Stimulation of MAPK-phosphatase 1 gene expression by glucocorticoids occurs through a tethering mechanism involving C/EBP.
Glucocorticoids (GCs) are known to inhibit mitogen-activated protein kinase (MAPK) signaling. This has been suggested to involve induced expression of MAPK-phosphatase 1 (DUSP1), which dephosphorylates and inactivates MAPKs. However, the mechanism for the transcriptional activation by GCs of DUSP1 or the identification of a GC-responsive region of the gene has so far not been described. To identify GC receptor (GR) binding to the human DUSP1 promoter in vivo, we used a chromatin immunoprecipitation (ChIP) assay and found GR to bind to a region approximately -1.4 kb upstream of the transcription start site. Using promoter deletion constructs, we identified a GC-responsive region between position -1266 and -1380 bp of the DUSP1 promoter. However, no direct binding of GR to this GC-responsive region was detected in an electrophoretic mobility shift assay (EMSA). Instead, we identified binding of CCAAT/enhancer-binding protein beta (C/EBPbeta) to a region between -1311 and -1304 bp of the DUSP1 promoter by EMSA and ChIP. Furthermore, mutation of the C/EBP binding site resulted in a dramatic loss of GC-inducible reporter gene expression, demonstrating the GC responsiveness of the DUSP1 gene to be located to a binding site for C/EBP in the DUSP1 promoter. Also, given that a GR mutant (GR(LS7)), incapable of transactivating through GC-responsive elements, still was able to bind to the DUSP1 gene in vivo and induce DUSP1 mRNA expression following treatment with GCs suggests the mode of GC activation to be mediated by a tethering mechanism involving the GR and the DUSP1 promoter-bound C/EBPbeta.
Johansson-Haque
Krishan
K
Department of Biosciences and Nutrition, Karolinska Institutet, Karolinska University Hospital Huddinge, Novum, SE-141 86 Huddinge, Sweden. krishan.johansson.haque@ki.se
Palanichamy
Elanchelian
E
Okret
Sam
S
D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D022762:CCAAT-Enhancer-Binding Proteins; D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D019950:Mitogen-Activated Protein Kinase 1