The involvement of lipid activators of protein kinase C in the induction of ZIF268 in PC12 cells exposed to lead

Lead (Pb) induces the expression of immediate early genes (IEG) in PC12 cells by a mechanism that involves protein kinase C (PKC). To define the mechanisms, the involvement of two commonly observed lipid activators of PKC, diacylglycerols, and phosphatidylinositols, were examined. A dose-dependent increase in the expression of the IEG zif268 was observed in PC12 cells exposed to Pb. The PKC inhibitor Ro-31-8220 blocked the induction. An increase in levels of diacylglycerols was observed in PC12 cells exposed to Pb, but the increase was inhibited by Ro-31-8220. The phosphatidylinositol 3-kinase inhibitor Wortmannin, but not the inhibitor LY 294002, blocked the induction zif268 in Pb-exposed cells. Small increases in phosphatidylinositol 3-kinase activity were observed after exposure to Pb. In summary, diacylglycerols are elevated in PC12 cells exposed to Pb by a mechanism that requires PKC. It is possible that diacylglycerols contribute to the induction of zif268 by Pb by sustaining PKC activation.     

Functional serum response elements upstream of the growth factor-inducible gene zif268.

The zif268 gene, which encodes a protein with three typical zinc finger sequences, is induced in mouse 3T3 cells by serum, phorbol 12-myristate 13-acetate platelet-derived growth factor, and fibroblast growth factor. The induction is coordinate with that of c-fos. The 5'-flanking region of zif268 contains sequences that resemble known regulatory elements, including four CC(A or T)6GG sequences similar to the core serum response elements (SREs) found upstream of c-fos and actin genes. To determine whether the zif268 SRE-like elements mediate induction, CAT (chloramphenicol acetyltransferase) plasmids with different lengths of zif268 upstream sequences were tested for inducibility in 3T3 cells by serum, platelet-derived growth factor, or phorbol 12-myristate 13-acetate. In addition, double-stranded oligonucleotides corresponding to each of the four zif268 putative SREs were tested individually for responsiveness when placed upstream of a thymidine kinase gene promoter. Each of the four SREs conferred inducibility by the agents tested, and multiple SREs resulted in greater inducibility than did a single element. Each of the zif268 SREs also competed with the c-fos SRE for binding by serum response factor present in HeLa cell nuclear extract. We conclude that the zif268 SRE-like sequences are functional and probably account for the coordinate induction of zif268 and c-fos.     

The kinase inhibitor iso-H7 stimulates rat satellite cell differentiation through a non-protein kinase C pathway by increasing myogenin expression level

We analysed the signaling pathways involved in myogenic differentiation of primary cultures of rat satellite cells using substances targeting the protein kinase C (PKC) and the cAMP protein kinase (PKA) pathways. We have previously shown that iso-H7, which putatively inhibits both PKC and PKA, strongly stimulates satellite cell differentiation, as well as the PKA inhibitor HA 1004. In the study reported here, the effects of iso-H7 on satellite cell differentation were compared to those observed in the presence of agents which reduce PKC activity. It was shown that treatments with the highly specific PKC inhibitor GF109203X or with 12-O-tetradecanoylphorbol 13-acetate (TPA) which induced a partial PKC downregulation, did not significantly alter myogenic differentiation. Northern blot analyses showed that iso-H7 activated the expression of myogenin but not that of MyoD mRNA. Concurrently, iso-H7 increased myosin light-chain mRNA expression. In contrast, TPA had no effect on these syntheses. Taken together, these results showed that iso-H7 did not act intracellularly as a PKC inhibitor but rather as a PKA inhibitor as previously suggested. Our results are compatible with the hypothesis that a reduction in PKA activity controls satellite cell myogenesis through an increased myogenin mRNA expression.Abbreviations PKC protein kinase C - PKA cAMP-dependent protein kinase - CK creatine kinase - iso-H7 1-(5-isoquinolinesulfonyl)-3-methylpiperazine - H7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - HA 1004 N-(3-hydroxyethyl)-1-piperazine ethanesulfonate - TPA 12-O-tetradecanoyl phorbol 13-acetate - MLC myosin light chain - GAPDH glyceraldehyde 3-phosphate deshydrogenase     

Pharmacologic evidence for the requirement of protein kinase C in IFN-induced macrophage Fc gamma receptor and Ia antigen expression.

