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.     

Protein kinase C activation and down-regulation in relation to phorbol ester-induced differentiation of SH-SY5Y human neuroblastoma cells

The role of protein kinase C activation in changes in muscarinic receptor functions and in the appearance of biochemical properties characteristic of neuronal cells was studied in SH-SY5Y human neuroblastoma cells induced to differentiate with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). A decrease in muscarinic receptor sensitivity with respect to agonist induced Ca2+ mobilization and receptor number parallelled the increase in membrane-associated protein kinase C (PK-C) activity. These changes occurred during the first 6 h of culture, and they were associated with rounding-up of cells. A subsequent decrease in particulate PK-C activity was followed by an increase in noradrenaline content, the appearance of an electrically excitable membrane, and an increase in the level of neuron-specific enolase. These changes were accompanied by a pronounced neurite outgrowth. 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine (H-7), an inhibitor of PK-C and cyclic nucleotide-dependent protein kinases, enhanced the morphological differentiation induced by TPA, whereas N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA-1004), which primarily inhibits cyclic nucleotide-dependent protein kinases, had no effect on the TPA-induced phenotypic differentiation. H-7 inhibited the decrease in muscarinic receptor sensitivity and receptor number, but had no effect on the appearance of the electrically excitable membrane or on the increase in the neuron-specific enolase level. Both H-7 and HA-1004 inhibited the TPA-induced increase in noradrenaline content.     

Protein kinase C-epsilon mediates phorbol ester-induced phosphorylation of connexin-43

We have used adenoviral vectors to express dominant negative variants of protein kinase C epsilon (PKCepsilon) or mitogen kinase kinase 1 (MKK1) to investigate their involvement in phorbol ester-induced connexin-43 (Cx43) phosphorylation in cardiomyocytes. Stimulation of cardiomyocytes with phorbol 12-myristate 13-acetate (PMA) increased the fraction of the slower migrating (> or = 45 kDa) and more extensively phosphorylated Cx43 species. Expression of dominant negative MKK1 did not prevent the effect of PMA on Cx43 phosphorylation. Selective inhibition of PKCE significantly decreased baseline levels of Cx43 phosphorylation and the PMA-induced accumulation of > or = 45 kDa Cx43. Thus, production of the more extensively phosphorylated species of Cx43 in cardiomyocytes by PMA requires activation of PKCepsilon.     

Dissociation of protein kinase C activation from phorbol ester-induced maturation of HL-60 leukemia cells

The role of C-kinase in the induction of maturation of HL-60 promyelocytic leukemia cells was examined using two activators of this kinase, 12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG). At 10(-8) M, a concentration that induced maturation, TPA effectively stimulated C-kinase activity in cell-free preparations by increasing the affinity of the enzyme for Ca2+. Similar activation was observed with 20 micrograms/ml of OAG. At these concentrations, addition of either compound to intact cells stimulated the phosphorylation of cellular proteins. Treatment with TPA resulted in an increased phosphorylation of 14 proteins, 9 of which also changed in response to OAG. In addition to the effects on protein phosphorylation, TPA and OAG both affected choline lipid metabolism. TPA at 10(-8) M stimulated the incorporation of [methyl-3H]choline into phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine. OAG at 20 micrograms/ml had quantitatively similar effects on the labeling of the former two lipids, but did not affect incorporation of choline into lysophosphatidylcholine. Despite the similar biochemical effects of TPA and OAG, the diglyceride was unable to induce HL-60 cell maturation as measured by inhibition of cell growth, development of nonspecific esterase activity, phagocytosis, adherence of cells to plastic, and loss of transferrin receptor activity. The lack of effect is not due to metabolism of OAG; maturation could not be induced by treating cells with fresh OAG every 2 h for a period of 12 h. These results suggest a dissociation of the activation of C-kinase and the induction of HL-60 cell maturation by TPA.     

