Interleukin-1β Activates Protein Kinase Cζ in Renal Mesangial Cells
1996; Elsevier BV; Volume: 271; Issue: 29 Linguagem: Inglês
10.1074/jbc.271.29.17241
ISSN1083-351X
AutoresDanuta M. Rzymkiewicz, Toshifumi Tetsuka, Dorit Daphna-Iken, Sunil Kumar Srivastava, Aubrey R. Morrison,
Tópico(s)Cell Adhesion Molecules Research
ResumoProtein kinase C (PKC) plays a role in signal transduction mediated by interleukin-1β (IL-1β) 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-1β 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-1β. Furthermore, incubation of mesangial cells with IL-1β causes translocation of PKCη 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-1β-stimulated binding of nuclear extracts to the NFκB motif. In contrast, calphostin C inhibited binding to the κB motif in a dose-dependent manner. Finally, antisense oligonucleotides to PKCη partially inhibited the IL-1β-induced PGE2 formation while control sense oligonucleotides were without effect. Taken together, these data suggest that PKCη is involved in the IL-1β signaling responses. Protein kinase C (PKC) plays a role in signal transduction mediated by interleukin-1β (IL-1β) 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-1β 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-1β. Furthermore, incubation of mesangial cells with IL-1β causes translocation of PKCη 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-1β-stimulated binding of nuclear extracts to the NFκB motif. In contrast, calphostin C inhibited binding to the κB motif in a dose-dependent manner. Finally, antisense oligonucleotides to PKCη partially inhibited the IL-1β-induced PGE2 formation while control sense oligonucleotides were without effect. Taken together, these data suggest that PKCη is involved in the IL-1β signaling responses. INTRODUCTIONActivation of protein kinase C (PKC) 1The abbreviations used are: PKCprotein kinase CIL-1βinterleukin-1βPMAphorbol 12-myristate 13-acetateNGFnerve growth factorPGE2prostaglandin E2. plays a major role in agonist-stimulated function in a variety of cell types (1Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (918) Google Scholar, 2Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1216) Google Scholar, 3Hata A. Akita Y. Suzuki K. Ohno S. J. Biol. Chem. 1993; 268: 9122-9129Abstract Full Text PDF PubMed Google Scholar). To date there are at least 12 isotypes of PKC described and identified as α, βI, βII, γ, δ, ε, η, η, θ, τ, λ, and µ. These PKC isotypes are unique with respect to their primary structure, expression patterns, subcellular localization, and responsiveness to extracellular ligands. Recent reviews have highlighted the evidence that the isotypes might have separate and unique functions in the cell (2Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1216) Google Scholar). Activation of PKC is associated with its translocation from the cytosolic (soluble) fraction to the particulate (membrane) fraction (4Woelf M. Cuatrecasas P. Sahyoun N. J. Biol. Chem. 1985; 260: 15718-15722Abstract Full Text PDF PubMed Google Scholar, 5Dang P.M.-C. Hakim J. Périanin A. FEBS Lett. 1994; 349: 338-342Crossref PubMed Scopus (66) Google Scholar). Furthermore, prolonged activation of some cells with phorbol esters is associated with the binding of phorbol esters to the kinase and resultant down-regulation. PKCη does not appear to bind phorbol esters (6Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3099-3103Crossref PubMed Scopus (413) Google Scholar) and is not associated with the down-regulation of PKCη (7Wooten M.W. Zhou G. Seibenhener M.L. Coleman E.S. Cell Growth & Differ. 