The p38/Reactivating Kinase Mitogen-activated Protein Kinase Cascade Mediates the Activation of the Transcription Factor Insulin Upstream Factor 1 and Insulin Gene Transcription by High Glucose in Pancreatic β-Cells

Abstract

Insulin upstream factor 1 (IUF1), a transcription factor present in pancreatic ␤-cells, binds to the sequence C(C/T)TAATG present at several sites within the human insulin promoter. Here we isolated and sequenced cDNA encoding human IUF1 and exploited it to identify the signal transduction pathway by which glucose triggers its activation. In human islets, or in the mouse ␤-cell line MIN6, high glucose induced the binding of IUF1 to DNA, an effect mimicked by serine/threonine phosphatase inhibitors, indicating that DNA binding was induced by a phosphorylation mechanism. The glucose-stimulated binding of IUF1 to DNA and IUF1-dependent gene transcription were both prevented by SB 203580, a specific inhibitor of stress-activated protein kinase 2 (SAPK2, also termed p38 mitogen-activated protein kinase, reactivating kinase, CSBP, and Mxi2) but not by several other protein kinase inhibitors. Consistent with this finding, high glucose activated mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase-2) (a downstream target of SAPK2) in MIN6 cells, an effect that was also blocked by SB 203580. Cellular stresses that trigger the activation of SAPK2 and MAP-KAP kinase-2 (arsenite, heat shock) also stimulated IUF1 binding to DNA and IUF1-dependent gene transcription, and these effects were also prevented by SB 203580.IUF1 expressed in Escherichia coli was unable to bind to DNA, but binding was induced by incubation with MgATP, SAPK2, and a MIN6 cell extract, which resulted in the conversion of IUF1 to a slower migrating form. SAPK2 could not be replaced by p42 MAP kinase, MAP-KAP kinase-2, or MAPKAP kinase-3. The glucose-stimulated activation of IUF1 DNA binding and MAPKAP kinase-2 (but not the arsenite-induced activation of these proteins) was prevented by wortmannin and LY 294002 at concentrations similar to those that inhibit phosphatidylinositide 3-kinase. Our results indicate that high glucose (a cellular stress) activates SAPK2 by a novel mechanism in which a wortmannin/LY 294002-sensitive component plays an essential role. SAPK2 then activates IUF1 indirectly by activating a novel IUF1-activating enzyme.

Effects of rosiglitazone and metformin on pancreatic beta cell gene expression

“…In the present study, rosiglitazone had no effect on the 200 bp section of the human proinsulin gene promoter analysed. This section of the promoter has been shown in previous studies to contain key regulatory elements essential for the control of proinsulin gene transcription [10,13,23]. Although our data do not exclude the possibility that further elements which may respond to rosiglitazone lie outside this region, our findings are consistent with previous studies which show 100 μmol/l rosiglitazone has no effect on proinsulin mRNA levels in INS1 cells over a 24-h period [24].…”

“…These data indicate that metformin rapidly stimulates an increase in IPF1 DNA binding activity in pancreatic beta cells, and that rosiglitazone has no effect over this shorter period. Probe specificity was confirmed by mutant oligonucleotide competition and antibody competition EMSA analysis (not shown) as in previous studies [13,23]. Using the same nuclear extracts, Western blotting analysis was performed to determine the level of nuclear IPF1 (Fig.…”

“…The construct pGL-Luc200 is derived from the pGL2 vector (Promega, Southampton, UK) and contains 200 bp of the human proinsulin gene promoter upstream of the firefly luciferase reporter gene, as previously described [13]. The control construct Luc lacks the proinsulin gene promoter fragment.…”

“…Electrophoretic mobility shift assays EMSA analysis was performed using the DIG Gel Shift Kit (Roche Molecular Biochemicals, Mannheim, Germany) using Probe B, containing the A3 site of the human proinsulin gene promoter [13]. Five micrograms of each nuclear extract, 0.4 ng of digoxigenin-labelled probe, 1× binding buffer, 1 μg (poly-deoxy-inosinic-deoxy-cytidylic acid) and 0.1 μg poly-L-lysine were incubated at room temperature for 20 min, fractionated on 6% polyacrylamide gels, and blotted onto nylon membrane, blocked for 30 min.…”

“…IPF1 accumulates in the beta cell nucleus in response to elevated glucose concentrations, events mediated by post-translational modification of the protein [13][14][15]. Expression of the Ipf1 gene is also regulated in response to glucose [16].…”