Transcription Factor Regulation by the HBP

In the case of PAI-1, our laboratory has examined the role of the transcription factor Spl. Although the PAI-1 promoter can be stimulated by TGF-P1 via a Smad-binding

Fig. 3. Inhibition of GFA by DON blocks high glucose but not Glc stimulation of the PAI-1 promoter. Rat mesangial cells were transfected with a PAI-1 promoter (nucleotides from -740 to +44) fused to the luciferase reporter gene and then exposed to 20 mM, high glucose (glucose) or 2 mM Glc, in the presence and absence of DON to inhibit GFA. Cells were harvested and luciferase activity determined. Values are expressed relative to basal glucose defined as 1.0. (Adapted from ref. 33.)

Fig. 3. Inhibition of GFA by DON blocks high glucose but not Glc stimulation of the PAI-1 promoter. Rat mesangial cells were transfected with a PAI-1 promoter (nucleotides from -740 to +44) fused to the luciferase reporter gene and then exposed to 20 mM, high glucose (glucose) or 2 mM Glc, in the presence and absence of DON to inhibit GFA. Cells were harvested and luciferase activity determined. Values are expressed relative to basal glucose defined as 1.0. (Adapted from ref. 33.)

site, we found that HBP flux could increase gene expression independent of TGF-P1 (33). Other PAI-1 promoter sequences had been identified, which bound the transcription factor specificity protein 1 (Sp1), a known target of O-glycosylation (56). We were able to demonstrate that a PAI-1 promoter luciferase reporter gene was activated by Glc and mutation of the Spl-binding sites abolished this stimulation (Fig. 4). In keeping with at least one function of O-glycosylation, O-glycosylated Sp1 showed enhanced DNA binding (33). It was also shown that under hyperglycemic conditions, there is increased glycosylation and reciprocally decreased overall phosphorylation of Sp1 (34). It has been proposed that one mechanism by which protein O-glycosylation alters function is by competition with phosphorylation which may occur on the same or adjacent sites (9) (Fig. 5). It is important to note, however, that in the case of Spl, with at least nine potential O-glycosylation sites, it is possible that specific sites may regulate different or even opposing functions.

Although we noted that the enhanced DNA binding was relatively modest (~30%), there was a marked stimulation of the promoter (33). Thus, the transactiva-tion function of Sp1 was examined by fusing the entire transcription factor (holo Sp1) or only the transactivation domain (TAD) with the yeast GAL4 DNA-binding domain. This chimeric, fused Sp1 GAL4 was cotransfected into mesangial cells with an expression vector encoding a luciferse reporter gene driven by a GAL4-binding promoter (GAL4-Luc). Both Glc and high glucose strongly stimulated luciferase gene expression and the glucose effect was blocked by DON, a GFA enzyme inhibitor (57) (Fig. 6). Thus, it appeared that increased flux through the HBP could alter gene expression by modulating transcription factor function in at least two ways, namely, DNA binding and transactivation. The mechanism of altered transactivation could be direct, i.e., mediated by Sp1 O-glycosylation, or indirect, for example, glycosylation-independent and mediated by an upstream HBP product such as Glc-6-P, and/or O-glycosylation of other targets that regulate Sp1 function such as signaling proteins or transcriptional coactivators/corepressors. To test whether "upstream" HBP metabolites were involved, we took advantage of cells derived from embryos of EMeg32 knockout mice (58). Glc-6-P N-acetyltransferase (EMeg32) catalyzes the acetylation of Glc-6-P. The targeted deletion of EMeg32 is embryonic lethal but embryonic fibroblasts could be generated. These cells have

Fig. 4. Activation of the PAI-1 promoter by high glucose and Glc is dependent on Sp1 binding sites. (A) Schematic representation of the transcription factor Sp1, which contains nine potential sites of O-glycosylation on Ser/Thr residues. Note the Ser/Thr rich regions in the TAD. (B) A portion of the PAI-1 promoter (nucleotides from -740 to +44) fused to a luciferase reporter (PAI-1, open bars) or the PAI-1 promoter -Luc with the Sp1 sites mutated (Sp1 mut, filled bars) was transfected into rat mesangial cells which were subsequently exposed to 2 mM Glc, 20 ng/mL TGF-P, or 20 mM glucose. Cells were harvested and luciferase activity measured. Although Glc and high glucose stimulated the wild-type PAI-1 promoter, mutation of the Sp1 sites abolished this effect. However, TGF-P 1 was able to stimulate both wild type and mutant promoters, presumably via the intact smad, transcription factor, binding site. *p < 0.05 vs stimulation of wild-type promoter. (Adapted from ref. 33.)

Fig. 4. Activation of the PAI-1 promoter by high glucose and Glc is dependent on Sp1 binding sites. (A) Schematic representation of the transcription factor Sp1, which contains nine potential sites of O-glycosylation on Ser/Thr residues. Note the Ser/Thr rich regions in the TAD. (B) A portion of the PAI-1 promoter (nucleotides from -740 to +44) fused to a luciferase reporter (PAI-1, open bars) or the PAI-1 promoter -Luc with the Sp1 sites mutated (Sp1 mut, filled bars) was transfected into rat mesangial cells which were subsequently exposed to 2 mM Glc, 20 ng/mL TGF-P, or 20 mM glucose. Cells were harvested and luciferase activity measured. Although Glc and high glucose stimulated the wild-type PAI-1 promoter, mutation of the Sp1 sites abolished this effect. However, TGF-P 1 was able to stimulate both wild type and mutant promoters, presumably via the intact smad, transcription factor, binding site. *p < 0.05 vs stimulation of wild-type promoter. (Adapted from ref. 33.)

extremely low levels of UDP-GlcNAc that do not rise on exposure to elevated glucose. When transfected with the PAI-1 promoter luciferase-expressing cDNA, basal levels of expression were low and did not increase in response to glucose or Glc (Fig. 7). However, addition of serum did lead to an increase demonstrating the specificity of a lack of the HBP end product to high glucose-induced gene expression (not shown).

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