1994; 5: 395-403PubMed Google Scholar). The Cys-rich region within the C1 domain of PKC consists of two zinc finger motifs with six cysteine residues each and a homologous DNA-binding motif found in transcription factors like GAL4 (8Pan T. Coleman J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2077-2081Crossref PubMed Scopus (161) Google Scholar). The use of deletion mutants of different PKC isotypes has revealed that the Cys-rich region is necessary for diacylglycerol and phorbol ester binding (9Burns D.J. Bell R.M. J. Biol. Chem. 1991; 266: 18330-18338Abstract Full Text PDF PubMed Google Scholar, 10Muramatsu M.A. Katbuchi K. Arai K. Mol. Cell. Biol. 1989; 9: 831-836Crossref PubMed Scopus (76) Google Scholar). PKCη contains only one zinc finger and does not bind diacylglycerol or phorbol ester (6Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3099-3103Crossref PubMed Scopus (413) Google Scholar).The observations that PKCη is resistant to staurosporine (11Tischler A.S. Ruzicka L.A. Dobner P.R. J. Biol. Chem. 1991; 266: 1141-1146Abstract Full Text PDF PubMed Google Scholar, 12Seynaeve C.M. Kazanietz M.G. Blumberg P.M. Sausville E.A. Worland P.J. Mol. Pharmacol. 1994; 45: 1207-1214PubMed Google Scholar), appears to be critical for mitogenic signal transduction (13Berra E. Diaz-Meco M.T. Dominguez I. Municio M.M. Sanz L. Lozano J. Chapkin R.S. Moscat J. Cell. 1993; 74: 555-563Abstract Full Text PDF PubMed Scopus (342) Google Scholar), and is expressed in the rat mesangial cell (14Huwiler A. Fabbro D. Stabel S. Pfeilschifter J. FEBS Lett. 1992; 300: 259-262Crossref PubMed Scopus (59) Google Scholar) led us to evaluate its role in the signal transduction process resulting in an up-regulation of PGE2 in this cell type and whether this pathway of signaling is relevant to transcriptional activation of the coxII gene by IL-1β.RESULTSTo assess the effects of protein kinase C inhibition on IL-1β-induced expression of coxII, we incubated mesangial cells with and without IL-1β and the protein kinase C inhibitors H7 and staurosporine. Fig. 1 demonstrates that IL-1β induces the message for coxII but not for coxI. In addition, staurosporine at both 10 and 100 nM produced a marked potentiation of coxII induction by IL-1β. Furthermore, staurosporine alone at a concentration of 100 nM induced coxII. Interestingly, H7 alone had no effect on coxII expression; however, it seemed to potentiate the effect of IL-1β on coxII expression although to a lesser extent than staurosporine. Because of these results, we carried out another series of experiments comparing the effects of staurosporine and calphostin C, and in addition, we added a PKC activator, PMA, as a positive control. Fig. 2 shows the results of a Northern analysis of such an experiment. As in Fig. 1, IL-1β stimulated coxII expression (lanes 2 and 3), which was potentiated by 100 nM staurosporine. PMA alone also enhanced expression of coxII (lane 4), but somewhat surprisingly, this effect was potentiated by staurosporine (lane 6). In contrast to the effects of staurosporine, calphostin C inhibited both the effects of IL-1β and PMA on coxII expression (lanes 8 and 9). We considered two possibilities to explain these observations. First, we hypothesized that IL-1β stimulated coxII expression through the participation of protein kinase C since it was calphostin C-inhibitable. We further hypothesized that this protein kinase C was staurosporine-insensitive. Another possibility was that staurosporine was exerting effects on the mesangial cells independent of its protein kinase C inhibitory activity. In an attempt to test whether protein kinase C was involved in IL-1β signaling, we assessed the effects of protein kinase C down-regulation by PMA on IL-1β induction of PGE2. Fig. 3 shows such an experiment. Mesangial cells were first incubated with 500 nM PMA for 24 h followed by IL-1β for an additional 24 h and then compared with untreated cells for their ability to increase PGE2 formation. This experiment demonstrates that down-regulation of PKC does not modify the cellular response to IL-1β. The results thus far were consistent with the involvement of a staurosporine-resistant (12Seynaeve C.M. Kazanietz M.G. Blumberg P.M. Sausville E.A. Worland P.J. Mol. Pharmacol. 1994; 45: 1207-1214PubMed Google Scholar), calphostin C-sensitive PKC that was not down-regulated by PMA (7Wooten M.W. Zhou G. Seibenhener M.L. Coleman E.S. Cell Growth & Differ. 