Phorbol 12-myristate 13-acetate-induced ectodomain shedding and phosphorylation of the human meprinbeta metalloprotease

Shedding of proteins localized at the cell surface is an important regulatory step in the function of many of these proteins. Human meprin (N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase, PPH, EC 3.4.24.18) a zinc-metalloendopeptidase of the astacin family is an oligomeric protein complex of alpha- and beta-subunits and is expressed abundantly in the intestine and kidney as well as in leukocytes of the lamina propria and in cancer cells. In transfected cells intracellular proteolytic removal of the membrane anchor results in the secretion of the meprin alpha-subunit. In rats and mice, the beta-subunit exists in a membrane-anchored form. In contrast, human meprinbeta is constitutively converted into a secretable form. We now show that phorbol 12-myristate 13-acetate (PMA) stimulates an increased release of hmeprinbeta from transfected COS-1 cells, whereas hmeprinalpha secretion is not influenced. This stimulatory effect is inhibited by the protein kinase C (PKC) inhibitor staurosporine, suggesting that activation of PKC mediates PMA-induced hmeprinbeta shedding. The use of different protease inhibitors shows that two different metalloprotease activities are responsible for the constitutive and the PMA-stimulated hmeprinbeta shedding. We identified tumor necrosis factor alpha-converting enzyme (TACE or ADAM17) as the protease that mediates the PMA-induced release. We also demonstrate that hmeprinbeta is phosphorylated by PMA treatment on Ser687 within a PKC consensus sequence in the cytosolic domain of the protein. This phosphorylation of hmeprinbeta is not, however, implicated in the enhanced secretion by PMA treatment.     

Anti-CD4 monoclonal antibodies enhance phorbol 12-myristate, 13-acetate-induced activation of human T cells

Evidence exists which indicates that the T cell differentiation molecule CD4 may interact with nonpolymorphic determinants of major histocompatibility complex (MHC) class II antigens on accessory cells to stabilize the formation of a ternary complex formed by the T cell receptor (CD3-TcR), antigen, and MHC class II restriction element. However, there is also evidence which suggests alternative or additional functional roles of CD4 in the delivery of signals to T cells independent of MHC class II recognition. In the present study, we examined different anti-CD4 monoclonal antibodies (mAbs) for their ability to influence lymphocyte proliferation induced by phorbol 12-myristate, 13-acetate (PMA). We found that the response of human peripheral blood mononuclear cells to PMA could be enhanced by some anti-CD4 mAbs (OKT4, OKT4A) but not by others (G17-2). This enhancement was due neither to a direct action of the mAbs on the monocytes nor to intercellular crosslinking through an Fc-Fc receptor interaction. We also found that the binding of anti-CD did not influence the down-regulation of CD4 expression induced by PMA, ruling out any correlation between increased stimulation and CD4 modulation. Our results, taken together with those recently published on the ability of a soluble anti-CD4 mAb (B66) to induce lymphocyte activation by itself, provide evidence that CD4 antigen plays a positive functional role in T cell stimulation in addition to stabilizing the antigen-antigen receptor interaction.     

Persistent effects of phorbol 12-myristate 13-acetate: Possible implication of vesicle traffic

Relative to their normal counterparts, transformed epithelial cells have a distinctive and quantifiable three-dimensional shape. Biophysical and mathematical methods are used to distinguish these extremes in cells from two lines, cultured from rat liver and tracheal epithelium, respectively. Cells adopted a more transformed-looking configuration transiently when exposed to phorbol 12-myristate 13-acetate (PMA) (Plummer and Heckman, [1990] Exp. Cell Res., 188:66–74). The purpose of the present work was to dissect the physiological processes involved in the shape change. Ruffling activity, known to be PMA-stimulated in other cells, was investigated. Although the ruffles appeared less robust than normal, PMA stimulated ruffling activity over a 5 h period. The number of sites where ruffling was initiated declined by 5 h, however, and suppression was seen by 10 h. Cells from both lines adopted the transformed shape configuration when exposed for 2 h to monensin. When the subset of shape features changed by this treatment was compared with those originally changed during transformation, it was found that monensin-treated cells mimicked the features of transformed cells. Its effect on ruffling was, however, unlike PMA's. Thus, the phenotype was unlikely to arise from ruffling itself but might be a process driven by ruffling. Chloroquine also stimulated cells to assume characteristics of transformed cells. Since both it and monensin could interfere with endosomes and with the processing of endocytosed contents, this was a likely site of action. Experiments were done to determine whether PMA also affected the processing of extracellular fluids. When the accumulation of horseradish peroxidase (HRP) was measured, the rate was found to be higher in PMA-treated cells from 5 min, the earliest time assayed, onward. The results suggest that the transformed type of cell in these cell lines showed a constitutive dilation and/or reorganization of some portion of the endosomal pathway. © 1996 Wiley-Liss, Inc.     

