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.     

Retinoic acid and phorbol ester synergistically up-regulate IL-8 expression and specifically modulate protein kinase C-epsilon in human skin fibroblasts.

Phorbol ester (TPA) and retinoic acid (RA) are two potent immunomodulatory agents whose actions are mediated through distinct signal transduction pathways involving protein kinase C (PKC) and nuclear RA receptors, respectively. We have investigated the interactions between these two pathways in the regulation of expression of the inflammatory cytokine IL-8 in human skin fibroblasts. TPA (as previously reported) and RA both induced IL-8 mRNA and protein in a time- and dose-dependent manner. IL-8 mRNA induction by TPA (10 nM) was maximal (15-fold) within 6 h, and returned to baseline within 24 h of treatment, although maximal induction (10-fold) by RA (1 microM) did not occur until 24 h posttreatment. Induction of IL-8 by TPA was blocked by 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine, which inhibits PKC and cAMP-dependent protein kinases (PKA), but not by N-(2-ganidinoethyl)-5-isoquinoline sulfonamide, which preferentially inhibits PKA, consistent with the participation of PKC in the induction of IL-8 by TPA. In contrast, induction of IL-8 by RA was inhibited by both 1-(5-isoquinoline sulfonamide and N-(2-gamidinoethyl)-5-isoquinoline sulfonamide, suggesting the participation of PKA in the induction of IL-8 by RA. However, activation of PKA by addition of cAMP analogues was not sufficient to induce IL-8 expression. Induction of IL-8 by RA also did not appear to be mediated indirectly through induction of IL-1, because addition of IL-1R antagonist did not block IL-8 induction by RA. RA and TPA added in combination synergistically enhanced expression of IL-8 mRNA, measured at 6 (2-fold) and 24 h (10-fold) posttreatment. To investigate the mechanism of this synergy, the effect of TPA and RA on fibroblast PKC activation and PKC isozyme levels were determined. TPA, either alone or together with RA, but not RA alone, stimulated phosphorylation of an endogenous 80-kDa PKC substrate. Dermal fibroblasts expressed three PKC isozymes (alpha, (delta, and (epsilon). TPA, but not RA, down-regulated PKC-alpha, neither TPA or RA affected the level of PKC-delta, and both TPA and RA down-regulated PKC-epsilon. This latter effect was enhanced 2-fold by addition of RA and TPA together. These data suggest that modulation of PKC-epsilon may be a common participant in the regulation of IL-8 expression by TPA and RA.     

Phorbol ester-mediated suppression of cytochrome P450 Cyp1a-1 induction in murine skin: involvement of protein kinase C.

Epidermal 7-ethoxyresorufin O-deethylase (EROD) activity was elevated greater than 100-fold within 4 to 7 h of topical treatment of SENCAR mice with 100 nmol dibenz[a,c]anthracene (DB[a,c]A). Treatment of skin with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) 2 to 8 h prior to DB[a,c]A application suppressed induction by 80%. Suppression was dose-dependent over the range of 0.01 to 5 micrograms TPA (ID50 approximately 0.6 nmol). EROD activities in normal and TPA-treated epidermis paralleled steady state P450 CYP1A1 mRNA content. Analogs of TPA incapable of activating or down-regulating protein kinase C (PKC) did not suppress induction. Pretreatment of skin with sn-1,2-didecanoylglycerol, an activator of PKC which causes translocation but no down-regulation, did not suppress EROD induction. However, induction was suppressed by chrysarobin, an anthralin analog that causes PKC down-regulation in the absence of prior activation. These studies suggest that PKC participates in the processes associated with Cyp1a-1 induction and that TPA effects Cyp1a-1 induction through its down-regulation of PKC.     

Dual effect of protein kinase C on the induction of DNA synthesis by colcemid in G0-arrested human diploid fibroblasts

G0-arrested human diploid fibroblasts, TIG-1, was stimulated to induce DNA synthesis by serum, epidermal growth factor (EGF), colchicine, colcemid, or 12-O-tetradecanoylphorbol-13-acetate (TPA). The induction of DNA synthesis was mediated by protein kinase C (PKC) when stimulated with TPA but not when stimulated with other agents. When TPA-stimulated cells were immediately treated with colcemid, induction of DNA synthesis was reduced. This reduction diminished when colcemid was added more than 6 h after TPA treatment. Conversely, when colcemid-stimulated cells were treated with TPA, induction of DNA synthesis was also reduced. This reduction was enhanced when the interval between the addition of two stimulants was extended. PKC-deprivation abolished both stimulatory and inhibitory effects of TPA on DNA synthesis. Staurosporine blocked an induction of DNA synthesis by TPA but appeared to be ineffective on the inhibitory action of TPA on DNA synthesis by colcemid. These results suggest that the inhibitory effect of TPA on the induction of DNA synthesis by colcemid is mediated by down regulation-sensitive and staurosporine-insensitive PKC.     