Regulation of VL30 gene expression by activators of protein kinase C

The mouse genome contains a retrovirus-like sequence, designated VL30, which is expressed at high levels in transformed cells and which can be induced by exogenously supplied epidermal growth factor (EGF). Binding of EGF to the EGF receptor produces changes in intracellular calcium levels and phospholipase activity which indirectly lead to activation of protein kinase C. We treated AKR-2B cells, Swiss 3T3 cells, and the 3T3 variants NR6 (EGF receptorless) and TNR9 (phorbol ester nonresponsive) with various phorbol ester tumor promoters and with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. Tumor-promoting phorbol esters (e.g. 12-O-tetradecanoyl phorbol acetate (TPA] increased the level of VL30 expression. Stimulation with either TPA or EGF produced a similar time course of VL30 expression. TPA induced VL30 expression in the EGF-receptorless NR6 cell line, indicating that neither EGF ligand-receptor binding nor phosphorylation of the EGF receptor was required for induction of VL30 expression. Protein synthesis was not required for the TPA-mediated increase in VL30 expression, as pretreatment with cycloheximide did not block or reduce the TPA effect. VL30 expression was also stimulated by treatment with sn-1,2-dioctanoylglycerol, an analog of a probable endogenous activator of protein kinase C. These results suggest that activation of protein kinase C plays a direct role in regulating VL30 expression.     

Protein kinase C β1 overexpression augments phorbol ester-induced increase in endothelial permeability

We studied the postulated involvement of the protein kinase C β₁ (PKCβ₁) isoform in the regulation of endothelial permeability using human dermal microvascular endothelial cell line (HMEC-1). We overexpressed the recombinant PKCβ₁ gene via retroviral-mediated transduction in these cells. PKCβ₁ gene transfer was stable, and PKCβ₁ protein production was persistent for at least 1 month posttransduction. Addition of 2 × 10⁻⁹ M and 2 × 10⁻⁸ M phorbol 12-myristate 13-acetate (PMA) to the control (nontransduced) HMEC-1 cells increased the transendothelial ¹²⁵I-albumin clearance rate (an index of endothelial permeability) from 2.5 ± 0.2 × 10⁻² μl/min to 5.4 ± 1.2 × 10⁻² μl/min and 16.8 ± 3.1 × 10⁻² μl/min, respectively. However, addition of 2 × 10⁻⁹ M PMA to PKCβ₁-overexpressing HMEC-1 cells produced a maximal increase in the transendothelial ¹²⁵I-albumin clearance rate of 15.9 ± 2.0 × 10⁻² μl/min. Challenge of these cells with 2 × 10 ⁻⁸ M PMA did not further augment the increase in permeability. Activation with PMA was associated with the translocation of the PKCβ₁ from the cytosol to the membrane. These data show that PKCβ₁ overexpression augments the increase in endothelial permeability in response to PKC activation, suggesting an important function for the PKCβ₁ isoform in the regulation of endothelial barrier. © 1996 Wiley-Liss, Inc.     

Protein kinase C activation in a 3T3 cell variant morphologically unresponsive to tumor-promoting phorbol esters

To investigate the mechanism of the morphological changes induced in cells by tumor-promoting phorbol esters, we isolated a 3T3 cell variant which was morphologically unresponsive to phorbol esters and analyzed the activation of protein kinase C induced by the phorbol esters in it. The variant resembled the parent cells in its activation and appeared to have been altered at some step distal to the early events of protein kinase C activation.     

Proteolytic degradation of protein kinase C in the phorbol ester-induced interleukin-2 secreting thymoma cells

Effects of phorbol 12-myristate 13-acetate (PMA) on the fate of protein kinase C in two mouse thymoma cell lines, which are either responsive (EL4) or unresponsive (IEL4) to PMA-induced interleukin-2 (IL-2) production, were investigated with polyclonal antibodies raised against rat brain enzyme. These antibodies immunoprecipitated completely the protein kinase C from both cell lines and detected mainly an 82-kDa protein by immunoblot analysis of the crude homogenates as well as the partially purified kinase preparations. PMA elicited a time- and dose-dependent redistribution of protein kinase C from cytosol to the particulate fraction and proteolytic degradation of the kinase from both cell lines. The dose of PMA required for half-maximum protein kinase C translocation and degradation was at least five times lower for EL4 than for IEL4. In the presence of 16 nM PMA the rates of protein kinase C translocation and degradation were faster in EL4 than in IEL4, and the half-lives of protein kinase C in EL4 and IEL4 were less than 5 min and greater than 2 h, respectively. Analysis of the tryptic fragments of the immunoprecipitated enzyme, previously phosphorylated in the presence of [gamma-32P]ATP, revealed minor structural differences between the protein kinase C from these two cell lines. In neither cell line did the PMA-induced degradation of protein kinase C result in an accumulation of the Ca2+/phospholipid-independent kinase (catalytic unit) as analyzed by immunoblotting and gel filtration chromatography. Thus, activation of protein kinase C through the proteolytic conversion to the effector-independent catalytic unit plays little role in IL-2 production. The role of protein kinase C translocation and degradation in the PMA-induced responses in EL4 cells is unknown. However, IL-2 production in EL4 cells was reduced when PMA-induced degradation of protein kinase C was retarded by exogenously added protease inhibitors.     

