Aim: The previous studies demonstrated that transforming growth factor-beta(1) (TGF-beta(1)) could upregulate the expression of aldose reductase (AR). The aim of this study is to clarify (investigate) the mechanism of TGF-beta(1)-induced AR expression.
Methods: Real-time polymerase chain reaction and western blot were used to analyse the AR expression in mRNA and protein levels in human mesangial cells, and reporter assay was used to analyse the function of various sites within 5'-flanking region of AR gene in AR expression.
Results: TGF-beta(1) (4 ng/mL) stimulation could upregulate AR expression. The cells pretreated with pharmacological inhibitors, U0126 and PD98059 for blocking extracellular signal-related kinase (ERK) signalling pathway or SP6000125 for blocking c-Jun N-terminal kinase (JNK) signalling pathway, respectively, showed reduced expression of AR after TGF-beta(1) stimulation. Similarly, the cells transiently transfected with pCMVTAM67, which is an expression plasmid for DN-c-Jun showed decreasing AR expression. Reporter assay revealed that the 5'-promoter region of AR consisting of an AP-1 site and two putative antioxidant response elements (ARE) was responsible for TGF-beta(1) stimulation. Mutation of either ARE did not affect the promoter activity in the reporter assay while mutation of AP-1 site caused a significant decrease in the responsiveness to TGF-beta(1).
Conclusion: TGF-beta(1) upregulate AR expression in both mRNA and protein levels. The results demonstrated that ERK and JNK are involved in the downstream signalling pathways and transcription factor AP-1 plays an important role in the regulation of TGF-beta(1)-induced AR expression in mesangial cells.