Phorbol esters inhibit phosphate uptake in opossum kidney cells: a model of proximal renal tubular cells

The effects of phorbol esters and diacylglycerol on phosphate uptake in opossum kidney (OK) cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on renal phosphate handling. OK cells are widely used as a model of proximal renal tubular cells and are reported to possess a Na+-dependent phosphate transport system. Phorbol-12,13-dibutyrate (PDBu) inhibited phosphate uptake. This inhibitory effect was synergistically enhanced with A23187. 4 beta-phorbol 12,13-didecanoate inhibited phosphate uptake, while 4 alpha-phorbol 12,13-didecanoate did not. 1-oleoyl-2-acetyl-glycerol (OAG), a synthetic diacylglycerol, also exhibited an inhibitory effect on phosphate uptake. These data suggest the possible involvement of protein kinase C in proximal renal tubular phosphate transport.     

Activators of Protein Kinase C Act at a Postreceptor Site to Amplify Cyclic AMP Production in Rat Pinealocytes

Activation of alpha 1-adrenoceptors appears to amplify beta-adrenergic stimulation of cyclic AMP (cAMP) accumulation in rat pinealocytes severalfold by a mechanism involving activation of a Ca2+-, phospholipid-dependent protein kinase (protein kinase C). The mechanism of action of protein kinase C was investigated in this report using intact cells. Activation of protein kinase C with 4 beta-phorbol 12-myristate 13-acetate (PMA; 10(-7) M) or the alpha 1-adrenergic agonist phenylephrine (PE; 10(-6) M) did not inhibit cAMP efflux in beta-adrenergically stimulated cells. The amplification of the beta-adrenergic cAMP response by these agents also occurred in the presence of isobutylmethylxanthine (10(-3) M) and Ro 20-1724 (10(-4) M), an observation suggesting that inhibition of cAMP phosphodiesterase activity is not the mechanism of action. Furthermore, although PMA (10(-7) M) caused a sixfold increase in the magnitude of the cAMP response to isoproterenol, it did not alter the EC50 of the response (1.7 X 10(-8) M), a result indicating that protein kinase C activation does not alter beta-adrenoceptor sensitivity. The cAMP response following cholera toxin pretreatment (60-120 min) was rapidly and markedly enhanced by alpha 1-adrenergic agonists (cirazoline greater than PE greater than methoxamine), by phorbol esters (PMA greater than 4 beta-phorbol 12,13,-dibutyrate much greater than 4 alpha-phorbol 12,13-didecanoate), and by synthetic diacylglycerols (1,2-dioctanoylglycerol greater than 1-oleoyl 2-acetylglycerol much greater than diolein). The cAMP response to forskolin (10(-5)-10(-3) M) was also increased by PE (3 X 10(-6) M) and PMA (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Contrasting effects of sn-1,2-dioctanoyl glycerol as compared to other protein kinase C activators in adrenal glomerulosa cells

Angiotensin II acts on adrenal glomerulosa cells to induce the phospholipase C-mediated generation of inositol trisphosphate and sn-1,2-diacylglycerol as the major products of inositol phospholipid breakdown. This last product is known to activate protein kinase C, but its role in the action of angiotensin II on steroidogenesis has not been defined. We report herein that, in bovine adrenal glomerulosa cells, protein kinase C activators, such as phorbol 12,13-dibutyrate, 12-O-tetradecanoylphorbol-13-acetate, mezerein and sn 1,2 oleoyl acetoylglycerol, each failed to increase steroidogenesis. These results contrast with our recent report on the enhancement of aldosterone output by sn-1,2-dioctanoylglycerol (DiC8) [J. Steroid Biochem. 35 (1990) 19-33]. In addition, the difference between DiC8 and the other protein kinase activators was also observed in the pattern of 86Rb efflux from preloaded glomerulosa cells; only DiC8 mimicked the effect of angiotensin II on ion fluxes. Furthermore, staurosporine, a potent inhibitor of protein kinase C, was capable of amplifying the aldosterone output induced by a maximally effective concentration of DiC8 or angiotensin II. These data suggest that the effect of the cell permeant DiC8 on aldosterone biosynthesis either is not mediated by protein kinase C activation, or is mediated by a phorbol ester-insensitive isoenzyme of protein kinase C.     

