Rosiglitazone, a PPAR-γ agonist, inhibits VEGF secretion by peripheral blood mononuclear cells and ROS production by human leukocytes.

OBJECTIVE: To investigate the effects of rosiglitazone, a peroxisome proliferator-activated receptor-γ agonist, on the secretion of vascular endothelial growth factor (VEGF) by peripheral blood mononuclear cells (PBMCs) and on the generation of reactive oxygen species (ROS) by leukocytes. METHODS: PBMCs and leukocytes were obtained from venous blood samples collected from 20 healthy individuals. VEGF secretion was evaluated using a commercial ELISA kit, while ROS production was determined using a luminol-dependent chemiluminescence assay. RESULTS: Rosiglitazone and calphostin C (a protein kinase C inhibitor) inhibited VEGF secretion by PBMCs by 63.7 and 62.3%, respectively. Both agents reduced ROS production in non-stimulated human leukocytes and down-regulated the enhanced generation of ROS in leukocytes that had been stimulated with the PKC activator phorbol 12,13-dibutyrate. CONCLUSION: The results support the involvement of PKC as a direct, and/or NADPH-oxidase as an indirect, target for the action of rosiglitazone on VEGF secretion by PBMCs and ROS production in human leukocytes.

Biochemical characterization of hyperactive beta2-chimaerin mutants revealed an enhanced exposure of C1 and Rac-GAP domains.

Recent studies established that the Rac-GAP beta2-chimaerin plays important roles in development, neuritogenesis, and cancer progression. A unique feature of beta2-chimaerin is that it can be activated by phorbol esters and the lipid second messenger diacylglycerol (DAG), which bind with high affinity to its C1 domain and promote beta2-chimaerin translocation to membranes, leading to the inactivation of the small G-protein Rac. Crystallographic evidence and cellular studies suggest that beta2-chimaerin remains in an inactive conformation in the cytosol with the C1 domain inaccessible to ligands. We developed a series of beta2-chimaerin point mutants in which intramolecular contacts that occlude the C1 domain have been disrupted. These mutants showed enhanced translocation in response to phorbol 12-myristate 13-acetate (PMA) in cells. Binding assays using [(3)H]phorbol 12,13-dibutyrate ([(3)H]PDBu) revealed that internal contact mutants have a reduced acidic phospholipid requirement for phorbol ester binding. Moreover, disruption of intramolecular contacts enhances binding of beta2-chimaerin to acidic phospholipid vesicles and confers enhanced Rac-GAP activity in vitro. These studies suggest that beta2-chimaerin must undergo a conformational rearrangement in order to expose its lipid binding sites and become activated.

Imaging of memory-specific changes in the distribution of protein kinase C in the hippocampus.

Activation of protein kinase C (PKC) can mimic the biophysical effects of associative learning on neurons. Furthermore, classical conditioning of the rabbit nictitating membrane (a form of associative learning) produces translocation of PKC activity from the cytosolic to the membrane compartments of the CA1 region of the hippocampus. Evidence is provided here for a significant change in the amount and distribution of PKC within the CA1 cell field of the rabbit hippocampus that is specific to learning. This change is seen at 1 day after learning as focal increments of [3H]phorbol-12,13-dibutyrate binding to PKC in computer-generated images produced from coronal autoradiographs of rabbit brain. In addition, 3 days after learning, the autoradiographs suggest a redistribution of PKC within CA1 from the cell soma to the dendrites.

Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors.

ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCalpha, PKCbetaII, PKCdelta and PKCepsilon, but not atypical PKCzeta, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCalpha-GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.

Role of the regulatory domain of protein kinase D2 in phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling.