Previous studies have implicated protein kinase C (PKC) as a mediator in the activation of macrophages by interferons. In order to probe further into the suspected role of protein kinase C in mouse peritoneal macrophage activation, the effects of protein kinase inhibitors in macrophage Fc gamma R and Ia Ag expression were studied. The protein kinase inhibitor, H7, reduced basal levels, and inhibited IFN-alpha-induced expression of Fc gamma R significantly. The concentration of H7 required to inhibit 50% of the Fc gamma R induction was approximately 12 microM, which reflects the previously reported affinity of this compound for PKC in vitro. H7 had only a minimal effect on IFN-gamma-induced Fc gamma R, suggesting different pathways of Fc gamma R induction by the two types of IFN. Ia induction by IFN-gamma was also inhibited by H7, indicating that both types of IFN can utilize PKC to mediate at least part of the signal required for Fc gamma R or Ia expression. HA-1004, a derivative of H7 which possesses high affinity for cyclic nucleotide-dependent protein kinases, but low affinity for PKC, did not alter induction, while H8, a slightly less effective PKC inhibitor than H7, was effective at higher concentrations. Another structurally distinct PKC antagonist, staurosporine, was also effective inhibiting IFN-alpha-induced Fc gamma R and IFN-gamma-induced Ia Ag expression, providing additional evidence that PKC is important. H7 was found to be effective when added as late as several hours after IFN treatment, indicating a prolonged or delayed requirement of PKC for optimal induction of Ia and Fc gamma R by IFN.     

Regulation of mitogen-stimulated human T-cell proliferation, interleukin-2 production, and interleukin-2 receptor expression by protein kinase C inhibitor, H-7

Recently published reports suggest that the activation of protein kinase C (PKC) plays an important role in the activation pathway of many cell types. In this study, we examined the role of PKC in human T-cell proliferation, IL-2 production, and IL-2R expression, when cultured with the mitogen PHA, the PKC inhibitor H-7, and H-7 control HA1004. H-7 inhibited the PHA-stimulated [3H]thymidine uptake, IL-2 production, and IL-2R expression in a dose-related manner. Further, we found H-7 inhibited T-cell proliferation, IL-2 production, IL-2 mRNA from PHA plus PMA-stimulated cultures. We also found that H-7 inhibited the early-stage activation of PHA-stimulated cells. The presence of exogenous purified human IL-2 or rIL-4 partly reversed the immunosuppression caused by H-7. In contrast, HA1004 had no effect on cell proliferation, IL-2 production, or IL-2R expression. Our results demonstrate that PKC activation is one major pathway through which T-cells become activated.     

Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C

Geldanamycin is an antitumor drug that binds HSP90 and induces a wide range of heat shock proteins, including HSP70s. In this study we report that the induction of HSP70s is dose-dependent in geldanamycin-treated human non-small cell lung cancer H460 cells. Analysis of the induction of HSP70s specific isoform using LC-ESI-MS/MS analysis and Northern blotting showed that HSP70-1/2 are the major inducible forms under geldanamycin treatment. Transactivation of hsp70-1/2 was determined by electrophoretic mobility-shift assay using heat shock element (HSE) as a probe. The signaling pathway mediators involved in hsp70-1/2 transactivation were screened by the kinase inhibitor scanning technique. Pretreatment with serine/threonine protein kinase inhibitors H7 or H8 blocked geldanamycin-induced HSP70-1/2, whereas protein kinase A inhibitor HA1004, protein kinase G inhibitor KT5823, and myosin light chain kinase inhibitor ML-7 had no effect. Furthermore, the protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor G?-6976 diminished geldanamycin-induced HSP70-1/2, suggesting an involvement of the PKC in the process. In addition, geldanamycin treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased geldanamycin-transactivated HSP70-1/2 expression. Taken together, our results demonstrate that geldanamycin-induced specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC.     

Induction of ice and inhibition of c-fos,jun D and zif 268 in 12 -month old spontaneously hypertensive rats

A semi-quantitative reverse transcprition-polymerase chain reaction (RT-PCR) assay was used to examine ICE, c-fos, jun D and zif 268 mRNA expression in the aortic and renal artery of 12-month old SHRs and wistar rats. Using this assay system, it was observed that the levels of aortic and renal artery expression of ICE were markedly higher in SHRs than in wistar rats. In contrast, the aortic and renal artery expression of immediate early genes (IEGs), c-fos, jun D and zif 268, were significant lower in SHRs than in wistar rats. Thus, our results suggest that differential regulation of death gene ICE and IEGs such as c-fos, jun D and zif 268 might be involved in the mechanism of pathogenesis of hypertension.     