Differential effects of phorbol ester and diacylglycerols on inositol phosphate formation in C62B glioma cells

Application of acetylcholine (ACh) to C62B glioma cells results in a rapid release of inositol phosphates. Since this response is transient, we evaluated the possible role of protein kinase C (PKC) in its desensitization. Pretreatment with 100 nM phorbol 12,13-dibutyrate (PDBu) significantly inhibited ACh-induced accumulation of [3H]inositol mono-, bis-, and trisphosphates. However, interpretation of this result as proof of PKC involvement was complicated by the failure of 1,2-dioctanoylglycerol, 1,2-didecanoylglycerol, or 1-oleoyl-2-acetylglycerol pretreatments to mimic the phorbol ester effect. Further evidence against PKC involvement was obtained using the PKC inhibitor sphingosine; PDBu inhibition of inositol phosphate formation was not reversed by sphingosine pretreatments at concentrations which blocked ACh-stimulated PKC activation of inositol trisphosphate phosphatase activity. These results suggest that there may be phorbol effects not mediated by PKC.     

Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.     

Protein kinase C-dependent and -independent mechanisms regulating the parotid substance P receptor as revealed by differential effects of protein kinase C inhibitors.

Substance P-induced inositol trisphosphate (InsP3) formation was inhibited by 1 microM-4 beta-phorbol 12,13-dibutyrate (PDBu) in rat parotid acinar cells. The inhibitory effect of PDBu was reversed by the protein kinase C inhibitors H-7 or K252a. Substance P also elicits a persistent desensitization of subsequent substance P-stimulated InsP3 formation. However, this desensitization was not inhibited by H-7. In addition, H-7 had no effect on the time course of substance P-induced InsP3 formation. These results suggest that, although activation of protein kinase C by phorbol esters can inhibit the substance P receptor-linked phospholipase C pathway, this mechanism apparently plays little, if any, role in regulating this system after activation by substance P.     

Mimicry of phorbol ester responses by diacylglycerols. Differential effects on phosphatidylcholine biosynthesis, cell-cell communication and epidermal growth factor binding

The biosynthesis of phosphatidylcholine (PC) in HEL-37 cells was followed by measuring the incorporation of [32P]Pi into PC. Incorporation was stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and by the synthetic diacylglycerol, sn-1,2-dioctanoylglycerol (diC8), but not by sn-1-oleoyl-2-acetylglycerol or sn-1,2-dihexanoylglycerol (diC6). DiC8 was rapidly metabolised by HEL-37 cells to the corresponding PC and phosphatidic acid derivatives. diC8, diC6 and oleoylacetylglycerol effectively displaced [3H]phorbol-12,13-dibutyrate bound to a soluble cell extract from HEL-37 cells, but only diC8 was able to displace the labelled phorbol ester from prelabelled cells. TPA, diC8, diC6 and oleoylacetylglycerol were all effective inhibitors of 125I-labelled epidermal growth factor binding to, and gap junctional communication between, HEL-37 cells. It is concluded that only cell-permeable diacylglycerols stimulate PC biosynthesis which may therefore require interaction with membranes other than the plasma membrane.     

