Studies on the Biological Responses and Signal Transduction Pathways of Advanced Glycation End-product in NRK-49F Cells

• Main Authors: Jau-Shyang Huang, 黃昭祥
• Other Authors: Lea-Yea Chuang
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/84917825090811422994

博士 === 高雄醫學大學 === 醫學研究所 === 88 === Advanced glycation end-product (AGE) is important in the pathogenesis of diabetic nephropathy, which is characterized by cellular hypertrophy/hyperplasia leading to renal fibrosis. However, the signal transduction pathways of AGE remain poorly understood. JAK (Janus kinase)/STAT (signal transducers and activators of transcription) pathway has been associated with cellular proliferation in some extra-renal cells. Because interstitial fibroblast proliferation may be important in renal fibrosis, we studied the role of the JAK/STAT pathway in NRK-49F (normal rat kidney fibroblast) cells cultured in AGE. We found that AGE dose-dependently increased cellular mitogenesis in NRK-49F cells at 5 and 7 d. AGE (100 g/ml) induced tyrosine phosphorylation of JAK2 (but not JAK1, JAK3, and TYK2) at 15-60 min. In addition, AGE also induced tyrosine phosphorylation of STAT1 and STAT3 at 1-2 h and 0.5-4 h, respectively. Being a transcription factor, AGE also increased the DNA-binding activities of STAT1 and STAT3. AG-490 (a specific JAK2 inhibitor) inhibited tyrosine phosphorylation of JAK2 and the DNA-binding activities of STAT1 and STAT3. The same results were obtained using the specific “decoy” oligodeoxynucleotides (ODNs) that prevented STAT1 and STAT3 from binding to DNA. Meanwhile, the STAT1 or STAT3 decoy ODN and AG-490 were effective in reversing AGE-induced cellular mitogenesis. To reveal the relationship between the JAK/STAT activity and cell cycle progression, we examined the effects of AGE on STAT5 activity and cell cycle progression in NRK-49F cells. We found that AGE rapidly induce tyrosine phosphorylation and protein-DNA binding activity of STAT5 at 5 min and 15 min, respectively. Among several G1 cyclins and cyclin-dependent kinases (cdks), cyclin D1 expression and cdk4 activity were both increased by AGE. Nevertheless, the levels of cdk inhibitors were not affected by AGE. Furthermore, our results showed that cyclin D1 expression and cdk4 activity were all inhibited by AG-490 and STAT5 decoy ODN. Interestingly, STAT5 decoy ODN and AG-490 both significantly blocked AGE-induced cell cycle progression and cellular mitogenesis. In this study, we also examined whether JAK/STAT are involved in AGE-regulated extracellular matrix (ECM) production in NRK-49F cells. We found that AGE time- and dose-dependently increased type I collagen and fibronectin productions in these cells. However, AGE did not increase TGF- bioactivity and TGF-1 mRNA expression. Additionally, AGE increased RAGE (receptor for AGE) protein expression and MAPK (ERK1/ERK2) activity. AGE-induced RAGE expression was dose-dependently inhibited by antisense RAGE ODN and captopril. AGE-induced type I collagen production and JAK2-STAT1/STAT3 activation were decreased by AG-490, antisense RAGE ODN and captopril. Meanwhile, STAT1 and STAT3 decoy ODNs also suppressed the induction of collagen by AGE. In contrast, they did not inhibit AGE-induced fibronectin synthesis. We concluded that the JAK2-STAT1/STAT3 signal transduction pathway is necessary for AGE-induced cellular mitogenesis in NRK-49F cells. In addition, STAT5 appears to modulate cyclin D1 synthesis and cdk4 activity, thereby contributing to AGE-induced cellular mitogenesis. On the other hand, we showed that RAGE-JAK2-STAT1/STAT3 pathway and MAPK kinase were involved in AGE-induced ECM production. Furthermore, renin-angiotensin system (RAS) may play an important role in AGE-induced biological effects.