Effect Of Phorbol 12-myristate 13-acetate Research Articles

Modulation of ileal apical Na+-dependent bile acid transporter ASBT by protein kinase C

Ileal apical Na(+)-dependent bile acid transporter (ASBT) is responsible for reabsorbing the majority of bile acids from the intestinal lumen. Rapid adaptation of ASBT function in response to physiological and pathophysiological stimuli is essential for the maintenance of bile acid homeostasis. However, not much is known about molecular mechanisms responsible for acute posttranscriptional regulation of ileal ASBT. The protein kinase C (PKC)-dependent pathway represents a major cell signaling mechanism influencing intestinal epithelial functions. The present studies were, therefore, undertaken to investigate ASBT regulation in intestinal Caco-2 monolayers by the well-known PKC activator phorbol 12-myristate 13-acetate (PMA). Our results showed that Na(+)-dependent [(3)H]taurocholic acid uptake in Caco-2 cells was significantly inhibited in response to 2 h incubation with 100 nM PMA compared with incubation with 4alpha-PMA (inactive form). The inhibitory effect of PMA was blocked in the presence of 5 microM bisindolylmaleimide I (PKC inhibitor) but not 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (Ca(2+) chelator) or LY-294002 (phosphatidylinositol 3-kinase inhibitor). PMA inhibition of ASBT function was also abrogated in the presence of myristoylated PKCzeta pseudosubstrate peptide, indicating involvement of the atypical PKCzeta isoform. The inhibition by PMA was associated with a significant decrease in the maximal velocity of the transporter and a reduction in ASBT plasma membrane content, suggesting a modulation by vesicular recycling. Our novel findings demonstrate a posttranscriptional modulation of ileal ASBT function and membrane expression by phorbol ester via a PKCzeta-dependent pathway.

P53 inhibition of AP1-dependent TFF2 expression induces apoptosis and inhibits cell migration in gastric cancer cells

Overexpression of trefoil factor 2 (TFF2) is associated with increased cell migration, resistance to apoptosis, and possibly increased gastric cancer invasion. Dysregulation of p53 is frequently observed in preneoplastic conditions of the stomach. Here, we investigated the effect of p53 on the expression and function of TFF2 in gastric cancer cell lines. Gene expression was determined by reverse transcription-polymerase chain reaction, and promoter activity was assessed by dual luciferase reporter assays. Apoptosis was detected by flow cytometry, and cell migration was evaluated by the Boyden chamber assay. Exogenous expression of p53 dose dependently inhibited endogenous TFF2 mRNA, protein, and promoter activity and resulted in induction of cell apoptosis and inhibition of cell migration. Downregulation of TFF2 by small interfering RNA sensitized gastric cancer cells to drug-induced p53-dependent apoptosis. Addition of human TFF2 peptide reversed p53-dependent apoptosis and inhibition of cell migration. The p53-responsive element was mapped to an AP-1-like cis-element at -182 bp upstream of the TFF2 transcription start site. Mutation of this AP-1-like element abrogated p53-mediated inhibition of TFF2 promoter activity. Gel shift and chromatin immunoprecipitation assays demonstrated that c-Jun and c-Fos bind to this AP-1-like element. Ectopic expression of c-Jun/c-Fos or p300 or treatment of cells with phorbol 12-myristate 13-acetate (PMA) stimulated endogenous TFF2 mRNA expression and promoter activity, and p53 inhibited the effects of AP-1 and PMA on TFF2. p53 induces cell apoptosis and inhibits cell migration in part by downregulating TFF2 expression through an AP-1-like site, suggesting that TFF2 may be an important downstream target of p53.