Okadaic acid,sphingosine, and phorbol ester reversibly modulate heat induction on protein kinase Fa/GSK-3α in A431 cells

Exposure of A431 cells to a rapid and sudden increase from 37°C to 46°C for 30 min could induce an increase in protein level and cellular activity of protein (kinase Fa /GSK-3α) up to ∼200% of control level. However, when cells were first treated with 500 nM tumor promoter phorbol ester TPA at 37°C for 30 min to activate cellular protein kinase C (PKC) or with 400 nM okadaic acid at 37°C for 30 min to inhibit cellular protein phosphatases followed by heat shock at 46°C for another 30 min, the heat induction on kinase Fa /GSK-3α was found to be completed blocked. In sharp contrast, when cells were first treated with 1 μM TPA at 37°C for 24 h or with 5 μM sphingosine at 37°C for 30 min to down-regulate cellular PKC, the heat induction on kinase Fa /GSK-3α was found to be reversely promoted up to ∼ 250% of control level, demonstrating that kinase Fa /GSK-3α may not represent a constitutively active/mitogen-inactivated protein kinase as previously conceived. Taken together, the results provide initial evidence that TPA/sphingosine and okadaic acid could reversibly modulate the heat induction on kinase Fa /GSK-3α in A431 cells, suggesting that phosphorylation/dephosphorylation mechanisms are involved in the regulation of the heat-shock induction of kinase Fa /GSK-3α, representing a new mode of signal transduction for the regulation of this multisubstrate protein kinase and a new mode of signaling pathway modulating the heat-induction process. © 1996 Wiley-Liss, Inc.     

Activation of distinct protein kinase C isozymes by phorbol esters: correlation with induction of interleukin 1 beta gene expression

Treatment of human promyelocytic leukemia cells U937 with phorbol 12-myristate 13-acetate (TPA) induces them to differentiate into monocytic cells [Harris, P., & Ralph, P. (1985) J. Leukocyte Biol. 37, 407-422]. Here we investigated the effects of TPA on interleukin 1 gene expression and the possible role of protein kinase C (PKC) in this process. Addition of TPA to serum-starved U937 cells induced the expression of the interleukin 1 beta (IL-1 beta) gene. This effect was apparent as early as 2 h and peaked at 24 h in the presence of 5 X 10(-8) M TPA. Higher concentrations of TPA, which partially or totally depleted protein kinase C levels in the cells (10(-9)-2 X 10(-5) M), had an inhibitory effect on IL-1 beta mRNA expression. Cell-permeable 1,2-dioctanoyl-sn-glycerol (diC8), a diacylglycerol that activates PKC in intact cells and cell-free systems, did not mimic the effect of TPA on the IL-1 beta mRNA induction. To determine the protein kinase C isozymes present in the control and TPA- (5 X 10(-8) M) treated U937 cells, we prepared antipeptide antibodies that specifically recognize the alpha, beta, and gamma isoforms of protein kinase C in rat brain cytosol and U937 cell extracts. In "control" U937 cells, 30% of PKC alpha was particulate, and PKC beta was cytosolic, while there was no detectable PKC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium/phospholipid-dependent protein kinases in rat Sertoli cells: regulation of androgen receptor messenger ribonucleic acid

The possibility that Sertoli cell responses to testosterone are modulated by the calcium/phospholipid-dependent protein kinase (protein kinase C; PKC) was examined in rat Sertoli cells in culture. Both soluble and particulate cell fractions showed low constitutive phosphotransferase activity. Incubation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-7) M) was associated with a transient induction in both cell fractions of calcium/phosphatidylserine-dependent PKC activity, which was elevated from 15 min to 1 h. Consistent with this, mRNAs for the calcium/phospholipid-dependent isomeric forms of PKC (alpha, beta, and gamma) were detected. The expression levels of mRNAs for PKCalpha and PKCbeta were also up-regulated (2.5- to 3-fold) by TPA (10(-7) M), but these effects were much slower (peaking after 12 h) than those on phosphotransferase activity. In the presence of TPA (10(-7) M), expression of androgen receptor (AR) mRNA showed a transient time-dependent down-regulation ( approximately 70%), in which the nadir was reached after 6 h and baseline expression was again obtained after 12 h. The regulatory effect of PKC activation on AR mRNA was confirmed by the absence of response to a biologically inactive phorbol ester. A concentration-dependent decrease (half-maximal effect at approximately 10(-8) M TPA) of AR mRNA was also observed. These data suggest that Sertoli cell responses to testosterone may be inhibited by a transiently active PKC with a wide intracellular distribution.     