1994; 5: 395-403PubMed Google Scholar). PKCη fits these requirements and is expressed in renal mesangial cells (14Huwiler A. Fabbro D. Stabel S. Pfeilschifter J. FEBS Lett. 1992; 300: 259-262Crossref PubMed Scopus (59) Google Scholar). Therefore, we evaluated the effect of IL-1β on PKCη translocation from the cytosol in renal mesangial cells by immunoblot analysis. Fig. 4 shows such an experiment. Fig. 4B shows a time course of immunologically detectable PKCη in the cytosol from cells treated with IL-1β for 0 (control), 5, 15, and 30 min. Fig. 4A shows a similar Western blot obtained from cells treated with IL-1β but that had been treated with 100 nM staurosporine 10 min prior to the addition of IL-1β. Fig. 4C shows a Western blot obtained from cells treated with IL-1β in the presence of 2 µM calphostin C 15 min prior to the addition of IL-1β. Fig. 4D shows the densitometric quantitation of the 68-kDa PKCη bands from Fig. 4, A-C (arrow). It can be seen that PKCη was translocated from the cytosol in response to IL-1β, and this translocation was unaffected by staurosporine. In contrast with pretreatment of cells with calphostin C, IL-1β failed to initiate translocation of PKCη. These experiments represent a mean of two in duplicate. Control experiments carried out with a 10-fold molar excess of PKCη carboxyl-terminal peptide completely inhibited antibody detection of the 67-kDa protein (data not shown).Fig. 2Northern analysis of total mesangial cell RNA probed with coxII (COX 2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 50 units/ml IL-1β, 100 nM PMA, 100 nM staurosporine (Stauro), and 5 µM calphostin C (Cal C) were used.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3PGE2 production by mesangial cells in response to IL-1β. Control () cells that were and cells PKC down-regulated by incubation with 100 nM PMA (□) for 24 h prior to stimulation with IL-1β are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Immunoblot analysis of mesangial cell cytosol. Cytosol was obtained from cells induced with IL-1β for 0, 5, 15, and 30 min. A, cells treated with IL-1β and 100 nM staurosporine; B, cells treated with IL-1β alone; C, cells treated with calphostin C and then IL-1β for 0, 10, and 30 min; and D, quantitative densitometry of 67-kDa PKCη bands from A, B, and C. ▴, IL-1/β+ staurosporine; ▵, IL-1β alone; ○, calphostin C + IL-1β.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since IL-1β induces the translocation of a factor to the nucleus that binds the κB motif in the proximal promoter of pghs2 (coxII), we determined the effect of staurosporine and calphostin C on the IL-1-stimulated binding by electromobility shift assays. Fig. 5A shows that IL-1β induces binding of a factor to the oligonucleotide κB motif, and this binding event was not influenced by staurosporine. In contrast, Fig. 5B shows that calphostin C produced a dose-dependent inhibition of binding, again suggesting the involvement of a staurosporine-insensitive but calphostin C-sensitive activation of NFκB in the renal mesangial cell stimulated with IL-1β.Fig. 5Gel shift assays of nuclear extracts. Electrophoretic mobility shift assays demonstrate: A, IL-1β-induced binding of nuclear extracts with κB motif that is not inhibited at 10 and 100 nM staurosporine (Stauro); B, similar experiments showing dose-dependent inhibition of binding with 2 and 5 µM calphostin C (Cal-C).