High glucose levels upregulate upstream stimulatory factor 2 gene transcription in mesangial cells

Abstract

Previously, we demonstrated that upstream stimulatory factor 2 (USF2) mediates high glucose-induced thrombospondin1 (TSP1) gene expression and TGF-beta activity in glomerular mesangial cells and plays a role in diabetic renal complications. In the present studies, we further determined the molecular mechanisms by which high glucose levels regulate USF2 gene expression. In primary rat mesangial cells, we found that glucose treatment time and dose-dependently up-regulated USF2 expression (mRNA and protein). By using cycloheximide to block the de novo protein synthesis, similar rate of USF2 degradation was found under either normal glucose or high glucose conditions. USF2 mRNA stability was not altered by high glucose treatment. Furthermore, high glucose treatment stimulated USF2 gene promoter activity. By using the luciferase-promoter deletion assay, site-directed mutagenesis, and transactivation assay, we identified a glucose-responsive element in the USF2 gene promoter (-1,740 to -1,620, relative to the transcription start site) and demonstrated that glucose-induced USF2 expression is mediated through a cAMP-response element-binding protein (CREB)-dependent transactivation of the USF2 promoter. Furthermore, siRNA-mediated CREB knock down abolished glucose-induced USF2 expression. Taken together, these data indicate that high glucose levels up-regulate USF2 gene transcription in mesangial cells through CREB-dependent transactivation of the USF2 promoter.

Fig. 1.

High glucose exposure dose-dependently upregulated USF2 protein levels in rat mesangial cells. Primary rat mesangial cells (p4) were cultured and made quiescent. Cells were then treated with different concentrations of d-glucose media for 24 h. USF2 protein levels in nuclear extracts were determined by Western blotting. Relative USF2 levels were determined by scanning densitometry of immunoblots and normalized to β-actin levels. Results are the mean ±SE (n = 3). *P < 0.05 versus mM.

Fig. 2.

High glucose exposure time-dependently upregulated USF2 protein levels in rat mesangial cells. Quiescent mesangial cells were treated with 5 mM (NG) or 30 mM (HG) glucose media for indicated time period. Then cells were harvested. USF2 protein levels in nuclear extracts were determined by Western blotting. Relative USF2 levels were determined by scanning densitometry of immunoblots and normalized to β-actin levels. Results are the mean ±SE (n = 3). *P < 0.05 versus NG.

Fig. 3.

High glucose exposure had no effect on USF2 protein stability. Quiescent rat mesangial cells were treated with 5 mM (NG) (A) or 30 mM (HG) (B) glucose media for 24 h. Cells were then treated with cycloheximide (10 μg/ml) for indicated time period. After treatment, nuclear extracts were prepared and USF2 protein levels were determined by Western blotting. (C) Relative USF2 levels were determined by scanning densitometry of immunoblots and normalized to β-actin levels. Measurement of the ratio of USF2/actin at time = 0 was assigned a relative value of 100%. The results are expressed as mean ±SE of three separate experiments.

Fig. 4.

High glucose exposure time-dependently stimulated USF2 mRNA levels in rat mesangial cells. Quiescent rat mesangial cells were treated normal (5 mM) or high glucose (30 mM) media for indicated time points. After treatment, cells were harvested and total RNA was extracted. USF2 mRNA levels were determined by real-time PCR as described in Materials and Methods. The results are expressed as mean ±SE of three separate experiments. *P < 0.05 versus NG.

Fig. 5.

High glucose exposure had no effect on USF2 mRNA stability. Quiescent rat mesangial cells were treated with actinomycin D (5 μg/ml) in the presence of normal (5 mM) or high glucose (30 mM) media for indicated period. After treatment, cells were harvested and total RNA was extracted. Northern analysis and hybridization for USF2 and β-actin were performed as described in Materials and Methods to measure the rate of decay of USF2 mRNA and the half-life. Measurement of the ratio of USF2/actin at time = 0 was assigned a relative value of 100%. The results are expressed as mean ±SE of three separate experiments.

Fig. 6.

High glucose exposure stimulated the USF2 gene promoter activity and the region between −1,742 and −1,620 was involved. Quiescent rat mesangial cells were transiently transfected with mouse USF2 promoter luciferase construct (USF2 (−2,400)) (A) and a series deletion of USF2 promoter constructs (B) and then treated with normal or high glucose media for 24 h. After 24 h, cells were harvested and lysed. The luciferase activity was measured and normalized to Renilla luciferase levels. Data are shown with the mean ±SE of triplicates of three separate experiments. *P < 0.05 for HG versus NG.

Fig. 7.

Glucose-stimulated increases in the USF2 promoter activity was mediated by a CRE site located between −1,742 and −1,620 of the USF2 promoter. Quiescent rat mesangial cells were transiently transfected with −1,742 bp USF2 promoter constructs bearing mutations of the HSF, c-Myb, and CRE sites in the presence of normal or high glucose media for 24 h. The plasmid pRL-SV40 was used as internal control. After 24 h, cells were harvested and lysed. The luciferase activity was measured and normalized to Renilla luciferase level. Data are shown with the mean ±SE of triplicates of three separate experiments.

Fig. 8.

Chromatin immunoprecipitation analysis demonstrated the interaction of CREB with the USF2 promoter. A: Schematic representation showing CREB binding site. Two arrows indicate primers used for amplifying the region from −1,742 to −1,620 spanning the CRE site. B: Formaldehyde-cross-linked chromatin isolated from primary mesangial cells was immunoprecipitated (IP) with anti-CREB antibody and subjected to PCR as described under “Materials and Methods.” PCR products were electrophoresed on 2% agarose gel. The experiments were repeated for three times, and the representative result is shown.

Fig. 9.

CREB mediated glucose-induced USF2 expression in mesangial cells. A: Quiescent rat mesangial cells were transiently cotransfected with −1,742 bp USF2 promoter construct and an empty control vector or an expression vector overexpressing a dominant-negative CREB mutant (A-CREB) in the presence of normal or high glucose media for 24 h. After 24 h, cells were harvested and lysed. The luciferase activity was measured and normalized to Renilla luciferase level. Data are shown with the mean ±SE of triplicates of three separate experiments. *P < 0.05 versus HG. B: Mesangial cells were transfected with control siRNA or CREB-siRNA in the presence of normal or high glucose for 24 h. Nuclear proteins were analyzed for USF2 and total CREB protein expression by Western blotting. β-actin was used as internal control. The representative blots were shown from three separate experiments.