Synergism between diacylglycerols and calcium ionophore in the induction of human B cell proliferation mimics the inositol lipid polyphosphate breakdown signals induced by crosslinking surface immunoglobulin

Resting human tonsillar B cells were stimulated to divide by heat killed Staphylococcus aureus Cowan strain 1 which was shown to induce hydrolysis of phosphatidylinositol 4, 5-bisphosphate known to give rise to diacylglycerol and an increase in cytosolic free calcium. Addition of the diacylglycerols, 1-oleoyl-2 acetyl glycerol or sn-1, 2-dioctanoylglycerol, together with the calcium ionophore ionomycin to B cell cultures induced marked cell proliferation whereas these agents were ineffective when used alone. Both diacylglycerols were shown to compete with [3H] phorbol 12,13 dibutyrate in binding to protein kinase C. These data support the hypothesis that synergism between cytosolic calcium and endogenous diacylglycerol, which activates protein kinase C, is involved in signal transduction in the proliferation of human B cells.     

Modulation of protein kinase C activity during inhibition of tumor cell growth by IFN-beta and -gamma

We investigated the effects of human interferon(IFN)-beta and -gamma on protein kinase C activity in human HEp-2 and KHm-14 tumor cells during IFN-induced inhibition of cell growth. Cytosolic protein kinase C activity in both cell lines was strikingly decreased following treatment with either IFN-beta or -gamma. In the particulate fraction, IFN-gamma decreased protein kinase C activity within 1 hr but it reappeared after 24 hr, whereas IFN-beta decreased the activity during the inhibition of cell growth. Furthermore, phorbol-12,13-dibutyrate(PDBu)-binding activity was altered in parallel with the changes in protein kinase C activity induced by the IFNs. In summary, we showed that IFN-beta and -gamma cause long-term modulation of protein kinase C activity in these cultured tumor cells.     

Postsynaptic densities contain a subtype of protein kinase C

Protein kinase C or an isoenzyme thereof appears to be a significant component of postsynaptic densities (PSDs) from rat brain. This cytoskeletal organelle binds 4 beta-phorbol 12,13-dibutyrate (PDBu) with a Bmax of about 20 pmol/mg protein and an apparent Kd of 3.3 nM. Ca2+ and phosphatidyl serine (PS) stimulated the endogenous phosphorylation of a subset of PSD polypeptides with Mr values between 16,000 and 22,000. Finally, a monospecific protein kinase C antibody reacted with a Mr 70,000 PSD polypeptide which migrated on SDS-PAGE slightly ahead of the Mr 77,000 purified enzyme. These data suggest that protein kinase C or a similar enzyme can be integrated into a cytoskeletal system and may play an important role in postsynaptic function.     

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.     

MAC-1 mediates adherence of human monocytes to endothelium via a protein kinase C dependent mechanism

Leukocyte adherence is mediated by a superfamily of glycoproteins denoted LFA-1 (the lymphocyte function-associated antigen-1), Mac-1 (macrophage antigen-1) and p150,95. The relative importance of these in mediating human monocyte adherence to endothelium, and the biochemical mechanisms which modulate these events, are not understood. In this report, the role of protein kinase C (pkC) in regulating human monocyte adherence to endothelial cells has been investigated. Addition of phorbol 12,13-dibutyrate (PDBu), which specifically stimulates pkC, caused a dose-dependent increase in their adherence to monolayers of bovine aortic endothelial cells. 4 alpha-phorbol didecanoate (4 alpha-PDD), a structural analogue of PDBu which does not stimulate pkC, failed to increase monocyte adhesion. PDBu also produced a dose-dependent increase in the expression of both Mac-1 and p150,95. The pkC-stimulated adherence of monocytes to endothelium was inhibited by the presence of a monoclonal antibody to Mac-1, while monoclonal antibodies to p150,95 and LFA-1 did not influence adherence. It is concluded that monocyte adherence to endothelial cells is regulated through a pkC-dependent mechanism; moreover, this process is mediated primarily via the Mac-1 adhesion glycoprotein.     