Patterns of MMP-2 and MMP-9 expression in human cancer cell lines

MMP-2 and MMP-9 secretion is elevated in several types of human cancers and their elevated expression has been associated with poor prognosis. Expression of MMPs is highly regulated by cytokines and signal transducation pathways, including those activated by phorbol 12-myristate 13-acetate (PMA). The aim of this study was to examine the effect of PMA on MMP-2 and MMP-9 secretion in 42 different human cancer cell lines, selected on the basis of their organ malignancies. They were cultured in the recommended media supplemented with 10% FBS and antibiotics in 24-well tissue culture plates. At near confluence, the cells were washed with PBS, 0.5 ml of medium was added, and the cultures were incubated. Parallel sets of cultures were also treated with PMA for induction of enzymes. After 24 h the media were collected and MMP-2 and MMP-9 levels were assayed by gelatinase zymography. Based on MMP-2 and MMP-9 secretion without and with PMA treatment, the various human cancer cell lines fell into one of two major groups. The first group characterized by low basal MMP-9 secretion fell into three different categories of susceptibility to PMA induction of MMP-9 expression: resistant, moderately susceptible and highly susceptible. High basal MMP-9 levels responsive to PMA induction characterized the second group. Most cancer cell lines examined exhibited basal levels of MMP-2, MMP-9 or both. MMP-2 secretion was not induced by PMA in any of the cancer cells examined.

Protein kinase C activation stabilizes LDL receptor mRNA via the JNK pathway in HepG2 cells

LDL is the most abundant cholesterol transport vehicle in plasma and a major prognostic indicator of atherosclerosis. Hepatic LDL receptors limit circulating LDL levels, since cholesterol internalized by the liver can be excreted. As such, mechanisms regulating LDL receptor expression in liver cells are appealing targets for cholesterol-lowering therapeutic strategies. Activation of HepG2 cells with phorbol esters enhances LDL receptor mRNA levels through transcriptional and posttranscriptional mechanisms. Here, we show that 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced stabilization of receptor mRNA requires the activity of protein kinase C and is accompanied by activation of the major mitogen activated protein kinase pathways. Inhibitor studies demonstrated that receptor mRNA stabilization is independent of the extracellular signal-regulated kinase or p38(MAPK), but requires activation of the c-Jun N-terminal kinase (JNK). An essential role for JNK in stabilizing receptor mRNA was further confirmed through small interfering RNA (siRNA) experiments and by activating JNK through two protein kinase C-independent mechanisms. Finally, prolonged JNK activation increased steady-state levels of receptor mRNA and protein, and significantly enhanced cellular LDL-binding activity. These data suggest that JNK may play an important role in posttranscriptional control of LDL receptor expression, thus constituting a novel mechanism to enhance plasma LDL clearance by liver cells.

The effects of phorbol ester activation and reactive oxygen species scavengers on the macrophage‐mediated foreign body reaction to polyurethanes

Phorbol myristate acetate, a protein kinase C activator, inhibited monocyte-derived macrophage (MDM)-mediated degradation of aliphatic (HDI) polycarbonate-based polyurethanes but not degradation of the aromatic polycarbonate-based polyurethane (MDI). The objectives of this study were to determine if reactive oxygen species are involved in the phorbol myristate acetate effect on esterase activity and MDM-mediated polycarbonate-based polyurethane degradation and to find a good marker of material-initiated activation of MDM. The phorbol myristate acetate-dependent effects of the material chemistry on cell activation and degradation were evaluated by adding reactive oxygen species scavengers, catalase plus superoxide dismutase to MDM and assaying possible markers of MDM activation: esterase activity, acid phosphatase activity, and high molecular weight group box 1 protein (HMGB1). All treatments reduced the esterase activity in MDM on HDI but not in MDM on MDI. Acid phosphatase was inhibited in MDM to varying degrees on all surfaces by phorbol myristate acetate or catalase plus superoxide dismutase either alone or together. Secretion of HMGB1 from MDM on HDI431 was higher than MDI; however only secretion from MDM on HDI was inhibited by phorbol myristate acetate. In MDM on HDI, catalase plus superoxide dismutase reduced intracellular HMGB1 levels +/- phorbol myristate acetate; whereas, catalase, superoxide dismutase plus phorbol myristate acetate increased intracellular HMGB1 in MDM on MDI, suggesting that esterase and HMGB1 are more specific markers of activation than acid phosphatase. Manipulation of signaling pathways may provide insight surrounding the mechanism of activation for oxidative and/or hydrolytic degradative pathways in the MDM response to material surface chemistry.