Protein kinase D2 (PKD2) belongs to the PKD family of serine/threonine kinases that is activated by phorbol esters and G protein-coupled receptors (GPCRs). Its C-terminal regulatory domain comprises two cysteine-rich domains (C1a/C1b) followed by a pleckstrin homology (PH) domain. Here, we examined the role of the regulatory domain in PKD2 phorbol ester binding, catalytic activity, and subcellular localization: The PH domain is a negative regulator of kinase activity. C1a/C1b, in particular C1b, is required for phorbol ester binding and gastrin-stimulated PKD2 activation, but it has no inhibitory effect on the catalytic activity. Gastrin triggers nuclear accumulation of PKD2 in living AGS-B cancer cells. C1a/C1b, not the PH domain, plays a complex role in the regulation of nucleocytoplasmic shuttling: We identified a nuclear localization sequence in the linker region between C1a and C1b and a nuclear export signal in the C1a domain. In conclusion, our results define the critical components of the PKD2 regulatory domain controlling phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling and reveal marked differences to the regulatory properties of this domain in PKD1. These findings could explain functional differences between PKD isoforms and point to a functional role of PKD2 in the nucleus upon activation by GPCRs.

A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.

Regulation of protein kinase C activity in neuronal differentiation induced by the N-ras oncogene in PC-12 cells.

Expression of the N-ras oncogene under the control of the glucocorticoid-responsive promoter in the pheochromocytoma cell line UR61, a subline of PC-12 cells, has been used to investigate the differentiation process to neuronal cells triggered by ras oncogenes (I. Guerrero, A. Pellicer, and D. E. Burstein, Biochem. Biophys. Res. Commun. 150:1185-1192, 1988). Using ras-inducible cell lines, we observed that expression of the oncogenic N-ras p21 protein interferes with the ability of phorbol esters to induce downregulation of protein kinase C. This effect was associated with the appearance of immunologically detectable protein kinase C as well as the activity of the enzyme as analyzed either by binding of [3H]phorbol-12,13-dibutyrate in intact cells or by in vitro kinase activity. These results indicate a relationship between ras p21 and protein kinase C in neuronal differentiation in this model system. Comparison to the murine fibroblast system suggests that this relationship may be functional.

Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides.

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.

Is phosphorylation of the alpha1 subunit at Ser-16 involved in the control of Na,K-ATPase activity by phorbol ester-activated protein kinase C?

The alpha1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufo alpha1 subunits. In cells incubated at 37 degrees C, phorbol 12, 13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally ( approximately 20-30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A(2), and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18 degrees C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing alpha1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase alpha1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.

Branched diacylglycerol-lactones as potent protein kinase C ligands and alpha-secretase activators.

Using as our lead structure a potent PKC ligand (1) that we had previously described, we investigated a series of branched DAG-lactones to optimize the scaffold for PKC binding affinity and reduced lipophilicity, and we examined the potential utility of select compounds as alpha-secretase activators. Activation of alpha-secretase upon PKC stimulation by ligands causes increased degradation of the amyloid precursor protein (APP), resulting in enhanced secretion of sAPPalpha and reduced deposition of beta-amyloid peptide (Abeta), which is implicated in the pathogenesis of Alzheimer's disease. We modified in a systematic manner the C5-acyl group, the 3-alkylidene, and the lactone ring in 1 and established structure-activity relationships for this series of potent PKC ligands. Select DAG-lactones with high binding affinities for PKC were evaluated for their abilities to lead to increased sAPPalpha secretion as a result of alpha-secretase activation. The DAG-lactones potently induced alpha-secretase activation, and their potencies correlated with the corresponding PKC binding affinities and lipophilicities. Further investigation indicated that 2 exhibited a modestly higher level of sAPPalpha secretion than did phorbol 12,13-dibutyrate (PDBu).

Odor increases [3H]phorbol dibutyrate binding to protein kinase C in olfactory structures of rat brain. Effect of entorhinal cortex lesion.