Phorbol esters inhibit low density lipoprotein processing by cultured human fibroblasts

A 24 h pretreatment of MRC5 fibroblasts with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) induced a marked decrease in low density lipoprotein (LDL) internalization and degradation; the maximal effect (about 55% decrease) was observed for 10(-7) M TPA. LDL binding was reduced about 35-40%. A significant decrease (about 25%) in LDL internalization was observed after a 2 h incubation of cells with the drug, but longer incubation times (4-6 h) led to a greater effect. Another tumor promoter, phorbol 12,13-dibutyrate decreased LDL internalization by about 35%, whereas the non-tumor promoting 4 alpha-phorbol 12,13-didecanoate had no effect. The protein kinase C inhibitor alpha-cobrotoxin partially antagonized the inhibitory effect of TPA on LDL internalization. The non-phorbol tumor promoter mezerein, another protein kinase C activator, decreased LDL uptake by about 50%. Finally, it was found that TPA had no significant effect on the affinity of the receptor for the LDL. These results suggest a role for protein kinase C in the LDL pathway in cultured human fibroblasts.     

Epidermal growth factor-induced phosphoinositide hydrolysis. Modulation by protein kinase C

A short-term treatment with phorbol 12,13-dibutyrate (PDBu) was found to inhibit totally the epidermal growth factor (EGF)-stimulated phosphoinositide hydrolysis in A431 cells, whereas long-term pretreatment with PDBu, which is known to down regulate protein kinase C, induced a greater accumulation of the EGF-triggered inositol phosphate accumulation, particularly of Ins(1,3,4,5)P4. The increased Ins(1,4,5)P3/Ins(1,3,4,5)P4 formation in the PDBu long-term pretreated cells was coincident with the increased Ca2+ influx stimulated by EGF in the same cells. Since long-term pretreatment with PDBu was found to enhance the EGF signals, an explanation for the synergism between EGF and phorbol esters in the induction of DNA synthesis is provided.     

Phospholipid functional groups involved in protein kinase C activation, phorbol ester binding, and binding to mixed micelles

The specificity of the phospholipid cofactor requirement of rat brain protein kinase C was investigated using Triton X-100 mixed micellar methods. Sixteen analogues of phosphatidylserine were prepared and tested for their ability to support protein kinase C activity, [3H]phorbol 12,13-dibutyrate binding, and protein kinase C binding to mixed micelles. Phosphatidylserinol, -L-serine methyl ester, -N-acetyl-L-serine, -2-hydroxyacetate, -3-hydroxypropionate, and -4-hydroxybutyrate did not activate protein kinase C in mixed micelles containing 2 mol % of sn-1,2-dioleoylglycerol. This indicates that both the carboxyl and amino moieties are important for activation. Phosphatidyl-D-serine and -L-homoserine were incapable of supporting full activation; this demonstrates stereospecificity and the importance of the distance between the phosphate and carboxyl and amino moieties. Since 1,2-rac-phosphatidyl-L-serine and 1,3-phosphatidyl-L-serine fully supported protein kinase C activity, the stereochemistry within the glycerol backbone at the interface was not necessary for maximal activation. Neither lysophosphatidyl-L-serine nor 1-oleoyl-2-acetyl-sn-glycero-3-phospho-L-serine supported protein kinase C activity implying that the interfacial conformation is critical to the activation process. The phospholipid dependencies of [3H]phorbol 12,13-dibutyrate binding and of protein kinase C binding to mixed micelles containing sn-1,2-dioleoylglycerol did not mirror those for activation. The data demonstrate that protein kinase C possesses a high degree of specificity with respect to phospholipid activation and implicate several functional groups within the phospho-L-serine polar head group in binding and activation.     