Mitochondrial translocation of protein kinase C delta in phorbol ester-induced cytochrome c release and apoptosis

Apoptosis is induced by the release of cytochrome c from mitochondria to the cytoplasm. The present studies demonstrate that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces translocation of protein kinase C (PKC) delta from the cytoplasm to mitochondria. The results also show that translocation of PKCdelta results in release of cytochrome c. The functional significance of this event is further supported by the demonstration that PKCdelta translocation is required for TPA-induced apoptosis. These findings demonstrate that translocation of PKCdelta to mitochondria is responsible, at least in part, for inducing cytochrome c release and apoptosis.     

Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters

A tumor-promoting phorbol ester, [3H]phorbol-12,13-dibutyrate, may bind to a homogeneous preparation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) in the simultaneous presence of Ca2+ and phospholipid. This tumor promoter does not bind simply to phospholipid nor to the enzyme per se irrespective of the presence and absence of Ca2+. All four components mentioned above appear to be bound together, and the quaternary complex thus produced is enzymatically fully active for protein phosphorylation. Phosphatidylserine is most effective. Various other phorbol derivatives which are active in tumor promotion compete with [3H]phorbol-12,13-dibutyrate for the binding, and an apparent dissociation binding constant of the tumor promoter is 8 nM. This value is identical with the activation constant for protein kinase C and remarkably similar to the dissociation binding constant that is described for intact cell surface receptors. The binding of the phorbol ester is prevented specifically by the addition of diacylglycerol, which serves as activator of protein kinase C under physiological conditions. Scatchard analysis suggests that one molecule of the tumor promoter may bind to every molecule of protein kinase C in the presence of Ca2+ and excess phospholipid. It is suggestive that protein kinase C is a phorbol ester-receptive protein, and the results presented seem to provide clues for clarifying the mechanism of tumor promotion.     

Protein kinase C isozyme expression in phorbol ester-sensitive and -resistant EL4 thymoma cells

To investigate whether differential protein kinase C isozyme expression in phorbol ester-sensitive and -resistant EL4 thymoma cells could account for the difference in phorbol ester responsiveness, we purified and characterized isozymes from the two cell lines. In both cell types, two peaks of protein kinase C activity were resolved on hydroxylapatite following DEAE-cellulose and phenyl-Superose chromatography. Western blot analysis showed that the first peak corresponded to protein kinase C-beta and the second to protein kinase C-alpha. Two-dimensional phosphotryptic mapping of the purified alpha and beta isozymes did not reveal any reproducible differences between sensitive and resistant EL4 cells. Nor were any differences between the cell types observed in the cytosolic versus membrane localization of alpha and beta protein kinase C. Northern blot analysis showed the expression of mRNA for protein kinase C-alpha, -beta, -delta and -epsilon in both cell lines, and the absence of mRNA for gamma or zeta. Although no major differences in expression of alpha, beta, or delta mRNA between sensitive and resistant EL4 cells were detectable, expression of protein kinase C-epsilon mRNA in resistant cells was only 20-25% of that in sensitive. Western blot analysis with anti-protein kinase C-epsilon antibodies showed the presence of the epsilon-isozyme in sensitive cells and the absence of detectable amounts in resistant cells. Although protein kinase C-epsilon constitutes only a small portion of the total protein kinase C in sensitive cells, the possibility is raised that decreased protein kinase C-epsilon expression may contribute to the failure of resistant EL4 cells to respond to phorbol esters.