Extracellular signal-regulated kinases in T cells. Anti-CD3 and 4 beta-phorbol 12-myristate 13-acetate-induced phosphorylation and activation.

Extracellular signal-regulated kinases (ERK) 1 and 2 are growth factor- and cytokine-sensitive serine/threonine kinases that are known to phosphorylate microtubule-associated protein 2 and myelin basic protein. The current studies examined whether ERK1 and/or ERK2 was present in T cells and whether they were phosphorylated and activated as a consequence of T cell activation. The data demonstrated that both ERK1 and ERK2 were present in Jurkat cells and peripheral blood T cells. In T cells, ERK2 was more prevalent than ERK1. The concentrations of ERK1 and ERK2 were not altered by stimulating the cells for 16 h with immobilized anti-CD3 mAb or anti-CD3 mAb and phorbol myristate acetate. mAb to CD3 and phorbol myristate acetate stimulated an increase in ERK1 and ERK2 MBP kinase activity. Anti-CD3 mAb triggered an increase their phosphate content which was detectable at 2 min but reached a maximum at 5 min. A portion of the increase in phosphate was caused by an increase in phosphotyrosine. We also examined the rate of ERK2 degradation. ERK2 was stable for up to 36 h, and its degradation was unaffected by the activation state of the cells. The data demonstrate that ERK1 and ERK2 are part of an anti-CD3 mAb-stimulated signal transduction cascade that is downstream of protein kinase C and, therefore, suggest that these kinases play an important role in T cell activation.     

Effects of epidermal growth factor and phorbol 12-myristate 13-acetate on protein phosphorylation in mouse embryo palate mesenchyme cells in vitro

Epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulated mouse embryo palate mesenchyme (MEPM) cells to incorporate [32P]O(3-)4 into a protein with an apparent molecular weight of 80 kDa, in vitro. Agents known to elevate intracellular levels of cyclic AMP did not stimulate phosphorylation of this phosphoprotein. Since there is a significant amount of evidence obtained with other cells indicating that phosphorylation of such an 80-kDa phosphoprotein reflects specifically the activation of protein kinase C in response to PMA and other agents, including mitogens, these findings raise the possibility that EGF may activate protein kinase C in MEPM cells.     

Phorbol 12-myristate 13-acetate-dependent protein kinase C delta-Tyr311 phosphorylation in cardiomyocyte caveolae

Protein kinase Cdelta (PKCdelta) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCdelta also is dynamically regulated through tyrosine phosphorylation in H(2)O(2)- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H(2)O(2) activates Src and related Src-family kinases (SFKs), which function as dual PKCdelta-Tyr(311) and -Tyr(332) kinases in vitro and contribute to H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCdelta-Tyr(311) phosphorylation, but this is not associated with SFK activation or PKCdelta-Tyr(332) phosphorylation. Rather, PMA increases PKCdelta-Tyr(311) phosphorylation by delivering PKCdelta to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCdelta-Tyr(311) phosphorylation, without blocking H(2)O(2)-dependent PKCdelta-Tyr(311) phosphorylation. The enzyme that acts as a PKCdelta-Tyr(311) kinase without increasing PKCdelta phosphorylation at Tyr(332) in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCdelta-Tyr(311) kinase, PMA-dependent PKCdelta-Tyr(311) phosphorylation persists in cardiomyocytes treated with the c-Abl inhibitor ST1571 and c-Abl is not detected in caveolae; these results effectively exclude a c-Abl-dependent process. Finally, we show that 1,2-dioleoyl-sn-glycerol mimics the effect of PMA to drive PKCdelta to caveolae and increase PKCdelta-Tyr(311) phosphorylation, whereas G protein-coupled receptor agonists such as norepinephrine and endothelin-1 do not. These results suggest that norepinephrine and endothelin-1 increase 1,2-dioleoyl-sn-glycerol accumulation and activate PKCdelta exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCdelta localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.     