Inhibition of DNA synthesis by phorbol esters through protein kinase C in cultured rabbit aortic smooth muscle cells

In cultured rabbit aortic smooth muscle cells (SMC), 12-O-tetradecanoylphorbol-13-acetate (TPA) induced DNA synthesis in the presence of plasma-derived serum to a small extent, but inhibited markedly the rabbit whole blood serum (WBS)-, platelet-derived growth factor (PDGF)- and epidermal growth factor-induced DNA synthesis. Phorbol-12,13-dibutyrate (PDBu) mimicked this antiproliferative action of TPA, but 4 alpha-phorbol-12,13-didecanoate was inactive in this capacity. Prolonged treatment of the cells with PDBu caused the partial down-regulation of protein kinase C. In these protein kinase C-reduced cells, WBS still induced DNA synthesis, but TPA did not inhibit the WBS-induced DNA synthesis. We have previously shown that protein kinase C is involved at least partially in the PDGF-induced DNA synthesis in rabbit aortic SMC. The present results together with this earlier observation suggest that protein kinase C has not only a proliferative but also an antiproliferative action in rabbit aortic SMC.     

Cisplatin resistance is associated with deregulation in protein kinase C-delta

Proteolytic activation of protein kinase C (PKC)-delta has been associated with cell death induced by the DNA damaging agent cisplatin. In the present study, we have examined if PKCdelta is affected when cells acquire resistance to cisplatin. The level of PKCdelta was elevated in cisplatin-resistant HeLa (HeLa/CP) cells compared to parental HeLa cells. Prolonged cellular exposure to the PKC activator phorbol-12,13-dibutyrate (PDBu), caused downregulation of PKCdelta in HeLa cells but not in HeLa/CP cells. Treatment of HeLa cells with PDBu resulted in the translocation of PKCdelta from the cytosol to the membrane but it failed to induce PKCdelta translocation in HeLa/CP cells. PDBu, however, induced translocation and downregulation of PKCalpha in both HeLa and HeLa/CP cells. The ability of PDBu to enhance cisplatin-induced cell death was attenuated in cisplatin-resistant HeLa cells. Thus, a deregulation in PKCdelta was associated with reduced cellular sensitivity to cisplatin.     

Stimulation of dopamine synthesis and activation of tyrosine hydroxylase by phorbol diesters in rat striatum

In rat striatal synaptosomes, 4 beta-phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PDBu), two activators of Ca2+-phospholipid-dependent protein kinase (protein kinase C) increased dopamine (DA) synthesis measured by following the release of 14CO2 from L-[1-14C] tyrosine. Maximal stimulation (21-28% increase of basal rate) was produced by 0.5 microM PMA and 1 microM PDBu. 4 beta-Phorbol and 4 beta-phorbol 13-acetate, which are not activators of protein kinase C, were ineffective at 1 microM. PMA did not change the release of 14CO2 from L-[1-14C]DOPA. Addition of 1 mM EGTA to a Ca2+-free incubation medium failed to affect PMA stimulation. KC1 (60 mM) enhanced DA synthesis by 25%. Exposure of synaptosomes to either PMA or PDBu prior to KC1 addition resulted in a more than additive increase (80-100%) of DA synthesis. A similar synergistic effect was observed when the phorbol diesters were combined with either veratridine or d-amphetamine but not with forskolin and dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with phorbol diesters produced an activation on of tyrosine hydroxylase (TH) associated with a 60% increase of the Vmax and a decrease of the Km for the pterine cofactor 6-methyl-5,6,7,8-tetrahydropterin. These results indicate that protein kinase C participates in the regulation of striatal TH in situ and that its activation may act synergistically with DA releasing agents in stimulating DA synthesis.     

Possible involvement of protein kinase C in interleukin-1 production by mouse peritoneal macrophages

Both lipopolysaccharide (LPS) and phorbol-12,13-dibutyrate (PDBu), a protein kinase C-activating phorbol ester, induced interleukin-1 (IL-1) production in mouse peritoneal macrophages. Prolonged treatment of the cells with PDBu led to the down-regulation and complete disappearance of protein kinase C. In these cells, PDBu did not increase IL-1 production, but LPS still stimulated IL-1 production although the maximum level was slightly reduced. These results suggest that protein kinase C and another unknown signal pathway are involved in LPS-induced IL-1 production.