ABSTRACT
Heme oxygenase-1 is the heme catabolic enzyme induced in human dermal fibroblasts by environmental stress. We report an increase of heme oxygenase-1 message in lens epithelial cells after exposure to UVA radiation, followed by a 10-fold increase of protein expression. The size of message was larger than previously demonstrated for fibroblasts. The relationship between heme oxygenase-1 activation and iron metabolism was investigated by measurement of activities of both cytosolic and mitochondrial cis-aconitase enzymes. A 2-fold increase in mitochondrial cis-aconitase activity in UVA-exposed cells coincided with the time of maximal heme oxygenase-1 expression. We propose that modulation of cis-aconitase activity at the translational level by an increase of cellular iron is an important consequence of heme oxygenase-1 activation. This might be a novel aspect of the protective role of heme oxygenase-1 in modulating the response of cells challenged with oxidative stress.
Subject(s)
Aconitate Hydratase/metabolism , Heat-Shock Proteins/biosynthesis , Heme Oxygenase (Decyclizing)/biosynthesis , Lens, Crystalline/enzymology , Lens, Crystalline/radiation effects , Animals , Cytosol/enzymology , Enzyme Induction , Epithelial Cells/enzymology , Epithelial Cells/radiation effects , Heme Oxygenase-1 , Mitochondria/enzymology , Rabbits , Ultraviolet RaysABSTRACT
Protein kinase C (PKC) plays a role in signal transduction mediated by interleukin-1beta (IL-1beta) leading to the increase in prostaglandin E2 (PGE2) production. In the present study we suggest that there are at least two distinct PKC isotypes involved in the signaling mechanism. Staurosporine potentiated the effect of IL-1beta on coxII mRNA expression while calphostin C totally inhibited mRNA expression. The down-regulation of PKC by growing mesangial cells in the presence of phorbol 12-myristate 13-acetate for 24 h failed to modify the up-regulated response in PGE2 formation by IL-1beta. Furthermore, incubation of mesangial cells with IL-1beta causes translocation of PKCzeta from cytosol to a presumed membrane compartment, and this translocation phenomenon was not inhibited by incubating the cells with staurosporine but was inhibited with calphostin C. Gel retardation assays also demonstrated that staurosporine did not inhibit the IL-1beta-stimulated binding of nuclear extracts to the NFkappaB motif. In contrast, calphostin C inhibited binding to the kappaB motif in a dose-dependent manner. Finally, antisense oligonucleotides to PKCzeta partially inhibited the IL-1beta-induced PGE2 formation while control sense oligonucleotides were without effect. Taken together, these data suggest that PKCzeta is involved in the IL-1beta signaling responses.
Subject(s)
Dinoprostone/metabolism , Glomerular Mesangium/metabolism , Interleukin-1/pharmacology , Prostaglandin-Endoperoxide Synthases/biosynthesis , Protein Kinase C/metabolism , Alkaloids/pharmacology , Animals , Base Sequence , Blotting, Northern , Cells, Cultured , Cytosol/metabolism , Enzyme Activation , Enzyme Inhibitors/pharmacology , Glomerular Mesangium/drug effects , Kinetics , Male , Molecular Sequence Data , NF-kappa B/metabolism , Naphthalenes/pharmacology , Oligonucleotide Probes , Oligonucleotides, Antisense/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/genetics , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Recombinant Proteins/metabolism , Restriction Mapping , Staurosporine , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic/drug effects , TransfectionABSTRACT
The pro-inflammatory cytokine, interleukin-1 beta, induces the mRNA for prostaglandin endoperoxide synthase II gene in renal mesangial cells. This inductive effect is selective for prostaglandin endoperoxide synthase II and not prostaglandin endoperoxide synthase I. In the present experiments IL-1 beta increased COX II mRNA, and this was inhibited by genistein and herbimycin A, both inhibitors of protein tyrosine kinases. The dose dependent effect of genistein on inhibition of mRNA for COX II correlated with the inhibition of the release of PGE2 into the media. Induction of COX II by interleukin-1 beta was mimicked by incubating the cells in the presence of a protein tyrosine phosphatase inhibitor, vanadate. These experiments also illustrate selective induction of COX II mRNA without induction of COX I mRNA. Western analysis utilizing antiphosphotyrosine antibodies demonstrated in whole lysates of mesangial cells treated with interleukin-1 beta that the transient phosphorylation of several proteins occurred. Interleukin-1 beta induced the transient phosphorylation of a protein of about 39/40 kD. Similarly, vanadate also produced a rapid and transient phosphorylation of a protein of about 39/40 kD in addition to other proteins. Immunoprecipitation of mesangial cell lysates with agarose conjugated antiphosphotyrosine antibody and Western analysis of precipitated proteins with anti-ERK2 antibody demonstrate that the 39/40 kD protein phosphorylated on tyrosine is ERK2 and suggests participation of one of the MAP kinase family of extracellular receptor kinases in IL-1 beta stimulated induction of the COX II gene.
Subject(s)
Glomerular Mesangium/drug effects , Interleukin-1/pharmacology , Prostaglandin-Endoperoxide Synthases/drug effects , RNA, Messenger/metabolism , Tyrosine/metabolism , Animals , Benzoquinones , Cells, Cultured , Dinoprostone/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Genistein , Glomerular Mesangium/enzymology , Isoflavones/pharmacology , Lactams, Macrocyclic , Male , Phosphorylation , Prostaglandin-Endoperoxide Synthases/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Quinones/pharmacology , RNA, Messenger/drug effects , Rats , Rats, Sprague-Dawley , Rifabutin/analogs & derivatives , Vanadates/pharmacologyABSTRACT
1. The form of Arrhenius plots of enzyme in mitochondria isolated from Drosophila melanogaster larvae exposed to heat shock, ethanol, or ethanol and heat shock, solubilized with charged detergents was analysed. 2. Heat shock and ethanol caused different changes in membrane microenvironment of the tyrosine transaminating activity, which found expression in different forms of Arrhenius plots, and different values of activation energy of enzyme. 3. The Arrhenius plots of the enzyme from mitochondria of larvae exposed both to ethanol and heat shock, solubilized with charged detergents, were similar to those observed for mitochondria from organisms exposed only to ethanol.