Interleukin-2 regulates the activity of ornithine decarboxylase in a cloned murine T lymphocytic cell line: evidence for a protein kinase C-dependent pathway

The role of protein kinase C (PKC) in the regulation of ornithine decarboxylase (ODC) activity during interleukin-2 (IL-2)-dependent cell growth was investigated. A large biphasic increase in the activity of ODC was observed after treatment of IL-2-deprived CTLL-2 cells with recombinant human IL-2 (rec IL-2). The PKC activators phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD), but not the inactive analog 4 alpha-PDD, induced ODC activity in exponentially growing cultures. Unlike IL-2, however, phorbol esters were poor inducers of IL-2-depleted cultures. H-7, a potent inhibitor of PKC and cyclic nucleotide-dependent protein kinases (CN-PK), suppressed the IL-2-induced ODC activity, while HA1004, a more potent inhibitor of CN-PK than of PKC, had opposite effects depending on its concentration. The results suggest that activation of PKC is involved in but is not the sole mechanism for the induction of ODC by rec IL-2. At concentrations which suppressed the induction of ODC activity by IL-2, H-7 inhibited DNA synthesis and HA1004 did not.     

Overexpression of Arabidopsis MAP kinase kinase 7 leads to activation of plant basal and systemic acquired resistance

There is a growing body of evidence indicating that mitogen-activated protein kinase (MAPK) cascades are involved in plant defense responses. Analysis of the completed Arabidopsis thaliana genome sequence has revealed the existence of 20 MAPKs, 10 MAPKKs and 60 MAPKKKs, implying a high level of complexity in MAPK signaling pathways, and making the assignment of gene functions difficult. The MAP kinase kinase 7 (MKK7) gene of Arabidopsis has previously been shown to negatively regulate polar auxin transport. Here we provide evidence that MKK7 positively regulates plant basal and systemic acquired resistance (SAR). The activation-tagged bud1 mutant, in which the expression of MKK7 is increased, accumulates elevated levels of salicylic acid (SA), exhibits constitutive pathogenesis-related (PR) gene expression, and displays enhanced resistance to both Pseudomonas syringae pv. maculicola (Psm) ES4326 and Hyaloperonospora parasitica Noco2. Both PR gene expression and disease resistance of the bud1 plants depend on SA, and partially depend on NPR1. We demonstrate that the constitutive defense response in bud1 plants is a result of the increased expression of MKK7, and requires the kinase activity of the MKK7 protein. We found that expression of the MKK7 gene in wild-type plants is induced by pathogen infection. Reducing mRNA levels of MKK7 by antisense RNA expression not only compromises basal resistance, but also blocks the induction of SAR. Intriguingly, ectopic expression of MKK7 in local tissues induces PR gene expression and resistance to Psm ES4326 in systemic tissues, indicating that activation of MKK7 is sufficient for generating the mobile signal of SAR.     

Role of protein kinase C in IFN-mediated Ly-6E antigen induction.

The YAC T cell lymphoma normally does not express Ly-6E mRNA or Ly-6E surface molecules but can be induced to do so on incubation with either IFN-gamma or IFN-alpha/beta. This system afforded a model to assess the possible role of protein kinase C (PKC) in IFN-mediated Ly-6E induction. First, we used various pharmacologic agents known to interfere with the function of PKC or other kinases. The PKC inhibitors H-7 and phloretin were found to block Ly-6E induction by IFN-gamma or IFN-alpha/beta both at the mRNA and protein levels. In contrast, inhibitors of cyclic nucleotide-dependent kinases (HA1004), of myosin L chain kinase (ML-9, A-3) or of calmodulin (R24157, W-7) failed to suppress this induction. Next, we investigated the effects of the PKC activators PMA and mezerein (MEZ) on Ly-6E expression. Although neither PMA nor MEZ by themselves could induce Ly-6E in YAC cells, both agents enhanced by up to fivefold the induction of Ly-6 mRNA and Ly-6E surface expression triggered by IFN-gamma. However, the induction of Ly-6E expression caused by IFN-alpha/beta was only marginally increased by cotreatment of YAC cells with PMA or MEZ. Altogether, these observations demonstrate that PKC or a related kinase is involved in the transduction mechanisms that lead to Ly-6E induction. However, activation of PKC is not sufficient for this induction and requires other unidentified signal(s) provided by IFN. Our data also indicate that IFN-gamma and IFN-alpha/beta induce Ly-6E through overlapping but distinct intracellular pathways with different sensitivities to PKC activators.     

Involvement of protein kinase C in the regulation of ornithine decarboxylase mRNA by phorbol esters in rat hepatoma cells

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.17; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl-L-carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +/- 1.6 h) is not significantly changed by either TPA (5.7 +/- 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters.