confirmed by the use of p21shRNA, effectively decreased p21 protein levels and reversed the effect of high glucose on inhibition cellular proliferation. Furthermore, high glucose treatment leads to an increase in p21 mRNA and promoter activity independent on osmotic pressure change. To further define the signaling pathway through which high glucose induces p21, we have performed a detailed functional analysis on the p21 promoter. Through both deletion and mutation analysis of the p21 promoter, these results indicate maximal p21 promoter activity requires both strong distal and proximal regions, suggesting synergistic interactions between transcription factors recognizing these sites. We focus on proximal regions of p21 promoter and define sp1-3 site has a pivotal role on the activation of high glucose. EMSA assays demonstrate that high glucose would increase sp1-3 element binding complex, two proteins of the complex are sp1 and Smads. To investigate the role of Smads involve the activation of p21 promoter, we found high glucose would increase Smad2/3 phosphorylation and binding activity by EMSA assays after 24 h exposure. Although TGFβ1 antibody and TGFβ type I receptor inhibitor (SB431542) could decrease high glucose induced p3TP-lux, they had no effect on the activation of p21 promoter by high glucose, suggesting independent on TGFβ1/ TGFβ type I receptor. From transient transfectioned dominant negative Smad3∆c plasmids, we got it can decrease hyperglycemia-induced p21 promoter activation and protein expression. We also treated with Smad3 antisense oligonucleotides for 10 days in STZ-induced diabetic mice and found this treatment could reduce p21 and TGFβRI protein expression and reduce the induction of kidney weight in vivo. Taken together, these results suggest that Smads activation plays a pivotal role in the transcriptional activation of the p21 gene by high glucose in vitro and in vivo.
Because Smad3 protein involved high glucose regulate p21 protein expression but the regulation independent on TGFβ1/ TGFβ type I receptor, so we tried to find out how high glucose activate Smad2/3 protein. We found PI3K, PKC, p38 and ROS signaling pathway involve high glucose activate p21 promoter and protein expression, but except PI3K signaling pathway, there are many researches about how high glucose activate PKC, p38, ROS signaling and regulate gene expression. So we focus on the relationship between high glucose and phosphoinositide-3 kinase (PI3K). We analysed PI3K and Akt kinase activity under high glucose condition, and got high glucose can increase their activity, otherwise, PI3K inhibitor LY294002 and dominant negative PI3K subunit p85 plasmid could decrease hyperglycemia-induced p21 promoter activation and protein expression. Furthermore, PI3K inhibitor LY294002 and dominant negative PI3K subunit p85 plasmid could reverse high glucose induced cellular hypertrophy. LY294002 also decreased p21 binding with cyclin E and cdk2. In molecular mechanism, we also confirmed by EMSA assays : LY294002 could decrease hyperglycemia-induced sp1-3 element binding activity and Smad binding element (SBE) binding activity. In another way, we found antioxidant reagent (N-acetyl-L-cystein) and NADPH oxidase inhibitor (DPI) could reverse hyperglycemia-induced p21 protein expression, and LY294002 could decrease H2O2 - induced p21 protein expression, this result suggested reactive oxygen species (ROS) generated by high glucose, mainly H2O2 , stimulates PI3K signaling pathway to induce p21 expression. Recently, PI3K/mTOR signaling pathway has been demonsted involve protein translation and play an important role on pathologic hypertrophy. Because we find high glucose can activate PI3K and induce cellular hypertrophy, so we use mTOR specific inhibitor rapamycin to investigate its role under high glucose condition. We got the mTOR specific inhibitor rapamycin can reverse high glucose induced p21 promoter, mRNA and protein expression. Otherwise, rapamycin also could reverse high glucose inhibit cellular proliferation and cellular hypertrophy. These result indicated PI3K/mTOR signaling pathway indeed involved high glucose induce cellular hypertrophy by regulating p21 protein expression.
Taken together, these results suggest that high glucose activated PI3K which related Smad3 and mTOR protein activation and played a pivotal role in the transcriptional activation of the p21 gene under high glucose condition, otherwise, PKC, p38 and ROS signaling pathway involve high glucose activate p21 promoter and protein expression.

並列關鍵字

p21waf1/cip1 ； PI3K signaling pathway ； HG ； cellular hypertrophy ； proximal tubular cell

參考文獻

參考文獻

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