Since protein kinase C (PKC) is known to be activated in the olfactory bulb and in several limbic areas related to odor processing, we determined whether an olfactory stimulus was able to modulate the activity of PKC in animals with bilateral entorhinal cortex lesion. The translocation of PKC from the cytosol to the membrane was studied using the phorbol ester 12,13-dibutyrate ([3H]PDBu) binding in control and bilateral entorhinal cortex (EC) lesioned rats. The lesion of EC per se did not significantly affect [3H]PDBu binding in any of the brain structures analyzed, while odor stimulation induced it in both control and EC-lesioned groups in the external plexiform layer of the olfactory bulb. In contrast, an odor-induced increase of [3H]PDBu binding in internal glomerular layer of the olfactory bulb was only observed in EC lesioned animals. Similar results were obtained in the piriform cortex. In both CA1 and CA3 hippocampal subfields, odor stimulation induced an increase of [3H]PDBu binding in both control and EC-lesioned animals, the increase being potentiated only in CA1 of lesioned rats. The dentate gyrus and the amygdala exhibited a similar pattern of [3H]PDBu binding, showing a significant increase exclusively in EC-lesioned animals after odor stimulation. The results strongly suggest that the EC plays a key role in odor processing. PKC appears to play an important role in responding to the activation of lipid second messengers, which have been described to be involved in the processing of odor stimuli in several structures of the olfactory pathway.

Induction of protein kinase C in mouse melanoma cells by retinoic acid.

Retinoic acid inhibits the proliferation of B16 mouse melanoma cells. It also eliminates the ability of these cells to grow in soft agar. These biological actions of retinoic acid have been shown to be accompanied by an increase in the amount of cyclic AMP-dependent protein kinase and an induction of a new isozyme form (RII beta). In this report we demonstrated that retinoic acid-treated B16 melanoma cells had large increases in protein kinase C activity. This increased enzyme activity was accompanied by increases in both the number of phorbol dibutyrate binding sites and the amount of immunoreactive protein kinase C. Other treatments (melanocyte-stimulating hormone, serum deprivation) which inhibited the growth of these cells did not increase protein kinase C activity. When B16 melanoma cells were treated for a prolonged time (72 h) with phorbol dibutyrate, protein kinase C activity was barely detectable. Under these conditions, melanin production was inhibited and cell growth was accelerated. When retinoic acid was added together with phorbol dibutyrate, it prevented the growth stimulatory effect of the phorbol ester and increased protein kinase C activity. However, the absolute activity of the enzyme was still below that found in control cells and very much lower than in cells treated with retinoic acid alone. Taken together with our previous findings, we propose that the increase in protein kinase C might be part of a differentiation program induced by retinoic acid.

Inhibition of rodent protein kinase C by the anticarcinoma agent dequalinium.

Dequalinium has previously been shown to be an anticarcinoma agent (M. J. Weiss et al., Proc. Natl. Acad. Sci. USA, 84: 5444-5448, 1987). The present study demonstrates that it can inhibit protein kinase C-beta 1 isolated from an overproducing cell line with a 50% inhibitory concentration of 8-15 microM. Further examination of the inhibition by using structural analogues of dequalinium reveals that the length of the methylene bridge between the two quinaldinium moieties, the presence of the ring substituents, and the bipartite character of the compound each contributes to the inhibitory potency. Related studies show that the analogues display the same rank order of inhibitory potency when tested with the trypsin-generated catalytic fragment of the enzyme, indicating that dequalinium inhibits kinase activity through an interaction with the catalytic subunit. Further studies argue that the ability of a given analogue to inhibit phosphotransferase activity correlates with its ability to compete with [3H]phorbol-12,13-dibutyrate binding on the intact enzyme (50% inhibitory concentration of 2-5 microM). This suggests that the inhibitor is either binding directly to the regulatory subunit as well, or that due to its interaction with the catalytic subunit, dequalinium produces an indirect effect on sites defined by phorbol ester binding. Kinetic analysis revealed that inhibition is noncompetitive with respect to ATP or phosphatidylserine. Studies conducted with types I, II, and III rat brain isozymes, resolved by hydroxylapatite chromatography, demonstrate that dequalinium inhibits each of them with similar potency (50% inhibitory concentration of 11 microM) and imply that the site of contact on the enzyme is a highly conserved region. Morphology studies with dequalinium in intact cells demonstrate that the inhibitor can protect control cells against phorbol ester-induced morphology changes but cannot protect protein kinase C-overproducing cells, suggesting that an elevation in protein kinase C levels alone is sufficient to overturn the protection conferred by dequalinium. On the basis of these results, we propose that protein kinase C could be a critical in vivo target of dequalinium.