Induction of Epstein-Barr virus lytic cycle by tumor-promoting and non-tumor-promoting phorbol esters requires active protein kinase C.

Exposure to the tiglian 12-O-tetradecanoylphorbol-13-acetate (TPA) represents one of the most efficient and widely used protocols for inducing Epstein-Barr virus (EBV)-infected cells from latent into lytic cycle. Since TPA is both a potent tumor promoter and a potent activator of the cellular protein kinase C (PKC), we sought to determine whether either of these activities was closely linked to EBV lytic cycle induction. A panel of TPA structural analogs, encompassing tiglians with different spectra of biological activities, was assayed on a number of EBV-positive B-lymphoid cell lines. Lytic cycle induction correlated with the capacity to activate PKC, not with tumor promoter status; some nonpromoting tiglians were as efficient as TPA in inducing lytic cycle antigen expression. We then sought more direct evidence for an involvement of PKC in the induction process. In initial experiments, 1-(5-isoquinolinyl sulphonyl)-2-methylpiperazine (H-7), the best available pharmacological inhibitor of PKC, completely blocked the induction of the lytic cycle by TPA and its active analogs. This is consistent with, but does not prove, a requirement for active PKC in the induction process, since H-7 targets PKC preferentially but also has some effects on other kinases. We therefore turned to the synthetic pseudosubstrate peptide PKC(19-36) as a means of specific PKC inhibition and to the closely related but inactive peptide PKC(19-Ser-25-36) as a control. Using the technique of scrape loading to deliver the peptides into cells of an adherent EBV-positive target line, we found that the pseudosubstrate peptide PKC(19-36) completely and specifically blocked tiglian-induced entry of the cells into the lytic cycle. The evidence both from TPA analogs and from enzyme inhibition studies therefore indicates that the pathway linking TPA treatment to lytic cycle induction involves active PKC. Interestingly, inhibition of PKC had no effect upon the spontaneous entry into lytic cycle which occurs in naturally productive cell lines, suggesting that spontaneous entry is signalled by another route.     

Involvement of p42/p44 mitogen-activated protein kinase in prostaglandin f(2alpha)-stimulated induction of heat shock protein 27 in osteoblasts

We previously reported that prostaglandin F(2alpha) (PGF(2alpha)) activates both phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells and then induces the activation of protein kinase C (PKC). In this study, we investigated the effect of PGF(2alpha) on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein, in these cells. PGF(2alpha) significantly induced the accumulation of HSP27 dose-dependently within the range of 10 nM to 10 microM. PGF(2alpha) stimulated the increase in the levels of mRNA for HSP27. A total of 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, induced the accumulation of HSP27. The stimulative effect of PGF(2alpha) was reduced in the PKC down-regulated cells. Calphostin C, a specific inhibitor of PKC, suppressed the PGF(2alpha)-induced HSP27 accumulation as well as that induced by TPA. HSP27 induction by PGF(2alpha) was reduced by U-73122, a phospholipase C inhibitor, or propranolol, a phosphatidic acid phosphohydrolase inhibitor. PGF(2alpha) and TPA stimulated p42/p44 mitogen-activated protein (MAP) kinase. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, suppressed the induction of HSP27 stimulated by PGF(2alpha) or TPA. PD98059 and calphostin C reduced the levels of mRNA for HSP27 increased by PGF(2alpha). These results indicate that PGF(2alpha) stimulates the induction of HSP27 via p42/p44 MAP kinase activation, which depends on upstream PKC activation in osteoblasts.     