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the activation of PKCη by IL-1β was involved in the up-regulation of PGE2 formation in the renal mesangial cell, we utilized antisense oligonucleotides to PKCη and transfected mesangial cells and assayed the PGE2 released into the media. Experiments using sense oligonucleotides transfected into mesangial cells were used as controls. Fig. 5 shows that the PKCη antisense oligonucleotide inhibited PGE2 formation by 37% (p < 0.05) while the sense control oligonucleotide did not inhibit PGE2 formation in response to IL-1β. Fig. 6 represents four experiments, and the data are expressed as the mean ± S.E. Fig. 7 shows a representative Western blot of cellular extracts obtained from control cells (nontransfected) and cells transfected with sense and with antisense oligonucleotides. It demonstrates that the antisense oligonucleotide inhibited CoxII protein expression by 40%.Fig. 6Transfection of mesangial cells with oligonucleotides. Effect of sense (S) and antisense (AS) PKCη oligonucleotides on mesangial cells transfected with this DNA. Figure shows percent inhibition of PGE2 formation after stimulation with IL-1β. n = 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Western blot of mesangial cell protein. Whole cell lysates of mesangial cells and cells transfected with sense (S) and antisense (AS) oligonucleotides were used. The effect of IL-1β on CoxII protein expression is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the antisense oligonucleotide interrupted the synthesis of PKCη, we performed Western blots on cytosolic extracts of mesangial cells and compared the expression of PKCη in control cells, cells treated with IL-1β for 30 min, and cells transfected with antisense oligonucleotides to PKCη as described previously. Fig. 8 shows such an experiment. Lane 1 shows the expression of PKCη in 10 µg of cytosolic protein in control cells. Lane 2 shows the levels detected in cytosol from similar amounts of protein from IL-1β-treated cells, and lane 3 represents cytosol from antisense transfected cells. These experiments demonstrate that the antisense oligonucleotide reduced the level of expression of PKCη in cell cytosol.Fig. 8Effect of antisense oligonucleotide on PKCη expression. Cytosol from mesangial cells (10 µg) in control (lane 1), cells treated with IL-1β for 30 min (lane 2), and cells transfected with antisense oligonucleotide to PKCη (lane 3) were blotted with antipeptide antibody to PKCη.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONIn an attempt to assess the functional contribution of PKC as a signaling mechanism recruited by IL-1β in the activation of coxII gene transcription, we have utilized a pharmacological approach using PKC inhibitors. These experiments demonstrate that staurosporine alone at 100 nM induced mRNA for coxII and markedly potentiated the effects of IL-1β and PMA on inducing coxII mRNA. In contrast, calphostin C alone had no effect on coxII mRNA expression but completely inhibited the ability of IL-1β and PMA to increase coxII mRNA. These surprising results could be explained by suggesting that a PKC isotype that was both calphostin C inhibitable and staurosporine resistant was involved in IL-1β-stimulated coxII expression.Since the renal mesangial cell expresses multiple isotypes including α, γ, ε, and η (14Huwiler A. Fabbro D. Stabel S. Pfeilschifter J. FEBS Lett. 1992; 300: 259-262Crossref PubMed Scopus (59) Google Scholar, 19Huwiler A. Fabbro D. Pfeilschifter J. Biochem. J. 1991; 279: 441-445Crossref PubMed Scopus (76) Google Scholar, 20Huwiler A. Fabbro D. Pfeilschifter J. Biochem. Biophys. Res. Commun. 1991; 180: 1422-1428Crossref PubMed Scopus (27) Google Scholar) and since PKCη is both staurosporine-insensitive (12Seynaeve C.M. Kazanietz M.G. Blumberg P.M. Sausville E.A. Worland P.J. Mol. Pharmacol. 1994; 45: 1207-1214PubMed Google Scholar) and calphostin C-sensitive (21Larivée P. Levine S.J. Martinez A. Wu T. Logun C. Shelhamer J.H. Am. J. Respir. Cell Mol. Biol. 1994; 11: 199-205Crossref PubMed Scopus (45) Google Scholar) and is not down regulated by PMA (7Wooten M.W. Zhou G. Seibenhener M.L. Coleman E.S. Cell Growth & Differ. 1994; 5: 395-403PubMed Google Scholar), we postulated that if PKCη was involved in the IL-1β signaling pathway leading to coxII expression, then down-regulation of PKC with PMA should not influence the response of the mesangial cell to IL-1β. Fig. 3 supports this thesis and demonstrates that exposure of mesangial cells to 500 nM PMA for 24 h does not influence the cellular response to IL-1β with respect to PGE2 formation. It should be stated that this experimental maneuver inhibited both nitrite and inducible nitric oxide synthase expression by 50% (data not shown). This experiment confirms that down-regulation of PKC by PMA does not influence the cellular response to IL-1β and again suggests the involvement of a PKC isotype that is not down-regulated by PMA. Fig. 4 indeed demonstrates that IL-1β initiates a translocation of PKCη from cytosol, presumably to some membrane component, and that incubating the cells with staurosporine does not prevent this translocation phenomenon while translocation was inhibited by calphostin C.Furthermore, staurosporine at 10 and 100 nM did not inhibit the IL-1β-stimulated binding of a nucleoprotein to the κB motif as determined by electromobility shift assays (Fig. 5A). In contrast, calphostin C inhibited the binding event in a dose-dependent manner, again confirming that IL-1β stimulates a staurosporine-insensitive, but calphositin C-sensitive translocation of NFκB to the nucleus.The experiments described thus far have demonstrated that IL-1β induces the mRNA for coxII and that this inductive phenomenon is dependent on a staurosporine-insensitive, calphostin C-sensitive PKC isotype. We have demonstrated that PKCη is translocated from the cytosol in response to IL-1β. Therefore, in an attempt to determine whether or not this activation of PKCη is relevant to CoxII induction of PGE2 formation, we reasoned that if we were able to decrease the activity of PKCη selectively with antisense oligonucleotides to PKCη, then we would be able to close the loop and demonstrate a functional consequence of PKCη in the signal transduction mechanisms involved in PGE2 production. The experiment illustrated by Fig. 6, therefore, demonstrates that while the antisense oligonucleotides to PKCη inhibit PGE2 formation in response to IL-1β by 37%, the sense oligonucleotides had no effect. Similarly, the antisense oligonucleotide inhibited CoxII protein expression by 40% while the sense oligonucleotide did not. Thus, we feel these experiments close the loop and draw a link between the activation of PKCη and the induction of coxII mRNA. Clearly the production of PGE2 in renal mesangial cells is the result of activation and induction of phospholipase A2 (22Pfeilschifter J. Schalkwijk C. Briner V.A. van den Bosch H. J. Clin. Invest. 1993; 92: 2516-2523Crossref PubMed Scopus (208) Google Scholar) plus the induction and expression of CoxII. The antisense oligonucleotides that only inhibit PGE2 and CoxII expression by about 40% suggest that the effects of PKCη are exerted entirely at the level of the cyclooxygenase.These experiments, while intriguing, clearly illustrate the complexity of the signal transduction process and point to the fact that there are multiple factors and pathways that are involved in IL-1β signal transduction mechanisms. One key feature of these experiments is the fact that while it could be argued that if the PKCη that is staurosporine-insensitive was the only isotype involved in this signal transduction process, then the use of staurosporine should not have potentiated the effect of IL-1β but rather should have had no effect on coxII message production. In reality, it did potentiate coxII mRNA and suggests that staurosporine was having an additional effect as well as that of failure to inhibit PKCη.We postulate that two additional mechanisms are possible to explain this phenomenon. The first is that staurosporine is having an additional effect that is unrecognized and that somehow leads to the potentiation of the effect of IL-1β on coxII mRNA production. While we cannot rule out this possibility since the use of staurosporine, a pharmacological agent, carries with it the possibility of certain unknown functions, we feel other plausible explanations for this phenomenon should be considered. In experiments not shown but previously reported, IL-1β does not affect coxI expression (16Coyne D.W. Nickols M. Bertrand W. Morrison A.R. Am. J. Physiol. 1992; 263: F97-F102PubMed Google Scholar, 25Rzymkiewicz D.M. DuMaine J. Morrison A.R. Kidney Int. 1995; 47: 1354-1363Abstract Full Text PDF PubMed Scopus (38) Google Scholar). Second, there may be other staurosporine-sensitive PKC isotypes that are involved in the signaling process and that are inhibited by staurosporine. Such a phenomenon could be linked to the activation of a phosphatase that regulates the activity of another signal transduction process mediated by IL-1β that is also coupled to transcriptional activation leading to an increase of mRNA for coxII. There is evidence from other investigators suggesting that activation of PKC decreases phosphorylation of c-jun at sites that negatively regulate its DNA binding activity (23Boyle W.J. Smeal T. Defize L.H.K. Angel P. Woodgett J.R. Karin M. Hunter T. Cell. 1991; 64: 573-584Abstract Full Text PDF PubMed Scopus (848) Google Scholar). In addition, there is evidence that activators of protein kinase C stimulate association of the Shc and the PEST tyrosine phosphatases (24Habib T. Herrera R. Decker S.J. J. Biol. Chem. 1994; 269: 25243-25246Abstract Full Text PDF PubMed Google Scholar). While we have no direct evidence that the Shc or PEST tyrosine phosphatases are involved in interleukin signaling mechanisms, we have provided evidence that vanadate alone, an inhibitor of protein tyrosine phosphatases, increases mRNA for coxII and that it potentiates the effect of IL-1β on coxII mRNA levels (25Rzymkiewicz D.M. DuMaine J. Morrison A.R. Kidney Int. 1995; 47: 1354-1363Abstract Full Text PDF PubMed Scopus (38) Google Scholar). The available data at least raise the issue of whether there may be a staurosporine-sensitive isotype that is coupled to the activation of a phosphatase that acts as a breaking phenomenon on the IL-1β stimulatory effect. The result of adding staurosporine to inhibit this protein kinase C would be to inhibit the effect of this phosphatase and, therefore, leave the positive effects mediated through PKCη unchecked. This would clearly explain a potentiation of the effect of IL-1β and will also explain why the drug alone at 100 nM was able to induce the message for the coxII gene. We are currently addressing this issue in the laboratory by designing experiments directed at this possibility. We suggest, therefore, that a PKC isotype that is coupled to the activation of a phosphatase could influence the expression of the mRNA for coxII. Similarly surprising observations have been described in PC12 cells where staurosporine markedly potentiated neurotensin and/or neuromedin mRNA accumulation in combination with other inducers (for example, NGF and PMA) and in some circumstances appeared to substitute for PMA (11Tischler A.S. Ruzicka L.A. Dobner P.R. J. Biol. Chem. 1991; 266: 1141-1146Abstract Full Text PDF PubMed Google Scholar). Furthermore, NGF-induced differentiation of PC12 cells utilizes the PMA-insensitive PKCη isotype. This was demonstrated by activation of PKCη by NGF and by attenuation of the effects of NGF on neurite outgrowth by antisense oligonucleotides to PKCη (26Coleman E.S. Wooten M.W. J. Mol. Neurosci. 1994; 5: 39-57Crossref PubMed Scopus (54) Google Scholar).In summary, we feel we have provided the evidence that PKCη is involved in the signaling mechanism for IL-1β. We feel that this pathway is involved in the ability of IL-1β to increase mRNA expression for coxII. The recent data suggesting that PKCη can phosphorylate IκB (27Diaz-Meco M.T. Dominguez I. Sanz L. Dent P. Lozano J. Municio M.M. Berra E. Hay R.T. Sturgill T.W. Moscat J. EMBO J. 1994; 13: 2842-2848Crossref PubMed Scopus (219) Google Scholar) and that inhibition of PKCη blocks the activation of NFκB-like activity in Zenopus laevis oocytes (28Dominguez I. Sanz L. Arenzana-Seisdedos F. Diaz-Meco M.T. Virelizier J.