title: Calphostin C induces AP1 synthesis and AP1-dependent c-jun transactivation in normal human chondrocytes independent of protein kinase C-alpha inhibition: possible role for c-jun N-terminal kinase.
abstract: Activator protein-1 (AP1) regulates the promoter activity of a large number of genes associated with developmental, proliferative, inflammatory, and homeostatic processes in human connective tissue cells. Some of these genes (e.g., cyclooxygenase-2) are regulated by the protein kinase C (PKC) inhibitor, calphostin C (CalC). We examined whether CalC could indeed induce AP1 and AP1 gene transactivation (c-jun) in human chondrocytes. Exploratory studies confirmed the anti-PKC effects of CalC, as equal molar concentrations of CalC blocked the PMA-induced translocation of PKC-alpha from the cytosolic to the membrane fraction. CalC induction of AP1, as judged by gel-shift analysis, using a consensus AP1 oligonucleotide, was biphasic with an initial increase (maximum 4 h), followed by a decline, reaching its nadir after 16 h, and finally a major upregulation phase at 24 h. Maximum induction of AP-1 was reached at a concentration of 250 nmol/L of CalC. CalC did not block PMA-induced AP1 synthesis. Gel-shift analysis in the presence of specific antibodies to c-Jun, JunB, JunD, c-Fos, and CREB/ATF showed that the AP1 complexes were probably c-Jun/c-Jun, c-Fos/c-Jun, c-Fos/JunB, or c-Jun/JunB dimers. Northern blot analysis confirmed that c-jun, junB, and c-Fos were the principal proto-oncogenes induced by CalC. To confirm that c-jun induction occurs at the transcriptional level and to examine the role of the AP1 site present in the c-jun promoter in the induction of c-jun by CalC, we performed transient transfections of c-jun promoter-CAT constructs harboring either wild-type (WT) AP1 regulatory element sites or mutant AP1 sites. CalC (250 nmol/L) induced a marked increase in CAT activity (i.e., promoter activation) with WT AP1 c-jun promoter-CAT plasmids, but the response was completely abrogated when using constructs where the AP1 site was mutated. PMA produced similar results, but the induction of the WT AP1 c-jun promoter-CAT plasmid was smaller. CalC (250 nmol/L) inhibited MAPK (p42/44) activity while stimulating c-Jun N-terminal kinase activity in a time-frame coincident with the activation of AP1. We conclude that CalC induces signaling pathways that activate AP1 and transactivate genes harboring AP1 enhancer sites independent of PKC-alpha.
authors.affiliation: Department of Medicine, University of Montreal, Montreal H3C 3J7, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Zhang
authors.forename: C
authors.initials: C
authors.lastname: Miller
authors.forename: Y
authors.initials: Y
authors.lastname: He
authors.forename: J
authors.initials: J
authors.lastname: Martel-Pelletier
authors.forename: J P
authors.initials: JP
authors.lastname: Pelletier
authors.forename: J A
authors.initials: JA
authors.lastname: Di Battista
mesh_terms: D001483:Base Sequence; D001693:Biological Transport, Active / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D016762:Genes, fos / Q000187:drug effects; D016758:Genes, jun / Q000187:drug effects*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051571:Protein Kinase C-alpha; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000096:biosynthesis*; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D017931:DNA Primers; D007527:Isoenzymes; D009281:Naphthalenes; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate