Differential effects of protein kinase C inhibitors on fibronectin-induced interleukin-beta gene transcription, protein synthesis and secretion in human monocytic cells.
Human monocytic cells express interleukin-1beta (IL-1beta) when stimulated with the extracellular matrix glycoprotein, fibronectin (FN). Protein kinase C (PKC) activation is considered important for this process; however, the metabolic steps at which PKC acts upon to mediate the FN-induced IL-1beta response remain unclear. We performed an analysis of the mechanisms by which two PKC inhibitors, Calphostin C and Staurosporine, prevent the FN-induced IL-1beta response. Both inhibitors blocked the secretion of IL-1beta protein into the media of peripheral blood mononuclear cells exposed to FN. Immunoprecipitation analysis revealed that under these circumstances, Calphostin C inhibited the production of IL-1beta protein, whereas Staurosporine allowed protein production, but inhibited its secretion. To determine the mechanisms responsible for these differences, we turned to human U937 promonocytic cells. U937 cells transfected with the human full-length IL-1beta promoter connected to a luciferase reporter gene were submitted to transcription assays, Northern blotting, and DNA electrophoresis mobility gel shift assays. These studies revealed that Calphostin C inhibited the nuclear translocation of the transcription factor activator protein-1 (AP-1) which is considered necessary for FN induction of IL-1beta gene transcription, and prevented the transcription of the IL-1beta gene. In contrast, Staurosporine alone induced AP-1 translocation and stimulation of the gene. Overall, our data indicate that Calphostin C prevents the transcription of the IL-1beta gene thereby inhibiting protein synthesis. Based on the high specificity of this compound for PKC, we conclude that PKC is necessary for FN-induced IL-1beta protein production. In contrast, Staurosporine prevented secretion of IL-1beta by unknown mechanisms.
Ritzenthaler
J
J
Pulmonary & Critical Care Division, Department of Medicine, Atlanta Veterans Affairs Medical Center and Emory University School of Medicine, Atlanta, GA, USA.
Roman
J
J
D015152:Blotting, Northern; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004586:Electrophoresis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005353:Fibronectins / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology*; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D019311:Staurosporine / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D020298:U937 Cells
D004791:Enzyme Inhibitors; D005353:Fibronectins; D007375:Interleukin-1; D009281:Naphthalenes; D018808:Transcription Factor AP-1; D011493:Protein Kinase C; D019311:Staurosporine; C058819:calphostin C