title: Phosphorylation of cAMP responsive element binding protein after treatment of mesangial cells with high glucose plus TGF beta or PMA.
abstract: We recently showed that mesangial cells treated with high glucose plus TGF beta or PMA demonstrated activation of a cAMP-response element (CRE) located in the 5' flanking region of the fibronectin gene. Gel shift mobility assays with a CRE oligonucleotide revealed multiple complexes that did not change in mobility or abundance under conditions of high glucose plus TGF beta or PMA. Here we show that treatment with cycloheximide to inhibit protein synthesis also did not change the DNA/protein complexes. These observations led us to conclude that post-translational modification of transcription factors may be responsible for the activation of the fibronectin gene observed under our experimental conditions. We identified the proteins complexed to CRE as CRE binding protein (CREB) and activating factor 1 (ATF1). This was accomplished by supershift assays and immunoblots. Two hours of high glucose plus TGF beta or 30 minutes of PMA caused a twofold elevation in phosphorylated CREB. Neither high glucose nor TGF beta alone caused phosphorylation of CREB. ATF-1 was not phosphorylated. We also show that high glucose plus TGF beta and PMA activated protein kinase C alpha; however, none of the agents tested stimulated intracellular cAMP levels, indicating that phosphorylation of CREB was independent of protein kinase A activation. These results demonstrate cross-talk between the protein kinase C and protein kinase A pathways in that agents which activate the protein kinase C pathway can stimulate phosphorylation of proteins that commonly serve as substrates for protein kinase A.
authors.affiliation: Audie Murphy Veteran's Administration Hospital, San Antonio, Texas, USA.
authors.forename: J I
authors.initials: JI
authors.lastname: Kreisberg
authors.forename: R A
authors.initials: RA
authors.lastname: Radnik
authors.forename: S H
authors.initials: SH
authors.lastname: Kreisberg
mesh_terms: D051697:Activating Transcription Factor 1; D000818:Animals; D000918:Antibody Specificity; D002273:Carcinogens / Q000494:pharmacology*; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D017362:Cyclic AMP Response Element-Binding Protein / Q000276:immunology / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis; D004586:Electrophoresis; D005353:Fibronectins / Q000235:genetics; D005920:Glomerular Mesangium / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D015151:Immunoblotting; D007527:Isoenzymes / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014157:Transcription Factors / Q000032:analysis / Q000276:immunology; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
chemical_list: D051697:Activating Transcription Factor 1; D002273:Carcinogens; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D005353:Fibronectins; D007527:Isoenzymes; D012333:RNA, Messenger; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; C497257:Prkce protein, rat; C087857:protein kinase C gamma; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate