Upstream stimulatory factor‐2 mediates quercetin‐induced suppression of PAI‐1 gene expression in human endothelial cells

The polyphenol quercetin (Quer) represses expression of the cardiovascular disease risk factor plasminogen activator inhibitor‐1 (PAI‐1) in cultured endothelial cells (ECs). Transfection of PAI‐1 promoter‐luciferase reporter deletion constructs identified a 251‐bp fragment (nucleotides ?800 to ?549) responsive to Quer. Two E‐box motifs (CACGTG), at map positions ?691 (E‐box1) and ?575 (E‐box2), are platforms for occupancy by several members of the c‐MYC family of basic helix‐loop‐helix leucine zipper (bHLH‐LZ) proteins. Promoter truncation and electrophoretic mobility shift/supershift analyses identified upstream stimulatory factor (USF)‐1 and USF‐2 as E‐box1/E‐box2 binding factors. ECs co‐transfected with a 251 bp PAI‐1 promoter fragment containing the two E‐box motifs (p251/luc) and a USF‐2 expression vector (pUSF‐2/pcDNA) exhibited reduced luciferase activity versus p251/luc alone. Overexpression of USF‐2 decreased, while transfection of a dominant‐negative USF construct increased, EC growth consistent with the known anti‐proliferative properties of USF proteins. Quer‐induced decreases in PAI‐1 expression and reduced cell proliferation may contribute, at least in part, to the cardioprotective benefit associated with daily intake of polyphenols. J. Cell. Biochem. 111: 720–726, 2010. © 2010 Wiley‐Liss, Inc.     

Upstream stimulatory factor (USF) proteins induce human TGF-beta1 gene activation via the glucose-response element-1013/-1002 in mesangial cells: up-regulation of USF activity by the hexosamine biosynthetic pathway

The hyperglycemia-enhanced flux through the hexosamine biosynthetic pathway (HBP) has been implicated in the up-regulated gene expression of transforming growth factor-beta1 (TGF-beta1) in mesangial cells, thus leading to mesangial matrix expansion and diabetic glomerulosclerosis. Since the -1013 to -1002 region of the TGF-beta1 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-beta1 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-beta1 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-GlcNAc-modified. 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-beta1 promoter activity.     

An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc.

The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.