Role of upstream stimulatory factors in regulation of renal transforming growth factor-beta1.

We previously identified an E-box to be implicated in high-glucose-induced transforming growth factor-beta1 (TGF-beta1) gene stimulation in murine mesangial cells. In the present study, we evaluated the role of upstream stimulatory factors (USFs) in mediating glucose-induced stimulation of TGF-beta1. Mesangial cells cultured in glucose concentrations exceeding 2.7 mmol/l D-glucose exhibited increased levels of USF1 and USF2 protein by Western analysis and electrophoretic mobility shift assay (EMSA). An E-box element from the murine TGF-beta1 promoter revealed USF1 and USF2 binding by EMSA. Chromatin immunoprecipitation assay revealed in vivo binding of USF1 to a glucose-responsive region of the TGF-beta1 promoter. Transient cotransfection studies of 293 cells with USF1 led to a twofold increase in TGF-beta1 promoter activity and a 46% increase in secreted TGF-beta1 protein levels. Wild-type and USF2 knockout mice exhibited a 2.5-fold stimulation of renal TGF-beta1 expression upon fasting and refeeding with a carbohydrate-rich diet, whereas USF1 knockout mice exhibited only a minimal increase of renal TGF-beta1 upon refeeding. USF1 mRNA levels were increased in mouse kidneys with carbohydrate refeeding, and USF1 protein was increased in diabetic rat kidneys compared with controls. We conclude that USF1 is stimulated by modest increases in glucose concentration in murine mesangial cells, bind to the murine TGF-beta1 promoter, contribute to carbohydrate-induced renal TGF-beta1 expression, and may play a role in diabetes-related gene regulation in the kidney.