Effects of phorbol 12-myristate 13-acetate on potassium transport in the red blood cells of frog Rana temporaria

Phorbol 12-myristate 13-acetate (PMA), a stimulator of PKC, was examined for its influence on K(+) ((86)Rb) influx in the frog erythrocyte. PMA, 0.1 microM, was found to accelerate ouabain-sensitive K(+) influx, which was suppressed by 73% with 1 mM amiloride, indicating secondary activation of the Na(+)-K(+)-pump due to stimulation of Na(+ )/H(+) exchange. PMA-induced stimulation of the sodium pump was completely inhibited with 1 microM staurosporine and by approximately 50% with 20 microM chelerythrine. In contrast to Na(+)-K(+)-pump, an activity of Cl(-)-dependent K(+) transport (K-Cl cotransport, KCC), calculated as the difference between K(+) influxes in Cl(-) and NO(3) (-)-media, was substantially decreased under the influence of PMA. Staurosporine fully restored the PMA-induced inhibition of KCC, whereas chelerythrine did not exert any influence. Osmotic swelling of the frog erythrocytes was accompanied by approximately twofold stimulation of KCC. Swelling-activated KCC was inhibited by approximately 50 and approximately 83% in the presence of PMA and genistein, respectively, but not chelerythrine. Exposure of the frog erythrocytes to 5 mM fluoride (F(-)) also reduced the KCC activity in isotonic and hypotonic media, with maximal suppression of K(+) influx in both media being observed upon simultaneous addition of PMA and F(-). Furosemide and [(dihydronindenyl)oxy] alkanoic acid inhibited the K(+) influx in both the media by approximately 50-60%. The results obtained show both the direct and indirect effects of PMA on the K(+) transport in frog erythrocytes and a complicated picture of KCC regulation in frog erythrocytes with involvement of PKC, tyrosine kinase and protein phosphatase.

Identification of an Autoinhibitory Mechanism That Restricts C1 Domain-mediated Activation of the Rac-GAP α2-Chimaerin

Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gained recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Like protein kinase C isozymes, chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified an autoinhibitory mechanism in alpha2-chimaerin that restricts access of phorbol esters and DAG, thereby limiting its activation. Although phorbol 12-myristate 13-acetate (PMA) caused limited translocation of wild-type alpha2-chimaerin to the plasma membrane, deletion of either N- or C-terminal regions greatly sensitize alpha2-chimaerin for intracellular redistribution and activation. Based on modeling analysis that revealed an occlusion of the ligand binding site in the alpha2-chimaerin C1 domain, we identified key amino acids that stabilize the inactive conformation. Mutation of these sites renders alpha2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Notably, in contrast to PMA, epidermal growth factor promotes full translocation of alpha2-chimaerin in a phospholipase C-dependent manner, but not of a C1 domain mutant with reduced affinity for DAG (P216A-alpha2-chimaerin). Therefore, DAG generation and binding to the C1 domain are required but not sufficient for epidermal growth factor-induced alpha2-chimaerin membrane association. Our studies suggest a role for DAG in anchoring rather than activation of alpha2-chimaerin. Like other DAG/phorbol ester receptors, including protein kinase C isozymes, alpha2-chimaerin is subject to autoinhibition by intramolecular contacts, suggesting a highly regulated mechanism for the activation of this Rac-GAP.

Phorbol-12-myristate 13-Acetate Acting through Protein Kinase Cε Induces Translocator Protein (18-kDa) Tspo Gene Expression

Translocator protein (TSPO) is an 18-kDa cholesterol-binding protein that is expressed at high levels in steroid synthesizing and several cancer cells where it is involved in steroidogenesis and cell proliferation, respectively. The factors regulating Tspo expression are unknown. We analyzed Tspo transcriptional responses to the tumor promoter, phorbol-12-myristate 13-acetate (PMA), in cells with varying TSPO levels. PMA induced Tspo promoter activity and Tspo mRNA levels in TSPO-poor nonsteroidogenic cells (NIH-3T3 fibroblasts and COS-7 kidney) but not in TSPO-rich steroidogenic cells (MA-10 Leydig) with high basal Tspo transcriptional activity. The stimulatory effect of PMA was mediated by an 805-515-bp region upstream of the transcription start site. Electrophoretic mobility shift assay (EMSA) revealed that PMA induced binding of c-jun and GA-binding protein transcription factor (GABP-alpha) to their respective activator protein 1 (AP1) and v-ets erythroblastosis virus E26 oncogene homologue (Ets) sites in this region. Protein kinase C (PKC)-specific inhibitors blocked PMA induction of Tspo promoter activity with an inhibition profile suggestive of involvement of PKCepsilon. PKCepsilon expression correlated with TSPO content in the three cell lines. In NIH-3T3 cells, PKCepsilon overexpression induced Tspo promoter activity and mRNA levels and enhanced PMA-induced up regulation of c-jun and TSPO. In MA-10 cells, a PKCepsilon-specific translocation inhibitor peptide reduced basal Tspo promoter activity. PKCepsilon siRNA pool reduced PKCepsilon and TSPO levels in MA-10 cells indicating a role for PKCepsilon in regulating TSPO expression. Taken together, these data suggest that elevated TSPO expression in steroidogenic cells may be due to high constitutive expression of PKCepsilon that renders them unresponsive to further induction while PMA activation of PKCepsilon drives inducible TSPO expression in nonsteroidogenic cells, likely through AP1 and Ets.