CD45 negatively regulates monocytic cell differentiation by inhibiting phorbol 12-myristate 13-acetate-dependent activation and tyrosine phosphorylation of protein kinase Cdelta

The protein-tyrosine phosphatase CD45 is expressed on all monocytic cells, but its function in these cells is not well defined. Here we report that CD45 negatively regulates monocyte differentiation by inhibiting phorbol 12-myristate 13-acetate (PMA)-dependent activation of protein kinase C (PKC) delta. We found that antisense reduction of CD45 in U937 monocytic cells (CD45as cells) increased by 100% the ability of PMA to enlarge cell size, increase cell cytoplasmic process width and length, and induce surface expression of CD11b. In addition, reduction in CD45 expression caused the duration of peak PMA-induced MEK and extracellular signal-regulated kinase (ERK) 1/2 activity to increase from 5 min to 30 min while leading to a 4-fold increase in PMA-dependent PKCdelta activation. Importantly, PMA-dependent tyrosine phosphorylation of PKCdelta was also increased 4-fold in CD45as cells. Finally, inhibitors of MEK (PD98059) and PKCdelta (rottlerin) completely blocked PMA-induced monocytic cell differentiation. Taken together, these data indicate that CD45 inhibits PMA-dependent PKCdelta activation by impeding PMA-dependent PKCdelta tyrosine phosphorylation. Furthermore, this blunting of PKCdelta activation leads to an inhibition of PKCdelta-dependent activation of ERK1/2 and ERK1/2-dependent monocyte differentiation. These findings suggest that CD45 is a critical regulator of monocytic cell development.     

Fate of immunoprecipitable protein kinase C in GH3 cells treated with phorbol 12-myristate 13-acetate

The effect of phorbol 12-myristate 13-acetate (PMA) on protein kinase C was studied by metabolically labeling GH3 cells with [35S]methionine and using a polyclonal antibody raised against rat brain protein kinase C to immunoprecipitate the enzyme. PMA accelerates the loss of immunologically reactive protein kinase C from the cells in a time- and dose-dependent manner. The half-life of the enzyme in cells treated with 400 nM PMA was 2 h whereas in control cells 60-70% of the enzyme was still detectable after 24 h. The concentration of PMA required to reduce cellular protein kinase C 50% after 24 h was 130 nM. PMA also induced the translocation of [35S]Met-labeled protein kinase C from the cytosol to the membranes in a concentration-dependent manner. Less protein kinase C was translocated to membranes when cells were treated with 20 nM PMA than when they were exposed to 400 nM PMA. In the latter case, most of the labeled protein kinase C became membrane-associated. Maximal translocation was evident after 15 min of incubation with either concentration of PMA and was followed by degradation of the membrane-associated enzyme. The rate of degradation of membrane-associated protein kinase C was the same with both concentrations of PMA. In cells treated with 20 nM PMA, disappearance of [35S]Met-labeled protein kinase C from the cytosolic fraction occurred in two phases, a rapid decrease characteristic of the membrane-associated enzyme, followed by a slower loss similar to that seen in control cells. The results indicate that turnover of protein kinase C is enhanced by membrane association.     

Effect of phorbol 12-myristate 13-acetate and its analogue 4 alpha-phorbol 12,13-didecanoate on protein phosphorylation and lysosomal enzyme release in rabbit neutrophils

The co-carcinogenic compound phorbol 12-myristate 13-acetate but not its inactive analogue 4 alpha-phorbol 12,13-didecanoate causes the phosphorylation of several rabbit neutrophil polypeptides whose molecular weights and isoelectric points (pI) are as follows: Mr = 40,000, pI = 6.4; Mr = 50,000, pI = 4.9; Mr = 55,000, pI = 6.3; Mr = 64,000, pI = 6.0; Mr = 70,000, pI = 5.6; Mr = 90,000, pI = 6.0. Most of these phosphorylated proteins are located exclusively in the cytosol; the 64,000 molecular weight protein is found both in the cytosol and the cytoskeleton, and the 40,000 molecular weight protein is found in the nuclear pellet. The 50,000 molecular weight protein is also phosphorylated in whole cells by the chemotactic peptide fMet-Leu-Phe and in cell-free systems by protein kinase C. Using limited proteolysis, one phosphopeptide fragment was phosphorylated by the three stimuli. In addition, phorbol 12-myristate 13-acetate but not 4 alpha-phorbol 12,13-didecanoate causes cell aggregation and the exocytotic release of the specific granules of rabbit neutrophils. In contrast, both compounds increase the amount of actin associated with the cytoskeleton. The divalent cation ionophore A23187 at low concentration and the compound phorbol 12-myristate 13-acetate act synergistically in causing neutrophil degranulation. Lysosomal enzyme release and the phosphorylation of the 50,000 molecular weight polypeptide produced by phorbl 12-myristate 13-acetate are inhibited by trifluoperazine, and these two responses seem to be causally related. These results are discussed in terms of the role of 1,2-diacylglycerol and activation of protein kinase C in specific granule release from rabbit neutrophils.     