Protein kinase C in adriamycin action and resistance in mouse sarcoma 180 cells.

Adriamycin has a wide variety of biological actions on susceptible cells, several of which may be integrally involved in cytotoxicity. In this paper, we present evidence that one of the alterations in cell function that occurs in the presence of Adriamycin is an elevation in the production of diacylglycerol. The effect is rapid, reaches a peak within 10 min of exposure of Sarcoma 180 cells to Adriamycin, and can thus be classified among the earliest alterations that occur in cells damaged by Adriamycin. Concomitant with the rise in diacylglycerol is an increase in cytosolic protein kinase C activity. Although Adriamycin does not appear to modulate the activity of this enzyme by direct binding, drug-exposed Sarcoma 180 cells have a 56% increase in intrinsic cytosolic protein kinase C (PKC) activity, with no change in the activity of the membrane form. Experiments with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate suggest that the PKC effect is linked to Adriamycin action, since activation of the enzyme by short 12-O-tetradecanoylphorbol-13-acetate exposure enhances Adriamycin's cytotoxicity as well as its ability to provoke DNA damage (measured by alkaline elution). Likewise, down-regulation of PKC by extended 12-O-tetradecanoylphorbol-13-acetate exposure partially protects the cells from Adriamycin-induced cytotoxicity as well as from DNA damage. Thus, the ability of cells to be injured by Adriamycin appears to be correlated with the activity of PKC. Multidrug-resistant subline Sarcoma 180A10 cells have the same total quantity of membrane-recruitable PKC as the sensitive parent Sarcoma 180 cells, as determined by [3H]phorbol-12,13-dibutyrate binding. However, the resistant cells have a significantly higher intrinsic PKC activity and an altered ability to translocate the enzyme to the cell surface. Taken together, the results raise the possibility that cell signaling mechanisms, particularly those involving protein kinase C, may play an important role in mediating the biological action of the anticancer drug Adriamycin.

Aflatoxin-transformed C3H/10T1/2 cells overexpress protein kinase C and have an altered response to phorbol ester treatments.

The aflatoxin B1-transformed C3H/10T1/2 (10T1/2) cell line 7SA has disordered growth in culture and is tumorigenic in syngeneic mice. Chronic exposure (14 days) of 10T1/2 and 7SA cells to phorbol 12,13-dibutyrate (PDBu) increased the saturation density of 10T1/2 cells but dramatically slowed the entry of 7SA cells into the log phase of growth without affecting their final saturation density. Similar PDBu treatment of low-density cultures dramatically decreased the size of 7SA colonies. Both cell lines bound [3H]PDBu in a specific and saturable manner. Analysis of this binding yielded linear Scatchard plots for both cell lines with distinctly different Kd values (10.7 nM for 10T1/2 versus 54.5 nM for 7SA). The total amount of [3H]PDBu bound was 2-fold greater in the 7SA cells versus the 10T1/2 cell line. Both cell lines released arachidonic acid following a 2-h exposure to PDBu; however, the magnitude of the response of the 7SA cells was only one-half that of the 10T1/2 cells. Western blot analysis of protein kinase C (PKC) using specific anti-PKC antibodies revealed a greater total amount of PKC alpha in the 7SA cells relative to an equal number of 10T1/2 cells. No immunoreactive PKC alpha was found in either cell line 16 h after exposure to 600 nM PDBu; however, PKC alpha returned to control levels in both cell lines 24 h after removal of the phorbol ester. These results suggest that an overexpression of PKC alpha may play a role in the altered biological properties of aflatoxin-transformed 10T1/2 cells.

Tumor-promoting phorbol ester and activated Ha-ras synergistically reduce the interleukin 3 requirement in a mast cell line.