Alkaline ceramidase 2 regulates β1 integrin maturation and cell adhesion

The polypeptide core of the integrin beta1 subunit (beta1) is glycosylated sequentially in the endoplasmic reticulum and the Golgi complex to form beta1 precursor and mature beta1, respectively. The beta1 precursor to mature beta1 conversion, termed beta1 maturation, regulates the cell surface levels and function of beta1-containing integrins, beta1 integrins. Here we demonstrate that the human alkaline ceramidase 2 (ACER2), a Golgi enzyme, regulates beta1 maturation by controlling the generation of sphingosine. ACER2 overexpression inhibited beta1 maturation, thus leading to a decrease in the levels of mature beta1 in T-REx HeLa cells, whereas RNA interference-mediated knockdown of ACER2 enhanced beta1 maturation in MCF-7 cells. ACER2 overexpression decreased the cell surface levels of beta1 integrins, thus inhibiting cell adhesion to fibronectin or collagen, whereas ACER2 knockdown has the opposite effects. Treatment with all-trans retinoic acid (ATRA) increased both the expression of ACER2 and the generation of sphingosine in HeLa cells and inhibited beta1 maturation. ACER2 knockdown attenuated the inhibitory effects of ATRA on both beta1 maturation and cell adhesion. In contrast, treatment with phorbol myristate acetate (PMA), a protein kinase C activator, decreased the expression of ACER2 and sphingosine in T-REx HeLa cells, thus enhancing beta1 maturation. ACER2 overexpression inhibited the stimulatory effects of PMA on both beta1 maturation and cell adhesion. These results suggest that the ACER2/sphingosine pathway plays an important role in regulating beta1 maturation and cell adhesion mediated by beta1 integrins.

“Functional mapping of the promoter region of the GNB2L1 human gene coding for RACK1 scaffold protein”

RACK1 (Receptor for Activated C Kinase 1) is a scaffold protein for different kinases and membrane receptors. Previously, we characterized an age-dependent decline of RACK1 protein expression which could be counteracted with DHEA (dehydroepiandrosterone) [Corsini, E., et al. 2002. In vivo dehydroepiandrosterone restores age-associated defects in the protein kinase C signal transduction pathway and related functional responses. J. Immunol. 168, 1753–1758. and Corsini, E., et al. 2005. Age-related decline in RACK-1 expression in human leukocytes is correlated to plasma levels of dehydroepiandrosterone. J. Leukoc. Biol. 77, 247–256.]. Hypothesizing a direct control of RACK1 expression by DHEA we studied the not yet characterized human promoter region of its coding gene GNB2L1. The FLOE (Fluorescently Labeled Oligonucleotide Extension) was used to map the transcription start site and a novel Gateway luciferase vector (GW luc basic; Del Vecchio, I., Zuccotti, A., Canneva, F., Lenzken, S.C., Racchi, M., 2007. Development of the first Gateway firefly luciferase vector and use of reverse transcriptase in FLOE (Fluorescently Labeled Oligonucleotide Extension) reactions. Plasmid 58, 269–274.) to obtain promoter region mutants. Human SH-SY5Y, THP1 and lymphoblastoid cells were used for transient transfections and treatments with lipopolysaccharide (LPS), phorbol myristate acetate (PMA), DHEA and cortisol (the first two molecules to differently activate NF-kB, a transcription complex able to regulate the murine Gnb2l1 gene expression, whereas DHEA and cortisol since they are known to be imbalanced during the aging and possess counteracting actions on the immune function). The primer extension demonstrated the existence of two alternative start sites of transcription respectively located at about 230 and 300 nt 5′ of the Genbank mRNA entry for GNB2L1. Moreover, as a result of the luciferase study we were able to demonstrate that a little region of approximately 300 nt conserved sufficient elements for reporter expression. We also reported that the DHEA modulation of GNB2L1 endogenous expression could not be recapitulated with the luciferase assays. Indeed, the promoter was significantly modulated by means of LPS and PMA treatments but not using DHEA. Differently the use of cortisol led us to demonstrate a biologically significant decrease of luciferase activity only in the presence of a binding site for nuclear receptors of glucocorticoids. Interestingly, other binding sites for transcriptional factors were identified in silico: different c-Rel (NF-kB) and some cardiomyocitic specific cis-acting elements. All this data suggest that the DHEA mediated GNB2L1 regulation is modulated by distant elements (enhancers/silencers), whereas LPS, PMA and cortisol effect can act directly on the mapped GNB2L1 promoter. In conclusion we hypothesize that the imbalance between DHEA and cortisol during aging could be important in the previously demonstrated recovery of the RACK1 expression.