Regulation of butyrate uptake in Caco-2 cells by phorbol 12-myristate 13-acetate

Butyrate and the other short-chain fatty acids (SCFAs) are the most abundant anions in the colonic lumen. Also, butyrate is the preferred energy source for colonocytes and has been shown to regulate colonic electrolyte and fluid absorption. Previous studies from our group have demonstrated that the HCO(3)(-)/SCFA(-) anion exchange process is one of the major mechanisms of butyrate transport across the purified human colonic apical membrane vesicles and the apical membrane of human colonic adenocarcinoma cell line Caco-2 and have suggested that it is mainly mediated via monocarboxylate transporter-1 (MCT-1) isoform. However, little is known regarding the regulation of SCFA transport by various hormones and signal transduction pathways. Therefore, the present studies were undertaken to examine whether hydrocortisone and phorbol 12-myristate 13-acetate (PMA) are involved in a possible regulation of the butyrate/anion exchange process in Caco-2 cells. The butyrate/anion exchange process was assessed by measuring a pH-driven [(14)C]butyrate uptake in Caco-2 cells. Our results demonstrated that 24-h incubation with PMA (1 microM) significantly increased [(14)C]butyrate uptake compared with incubation with 4alphaPMA (inactive form). In contrast, incubation with hydrocortisone had no significant effect on butyrate uptake in Caco-2 cells compared with vehicle (ethanol) alone. Induction of butyrate uptake by PMA appeared to be via an increase in the maximum velocity (V(max)) of the transport process with no significant changes in the K(m) of the transporter for butyrate. Parallel to the increase in the V(max) of [(14)C]butyrate uptake, the MCT-1 protein level was also increased in response to PMA incubation. Our studies demonstrated that the butyrate/anion exchange was increased in response to PMA treatment along with the induction in the level of MCT-1 expression in Caco-2 cells.     

Stimulation of phosphatidylethanolamine synthesis in isolated rat hepatocytes by phorbol 12-myristate 13-acetate

Incubation of freshly isolated rat hepatocytes in the presence of phorbol 12-myristate 13-acetate stimulates the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamines. This stimulation is strongly dependent on the ethanolamine concentration in the medium and becomes apparent at ethanolamine concentrations above 25 microM. Treatment of hepatocytes with phorbol 12-myristate 13-acetate results in a decreased labelling of intracellular ethanolamine, ethanolaminephosphate and CDPethanolamine. Exposure of cells to phorbol 12-myristate 13-acetate induces an increase of the activity of the enzymes CTP: ethanolaminephosphate cytidylyltransferase and ethanolaminephosphotransferase. These effects are accompanied by a decrease of the pool size of ethanolaminephosphate and CDPethanolamine and an increase of the level of diacylglycerols after 30 min of incubation in the presence of phorbol 12-myristate 13-acetate. Upon prolonged incubation, the CDPethanolamine and diacylglycerol pools are restored to the level found in untreated cells. These results indicate that stimulation of phosphatidylethanolamine synthesis by phorbol 12-myristate 13-acetate is probably exerted at the level of CTP : ethanolaminephosphate cytidylytransferase, although there may be an additional effect on the subsequent step of phosphatidylethanolamine synthesis, the formation of phosphatidylethanolamines from CDPethanolamine and diacylglycerols.     