-L. Moscat J. Mol. Cell. Biol. 1993; 13: 1290-1295Crossref PubMed Google Scholar) suggest that this isotype of PKC may be very relevant to the effect of the transcriptional factor NFκB on PGE2 production in response to IL-1β. This mechanism also has support in the observation that PKCη mediates NFκB activation in human immunodeficiency virus-infected monocytes (29Folgueira L. McElhinny J.A. Bren G.D. MacMorran W.S. Diaz-Meco M.T. Moscat J. Paya C.V. J. Virol. 1996; 70: 223-231Crossref PubMed Google Scholar). INTRODUCTIONActivation of protein kinase C (PKC) 1The abbreviations used are: PKCprotein kinase CIL-1βinterleukin-1βPMAphorbol 12-myristate 13-acetateNGFnerve growth factorPGE2prostaglandin E2. plays a major role in agonist-stimulated function in a variety of cell types (1Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (918) Google Scholar, 2Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1216) Google Scholar, 3Hata A. Akita Y. Suzuki K. Ohno S. J. Biol. Chem. 1993; 268: 9122-9129Abstract Full Text PDF PubMed Google Scholar). To date there are at least 12 isotypes of PKC described and identified as α, βI, βII, γ, δ, ε, η, η, θ, τ, λ, and µ. These PKC isotypes are unique with respect to their primary structure, expression patterns, subcellular localization, and responsiveness to extracellular ligands. Recent reviews have highlighted the evidence that the isotypes might have separate and unique functions in the cell (2Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1216) Google Scholar). Activation of PKC is associated with its translocation from the cytosolic (soluble) fraction to the particulate (membrane) fraction (4Woelf M. Cuatrecasas P. Sahyoun N. J. Biol. Chem. 1985; 260: 15718-15722Abstract Full Text PDF PubMed Google Scholar, 5Dang P.M.-C. Hakim J. Périanin A. FEBS Lett. 1994; 349: 338-342Crossref PubMed Scopus (66) Google Scholar). Furthermore, prolonged activation of some cells with phorbol esters is associated with the binding of phorbol esters to the kinase and resultant down-regulation. PKCη does not appear to bind phorbol esters (6Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3099-3103Crossref PubMed Scopus (413) Google Scholar) and is not associated with the down-regulation of PKCη (7Wooten M.W. Zhou G. Seibenhener M.L. Coleman E.S. Cell Growth & Differ. 1994; 5: 395-403PubMed Google Scholar). The Cys-rich region within the C1 domain of PKC consists of two zinc finger motifs with six cysteine residues each and a homologous DNA-binding motif found in transcription factors like GAL4 (8Pan T. Coleman J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2077-2081Crossref PubMed Scopus (161) Google Scholar). The use of deletion mutants of different PKC isotypes has revealed that the Cys-rich region is necessary for diacylglycerol and phorbol ester binding (9Burns D.J. Bell R.M. J. Biol. Chem. 1991; 266: 18330-18338Abstract Full Text PDF PubMed Google Scholar, 10Muramatsu M.A. Katbuchi K. Arai K. Mol. Cell. Biol. 1989; 9: 831-836Crossref PubMed Scopus (76) Google Scholar). PKCη contains only one zinc finger and does not bind diacylglycerol or phorbol ester (6Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3099-3103Crossref PubMed Scopus (413) Google Scholar).The observations that PKCη is resistant to staurosporine (11Tischler A.S. Ruzicka L.A. Dobner P.R. J. Biol. Chem. 1991; 266: 1141-1146Abstract Full Text PDF PubMed Google Scholar, 12Seynaeve C.M. Kazanietz M.G. Blumberg P.M. Sausville E.A. Worland P.J. Mol. Pharmacol. 1994; 45: 1207-1214PubMed Google Scholar), appears to be critical for mitogenic signal transduction (13Berra E. Diaz-Meco M.T. Dominguez I. Municio M.M. Sanz L. Lozano J. Chapkin R.S. Moscat J. Cell. 1993; 74: 555-563Abstract Full Text PDF PubMed Scopus (342) Google Scholar), and is expressed in the rat mesangial cell (14Huwiler A. Fabbro D. Stabel S. Pfeilschifter J. FEBS Lett. 1992; 300: 259-262Crossref PubMed Scopus (59) Google Scholar) led us to evaluate its role in the signal transduction process resulting in an up-regulation of PGE2 in this cell type and whether this pathway of signaling is relevant to transcriptional activation of the coxII gene by IL-1β.
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