Protein Kinase C Regulates the Expression of M1 Receptors and BDNF in Rat Retinal Cells

Protein kinase C (PKC) plays a key role in cellular events including proliferation, survival and differentiation. Our previous study showed the effect of phorbol 12-myristate 13-acetate (PMA), a PKC activator, inducing a decrease in retinal cells proliferation. This effect was mediated by muscarinic type 1 receptors (M1) activation and brain derived neurotrophic factor (BDNF) treatment also induced a decrease in cell proliferation. Based on these results we analyzed the expression of either M1 receptors or BDNF following PMA treatment of retinal cell cultures. Our data demonstrated that PMA induced a decrease in both protein expressions after 48 h in culture. However, after 45 min, PMA induced a transient increase in BDNF expression and a decrease in M1 receptors expression. Analyzing the expression of M1 receptors and BDNF during the postnatal development in vivo, we observed a decrease in both proteins. Taken together our results suggest the involvement of PKC in the control of M1 expression in retinal cells.

The regulation of glycine transporter GLYT1 is mainly mediated by protein kinase Cα in C6 glioma cells

Glycine has been shown to possess important functions as a bidirectional neurotransmitter. At synaptic clefts, the concentration of glycine is tightly regulated by the uptake of glycine released from nerve terminals into glial cells by the transporter GLYT1. It has been recently demonstrated that protein kinase C (PKC) mediates the downregulation of GLYT1 activity in several cell systems. However, it remains to be elucidated which subtypes of PKC might be important in the regulation of GLYT1 activity. In this study, we attempted to make clear the mechanism of the phorbol 12-myristate 13-acetate (PMA)-suppressed uptake of glycine in C6 glioma cells which have the native expression of GLYT1. In C6 cells, the expression of PKCα, PKCδ, and PKCɛ of the PMA-activated subtypes was detected. The PMA-suppressed action was fully reversed by the removal of both extracellular and intracellular Ca 2+. Furthermore, the inhibitory effects of PMA or thymeleatoxin (THX), which is a selective activator of conventional PKC (cPKC), were blocked by the downregulation of all PKCs expressed in C6 cells by long-term incubation with THX, or pretreatment with GF109203X or Gö6983, which are broad inhibitors of PKC, or Gö6976, a selective inhibitor of cPKC. On the other hand, treatment of C6 cells with ingenol, a selective activator of novel PKCs, especially PKCδ and PKCɛ, did not affect the transport of glycine. Silencing of PKCδ expression by using RNA interference or pretreatment with the inhibitor peptide for PKCɛ had no effect on the PMA-suppressed uptake of glycine. Together, these results suggest PKCα to be a crucial factor in the regulation of glycine transport in C6 cells.