Effects of phorbol 12-myristate 13-acetate on triglyceride and cholesteryl ester synthesis in cultured coronary smooth muscle cells and macrophages

In cultured pig coronary smooth muscle cells phorbol 12-myristate 13-acetate (PMA) stimulated the conversion of [4-14C]cholesterol into cholesteryl esters and the incorporation of [2-3H]glycerol into triglycerides 6.4- and 4.5-fold, respectively. The maximal effects occurred after 3 h of treatment and there was a return to basal values after 72 h. In the presence of 400 microM oleic acid, PMA stimulated the conversion of [4-14C]cholesterol into cholesteryl esters and that of [2-3H]glycerol into triglycerides 5.3- and 2.3-fold, respectively. The stimulatory effects were more sustained (still significant after 72 h) and their maxima were delayed (peaks after 24 h). PMA was also found to increase 2-fold the amount of triglyceride that accumulated in the cells in the presence of oleic acid after 24 h. In macrophages IC-21, the effects of PMA were observed only in the presence of oleic acid. They consisted of a 1.9-fold stimulation in the conversion of [4-14C]cholesterol into cholesteryl esters after 72 h and of a 1.7-fold stimulation in the incorporation of [2-3H]glycerol into triglycerides after 24 h. PMA also increased the amount of triglyceride that accumulated in the cells 1.9-fold after 72 h. It is concluded that PMA, and possibly growth factors, may promote lipid storage in smooth muscle cells and that fatty acids favor long lasting effects of PMA in smooth muscle cells and are necessary for any effect of PMA in macrophages.     

Stimulation of a calcium-dependent actin nucleation activity by phorbol 12-myristate 13-acetate in rabbit macrophage cytoskeletons

Cytoskeletons of detergent-extracted quiescent macrophages have nucleation sites that increase the rate of pyrene-labeled actin assembly in vitro. Cytochalasin D, which inhibits actin assembly at the fast-exchanging ends of filaments (barbed with respect to heavy meromyosin decorated filaments), only partially inhibits the increased assembly rate, demonstrating that pyrene-actin monomers add to both ends of filaments present in the cytoskeletons. Cytoskeletons prepared from macrophages treated with phorbol 12-myristate 13-acetate for 20-30 s before permeabilization, markedly stimulated (300% of control) the rate of actin assembly, and this increment was completely cytochalasin-sensitive, indicating that exposure to phorbol leads to formation of free barbed ends. Nucleation activity required more than 5 nM free calcium only in the assay and was maximal in the presence of 200 nM calcium. Concentrations of calcium of at least 30 nM dissociate the nucleation activity from the cytoskeleton, and it is recovered fully active in the calcium wash.     

Insulin and phorbol ester stimulate phosphorylation of a 40-kDa protein in adipocyte plasma membranes

Several substrates of endogenous Ca2+- and phospholipid-sensitive protein kinase have been identified in plasma membranes and cytosol from rat adipocytes. Specifically, Ca2+ stimulates phosphorylation of a 40-kDa protein in isolated plasma membranes, an effect which is further enhanced by the addition of the phorbol ester tetradecanoylphorbol acetate and phospholipase C. The 40-kDa phosphoprotein is also present in the cytosol, and its phosphorylation is stimulated in a Ca2+-dependent manner by phosphatidylserine, diacylglycerol, and phorbol ester. Direct addition of insulin to adipocyte plasma membranes stimulates phosphorylation of the 40-kDa protein in a concentration-dependent manner. Maximal stimulation was observed at 10(-8) M insulin. At 6.7 X 10(-8) M insulin, phosphorylation of the 40-kDa protein was stimulated by 68 +/- 9% (n = 6). Addition of phorbol ester (1, 10, and 100 ng/ml) plus insulin further enhanced the phosphorylation (286 +/- 39, n = 3; 350 +/- 65, n = 4; and 323 +/- 42%, n = 5, stimulation, respectively). Analysis of the 40-kDa phosphoprotein by two-dimensional polyacrylamide gel electrophoresis revealed that incubations containing no additions, insulin, and/or phorbol ester all resulted in the generation of a single and apparently identical phosphorylated 40-kDa species. These studies indicate that insulin and Ca2+- and phospholipid-dependent protein kinase stimulate phosphorylation of a 40-kDa protein in adipocyte plasma membranes.     

Production of nuclease activity in U937 cells by phorbol 12-myristate 13-acetate and lipopolysaccharide

The proliferation and differentiation signals of myelogeneous U937 cells are provided by extracellular stimuli, such as lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA). In a DNA-native-polyacrylamide gel assay system, we demonstrated that a particular nuclease activity is expressed in PMA-stimulated U937 cells and secreted into the culture medium. The nuclease activity was induced in U937 cells by LPS treatment, while the secretion of the enzyme was undetected in the culture medium. Therefore, it is likely that the expression and secretion of the particular nuclease in U937 cells are controlled by extracellular stimulations, such as PMA and LPS treatment.