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

Abstract

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.