Upstream Stimulatory Factor (USF) Proteins Induce Human TGF-β1 Gene Activation via the Glucose-response Element–1013/–1002 in Mesangial Cells

Abstract

The hyperglycemia-enhanced flux through the hexosamine biosynthetic pathway (HBP) has been implicated in the up-regulated gene expression of transforming growth factor-␤1 (TGF-␤1) in mesangial cells, thus leading to mesangial matrix expansion and diabetic glomerulosclerosis. Since the ؊1013 to ؊1002 region of the TGF-␤1 promoter shows high homology to glucose-response elements (GlRE) formerly described in genes involved in glucose metabolism, we studied the function of the GlRE in the high glucose-induced TGF-␤1 gene activation in mesangial cells. We found that high glucose concentrations enhanced the nuclear amount of upstream stimulatory factors (USF) and their binding to this sequence. Fusion of the GlRE to the thymidine kinase promoter resulted in glucose responsiveness of this promoter construct. Overexpression of either USF-1 or USF-2 increased TGF-␤1 promoter activity 2-fold, which was prevented by mutation or deletion of the GlRE. The high glucose-induced activation of the GlRE is mediated by the HBP; increased flux through the HBP induced by high glucose concentrations, by glutamine, or by overexpression of the rate-limiting enzyme glutamine:fructose-6-phosphate aminotransferase (GFAT) particularly activated USF-2 expression. GFAT-overexpressing cells showed higher USF binding activity to the GlRE and enhanced promoter activation via the GlRE. Increasing O-GlcNAc modification of proteins by streptozotocin, thereby mimicking HBP activation, also resulted in increased mRNA and nuclear protein levels of USF-2, leading to enhanced DNA binding activity to the GlRE. USF proteins themselves were not found to be O-GlcNAcmodified. Thus, we have provided evidence for a new molecular mechanism linking high glucose-enhanced HBP activity with increased nuclear USF protein levels and DNA binding activity and with up-regulated TGF-␤1 promoter activity.The adverse effects of hyperglycemia in human and experimental diabetic nephropathy have been linked to the enhanced expression and bioactivity of the prosclerotic cytokine transforming growth factor-␤1 (TGF-␤1) 1 (1-3). The hyperglycemiainduced TGF-␤1 stimulates the production of extracellular matrix proteins in mesangial cells and other renal cells (4 -7), thus leading to the thickening of glomerular and tubular basement membranes and the progressive expansion of the glomerular mesangium and the tubulointerstitium (8, 9). Increased renal expression of TGF-␤1 has been found in experimental and human diabetes (2, 3) and has also been demonstrated in high glucose-treated renal mesangial and tubular cells (5, 6). The molecular mechanism of up-regulated human TGF-␤1 gene expression involves protein kinase C-and p38 MAPK-dependent pathways (10 -12) leading to AP-1 activation (13, 14) and subsequently enhanced TGF-␤1 promoter activity via two adjacent AP-1 binding sites located at Ϫ418/Ϫ412 and Ϫ371/ Ϫ363, respectively (13).Moreover, increased synthesis of amino sugars through the hexosamine biosynthetic pathway (HBP) has also been implicated in hyperglycemia-induced TGF-␤1 s...

Stimulation of Urinary TGF-β and Isoprostanes in Response to Hyperglycemia in Humans

“…Recent studies that evaluated glucose-induced TGF-␤1 production demonstrated a key role for the upstream stimulatory factor family of transcription factors in vitro as well as in vivo (9,10). The causative role of TGF-␤ in stimulating early gene expression of matrix molecules as well as mesangial matrix accumulation is supported by experiments with neutralizing antibodies in streptozotocin-induced diabetic mice and in db/db mice (11,12).…”

Glucose increases hepatic lipase expression in HepG2 liver cells through upregulation of upstream stimulatory factors 1 and 2

“…In various non-hepatoma cells, glucose has been shown to increase either nuclear USF1 or USF2 or both [32][33][34]. In HepG2 cells, glucose increases predominantly nuclear USF1, and silencing of USF1 but not USF2 abolished the increase in HL promoter activity.…”

“…In USF1 a number of polymorphisms have been reported [36,38,40], some of which are associated with unfavourable results in oral glucose and fat tolerance tests [41], increased adipocyte lipolysis [42] and decreased expression of USF target genes in fat biopsies [40]. In nonhepatic cells, glucose has been shown to increase nuclear expression of either USF1 or USF2 [31][32][33][34]. We hypothesised therefore that expression of USF1 or USF2 itself is subject to regulation by glucose.…”

“…In the liver, expression of the genes coding for glucokinase, fatty acid synthase, apolipoprotein (Apo)A-II, ApoA-V, ApoC-III and ApoE is upregulated by USF [24][25][26][27][28][29]. In liver as well as in other tissues, USFs play an important role in the regulation of genes by insulin [24][25][26] or glucose [30][31][32][33][34]. Interestingly, the USF1 gene on chromosome 1q21 has been linked with type 2 diabetes [35], FCHL [36,37] and both cardiovascular disease and all-cause mortality among women [38].…”

Rat strain-dependent susceptibility to ischemia-induced retinopathy associated with retinal vascular endothelial growth factor regulation

“…It has been reported that USF1 is associated with the familial combined hyperlipidemia and affects the complex lipid phenotype (Pajukanta et al 2004, Coon et al 2005. USF proteins induce human TGF-b1 gene activation via the glucose-response element (Weigert et al 2004). In order to determine whether the genetic variation of the USF transcription factor-binding site in the VEGF promoter region affects VEGF expression in OIR-BN rats, we performed genotyping of USF-binding site SNP allele and measured the retinal VEGF levels and vascular permeability in 97 F2 rats in response to ischemia.…”