Infection of the bone marrow-derived mast cell line PB-3c with a retrovirus carrying oncogenic c-Ha-ras or v-Ha-ras reduced the interleukin 3 (IL-3) growth requirement and induced a state of tumorigenicity. In contrast, normal c-Ha-ras had no effect on the IL-3 requirement of this cell line nor did the cells become tumorigenic. A factor reduction similar to that caused by activated Ha-ras was transiently obtained with 12-O-tetradecanoylphorbol-13-acetate in the PB-3c cells expressing normal c-Ha-ras. The analogous stimulation of protein kinase C (PKC) in PB-3c cells producing oncogenic Ha-ras led to an additional reduction of the IL-3 requirement during the first 24 h. In the absence of IL-3, the prolonged exposure of the cells to 12-O-tetradecanoylphorbol-13-acetate for 72 h resulted in a stimulation of growth when activated but not when normal Ha-ras was expressed. PB-3c cell lines expressing activated Ha-ras neither revealed differences in the amounts nor in the subcellular distribution of PKC activity but displayed elevated levels of immunoreactive beta-PKC compared to the parental PB-3c cells. Upon 12-O-tetradecanoylphorbol-13-acetate treatment, a protracted down-regulation of the immunodetectable alpha-PKC as well as constitutively high levels of c-fos mRNA were observed when oncogenic Ha-ras was expressed. These data suggest the involvement of specific PKC subtypes and of c-fos in the reduction of the IL-3 requirement caused by activated Ha-ras in this particular hematopoietic cell line.

Protein kinase C down-regulation, and not transient activation, correlates with melanocyte growth.

The nontumorigenic, immortal line of murine melanocytes, Mel-ab, requires the continual presence of biologically active phorbol esters for growth (R.E. Wilson et al., Cancer Res., 49:711-716, 1989). Comparable treatments of B16 murine melanoma cells result in partial inhibition of cell proliferation. The role of protein kinase C (PKC) in the modulation of growth of cells from these two melanocytic cell lines has been investigated. Significant levels of PKC were present in quiescent Mel-ab cells as determined by Western blotting, whereas no immunoreactive protein was detected in cell extracts from either proliferating Mel-ab or B16.F1 cells. Phosphorylation of a Mr 80,000 protein, which by one- and two-dimensional gel analysis comigrated with the known Mr 80,000 protein substrate of PKC in fibroblasts, was induced in 12-O-tetradecanoylphorbol-13-acetate-stimulated quiescent Mel-ab cells but not in proliferating Mel-ab cells or B16.F1 melanoma cells. Direct measurement of PKC activity in these cells demonstrated a 10-fold greater level of activity in quiescent Mel-ab cells (262 +/- 50 pmol/min/mg SD) compared with growing cells (22.8 +/- 11.8 pmol/min/mg SD). An intermediate level of activity was detected in proliferating B16.F1 melanoma cells (148.5 +/- 20.4 pmol/min/mg SD). The subcellular distribution of PKC was dependent upon the growth state of the cells such that quiescent Mel-ab cells displayed a higher level of activity in the cytosol, whereas growing Mel-ab cells displayed greater activity in the particulate fraction. Like many other transformed lines, B16.F1 melanoma cells constitutively expressed the majority of enzyme activity in the particulate fraction. Measurement of [3H]phorbol ester binding in intact cells paralleled the PKC activation data such that quiescent Mel-ab cells displayed binding of 1612 +/- 147 cpm/10(6) cells, whereas proliferating Mel-ab and B16.F1 melanoma cells displayed binding of 652 +/- 28 and 947 +/- 81 cpm/10(6) cells, respectively. Membrane-permeant diacylglycerol analogues, which activated but did not down-regulate PKC, were devoid of growth-stimulating effects on melanocytes, even in the presence of the specific diacylglycerol kinase inhibitor, R59022. Together, these data show that PKC down-regulation, and not activation, correlates with the growth of melanocytes in culture.

Regulation of protein kinase C isozymes in kidney regeneration.