Protein kinase C and CYPlAl induction in rainbow trout (Oncorhynchus mykiss) hepatocyte culture

1. The role of protein kinase C (PKC) in B-naphthoflavone (BNF) induction of CYP1A1 in rainbow trout hepatocytes was investigated.2. Primary cultures of rainbow trout hepatocytes treated with BNF for 24 hr showed an increase in microsomal 7-ethyoxyresorufm-O-deethylase (EROD) activity compared to cells treated with vehicle (DMSO) only.3. Increases in EROD activities were proportional to increased concentrations of BNF from 1 to 10 nM reaching a plateau at higher concentrations (20–100 nM) of BNF.4. Western blot analysis using specific antibody (LM4b) against CYP1A1 showed that changes in microsomal CYP1A1 protein paralleled that of EROD activity.5. The induction of EROD activity by BNF required both protein and RNA synthesis since the process was blocked by both cycloheximide and actinomycin D.6. Pretreatment of hepatocytes with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to a dose dependent suppression of BNF-induced EROD activity and CYP1A1 content. TPA alone had no effect on hepatic EROD activity and CYP1A1 protein level.7. Pretreatment with sn-1,2 didecanoylglycerol, a PKC activator, had no effect on BNF-induced EROD activity in these cells.8. Pretreatment of cells with staurosporine, a PKC inhibitor, effectively blocked BNF-induced EROD activity.9. PKC may play a role in the induction of CYP1A1 gene expression in fish liver by BNF.     

c-Src-dependent tyrosine phosphorylation of IKKbeta is involved in tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 expression

The signaling pathway involved in tumor necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule-1 (ICAM-1) expression was further studied in human A549 epithelial cells. TNF-alpha- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ICAM-1 promoter activity was inhibited by a protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or an Src-specific tyrosine kinase inhibitor (PP2). TNF-alpha- or TPA-induced IkappaBalpha kinase (IKK) activation was also blocked by these inhibitors, which slightly reversed TNF-alpha-induced but completely reversed TPA-induced IkappaBalpha degradation. c-Src and Lyn, two members of the Src kinase family, were abundantly expressed in A549 cells, and their activation by TNF-alpha or TPA was inhibited by the same inhibitors. Furthermore, the dominant-negative c-Src (KM) mutant inhibited induction of ICAM-1 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKC or wild-type c-Src plasmids induced ICAM-1 promoter activity, this effect being inhibited by the dominant-negative c-Src (KM) or IKKbeta (KM) mutant but not by the nuclear factor-kappaB-inducing kinase (NIK) (KA) mutant. The c-Src (KM) mutant failed to block induction of ICAM-1 promoter activity caused by overexpression of wild-type NIK. In co-immunoprecipitation and immunoblot experiments, IKK was found to be associated with c-Src and to be phosphorylated on tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr188 and Tyr199, near the activation loop of IKKbeta, were identified as being important for NF-kappaB activation. Substitution of these residues with phenylalanines abolished ICAM-1 promoter activity and c-Src-dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA. These data suggest that, in addition to activating NIK, TNF-alpha also activates PKC-dependent c-Src. These two pathways converge at IKKbeta and go on to activate NF-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate ICAM-1 expression.     

Novel protein kinase C isoforms and mitogen-activated kinase kinase mediate phorbol ester-induced osteopontin expression

BACKGROUND AND AIMS: The expression of osteopontin (OPN), a protein postulated to play a role in tumorigenesis, is induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) in vivo and in the in vitro initiation-promotion skin carcinogenesis model (JB6 cells). Although TPA-induced OPN expression in JB6 cells has been suggested to involve protein kinase C (PKC), the PKC isoforms and the downstream pathway mediating OPN expression have not been extensively studied. METHODS: Using the JB6 cell model, we determined the involvement of PKC isoforms, mitogen-activated protein kinase kinase (MAPK kinase/MEK) and MAPK in TPA-induced OPN expression using inhibitors specific to PKC isoforms and MEK and performing Northern blot analyses. Western blot analyses of cells treated with specific inhibitors were also performed to determine whether PKC isoforms or MEK were involved in activation of MAPK. KEY RESULTS: TPA increased the steady-state level of OPN mRNA as early as 2-4h and this expression persisted for at least 4 days. TPA induction of OPN expression in JB6 cells is mediated through PKC epsilon and PKC delta, which also mediated the phosphorylation of MAPK. Additionally, inhibition of MEK activity, which activates MAPK, attenuated TPA-induced OPN expression. These findings suggest that activation of MAPK is important in mediating OPN expression. CONCLUSION: TPA-induced steady-state OPN mRNA expression in mouse JB6 cells involves the activation of MAPK mediated through PKC epsilon and/or PKC delta.