Tissue damage and repair processes are important factors in renal tumor progression. To determine whether protein kinase C (PKC) is involved in these processes, we characterized PKC isozymes during rat kidney regeneration using 3 models: (a) diffuse cortical hyperplasia and hypertrophy induced by folic acid; (b) focal necrosis of the S3 segments induced by S-(1,2-dichlorovinyl)-L-cysteine; and (c) compensatory renal hypertrophy. Immunoblot analyses demonstrated that 5 PKC isozymes, alpha, beta, delta, epsilon, and zeta, were expressed in rat kidney. Six h after folic acid treatment, phorbol ester receptors were down-modulated. Down-modulation preceded an increase in DNA synthesis which was maximal at 24 h. The reduction in phorbol ester receptors was due largely to a decrease in alpha-PKC. zeta-PKC, which is not a phorbol ester receptor, was also decreased. delta- and epsilon-PKCs were not changed. However, alpha-PKC was not down-modulated during compensatory hypertrophy induced by unilateral nephrectomy. Thus, the observed decrease of alpha-PKC after folic acid treatment is most likely associated with the hyperplastic and not the hypertrophic effects of this renal toxin. These results demonstrate that activation-associated down-modulation of PKC, in particular alpha-PKC, occurs during chemical-induced renal regeneration and suggests a general role for PKC activation in non-phorbol ester tumor promotion.

Specific binding to protein kinase C by ingenol and its induction of biological responses.

We have examined the ability of ingenol to bind to and activate protein kinase C and to induce similar responses to the phorbol esters in biological systems. The rationale was that ingenol possesses the critical functionalities of the phorbol ester pharmacophore with the exception of the hydrophobic domain; it might therefore possess weak potency, although previous reports had indicated that ingenol was biologically inactive. Our data demonstrate that ingenol indeed binds to protein kinase C with a Ki of 30 microM and activates the enzyme. In addition, ingenol was biologically active in 3 separate cell systems, showing effects similar to the phorbol esters on morphological change, cell-cell communication, epidermal growth factor binding, arachidonic acid metabolite release, and ornithine decarboxylase activity. The 50% effective concentration values for the biological activity of ingenol were between 30 microM and 1 mM, varying somewhat with the cell system and type of response. The biological activity of ingenol in general supports the proposed models of the phorbol ester pharmacophore and imposes additional experimental constraints that the modeling must satisfy.

Differential roles of the tandem C1 domains of protein kinase C delta in the biphasic down-regulation induced by bryostatin 1.

Bryostatin 1 (Bryo), currently in clinical trials, has been shown to induce a biphasic concentration-response curve for down-regulating protein kinase C (PKC) delta, with protection of the enzyme from down-regulation at high Bryo doses. In our ongoing studies to identify the basis for this unique behavior of PKCdelta, we examined the participation of the two ligand binding sites (C1a and C1b) in the regulatory domain of the enzyme. Three mutants of PKCdelta prepared by introducing a point mutation in either C1a or Clb or both C1a and Clb were overexpressed in NIH 3T3 cells. All of the constructs retained a biphasic response to down-regulation assessed after 24-h treatment with Bryo. However, the roles of the individual C1 domains were different for the two phases of the response. For down-regulation, both the C1a and the C1b mutants displayed equivalent 3-4-fold reductions in their affinities for the ligand. For protection from down-regulation, a reduced protection was observed for the C1a mutant, which showed a broader biphasic curve compared with those for wild-type PKCdelta and the Clb mutant. Like wild-type PKCdelta, all of the mutants showed the same subcellular partitioning of the protected enzyme to the particulate fraction of the cells, arguing against changes in sensitivity to Bryo due to differences in localization. Likewise, relatively similar patterns of localization were observed using green fluorescent protein-PKCdelta constructs. We conclude that the C1 domains of PKCdelta do not have equivalent roles in inducing protection against Bryo-induced down-regulation. The C1a domain plays a critical role in conferring the degree of protection at high concentrations of Bryo. Elucidation of the differential effect of Bryo on PKCdelta may suggest strategies for the design of novel ligands with Bryo-like activities.