A pH-stabilizing role of voltage-gated proton channels in IgE-mediated activation of human basophils

Eosinophils and other phagocytes use NADPH oxidase to kill bacteria. Proton channels in human eosinophils and neutrophils are thought to sustain NADPH oxidase activity, and their opening is greatly enhanced by a variety of NADPH oxidase activators, including phorbol myristate acetate (PMA). In nonphagocytic cells that lack NADPH oxidase, no clear effect of PMA on proton channels has been reported. The basophil is a granulocyte that is developmentally closely related to the eosinophil but nevertheless does not express NADPH oxidase. Thus, one might expect that stimulating basophils with PMA would not affect proton currents. However, stimulation of human basophils in perforated-patch configuration with PMA, N-formyl-methionyl-leucyl-phenylalanine, or anti-IgE greatly enhanced proton currents, the latter suggesting involvement of proton channels during activation of basophils by allergens through their highly expressed IgE receptor (Fc epsilonRI). The anti-IgE-stimulated response occurred in a fraction of cells that varied among donors and was less profound than that to PMA. PKC inhibition reversed the activation of proton channels, and the proton channel response to anti-IgE or PMA persisted in Ca(2+)-free solutions. Zn(2+) at concentrations that inhibit proton current inhibited histamine release elicited by PMA or anti-IgE. Studied with confocal microscopy by using SNARF-AM and the shifted excitation and emission ratioing of fluorescence approach, anti-IgE produced acidification that was exacerbated in the presence of 100 microM Zn(2+). Evidently, proton channels are active in basophils during IgE-mediated responses and prevent excessive acidification, which may account for their role in histamine release.

Effect of phorbol myristate acetate on T cell colony formation, interleukin-2 (IL-2) receptor expression and IL-2 production by cells from patients at all stages of HIV infection

We and others have shown that several T cell responses induced by the mitogen phytohaemagglutinin (PHA), including T cell colony formation, IL-2 receptor (IL-2R) expression, and IL-2 production are impaired in patients with AIDS and lymphadenopathy syndrome (LAS). We investigated whether phorbol myristate acetate (PMA) could act in synergy with PHA (as it does in healthy subjects) to enhance in vitro T cell responses of patients at all stages of infection by HIV. In AIDS patients with opportunistic infections (AIDS/OI), PHA + IL-2 + PMA led to a total disappearance of T cell colonies in 10/11 patients, among whom six already displayed very low numbers of colonies induced by PHA + IL-2 (less than 50 colonies/5 x 10(4) cells). In contrast, T cell colony formation induced by PHA + IL-2 + PMA was maintained or increased, compared with that induced by PHA + IL-2, in five out of six AIDS patients with Kaposi's sarcoma (AIDS/KS), 10/14 LAS and six out of seven HIV-seropositive asymptomatic (HIV+/AS) homosexuals. In these three groups of patients, a low percentage of colony cells induced by PHA + IL-2 + PMA expressed CD3 and CD4 molecules, but 50-89% of cells were IL-2R (Tac) positive, as in healthy controls. Studies on T cell activation and IL-2 production were performed on a selected group of 12 HIV-infected patients for whom sufficient numbers of lymphocytes could be obtained. PMA induced CD4 down-modulation in controls and in HIV-infected patients. However, CD3 down-modulation and induction of the Tac chain of IL-2R by PMA were significantly impaired in patients, compared with controls, and these two parameters were correlated. Although PHA alone induced virtually normal levels of Tac antigen on patients' cells, Tac induction by PHA + PMA was significantly decreased in patients versus controls. Cells from five out of 10 patients tested failed to produce detectable amounts of IL-2 after PHA stimulation, whereas IL-2 production increased significantly in all patients tested (n = 9) after PHA + PMA, with a level of IL-2 activity significantly higher than in controls. No correlation was found in this group of patients between the effects of PMA + PHA on T cell colony formation, Tac expression, or IL-2 production, as compared with PHA alone. Taken together, our results indicate that in vitro T cell functional studies with PMA may be useful to evaluate better the defects of T cell activation in HIV-infected patients.

Phorbol 12-Myristate 13-Acetate-dependent Protein Kinase Cδ-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.

Protein kinase C-mediated protection of motoneurons from excitotoxicity

Amyotrophic lateral sclerosis (ALS) is a progressive motoneuron disease, whose pathogenesis remains unclear. Phorbol 12-myristate 13-acetate (PMA) has been shown to be neurotrophic and neuroprotective in several types of neurons in culture. However, the survival effect of PMA on motoneurons has not been reported. Therefore, we investigated this using purified rat motoneurons. PMA showed a small but significant neurotrophic activity, which was inhibited by GF109203X, a protein kinase C (PKC) inhibitor. Under the conditions in which the survival of motoneurons is supported by brain-derived neurotrophic factor (BDNF), excitotoxicity was induced by glutamate. PMA markedly protected motoneurons against excitotoxicity. The up-regulation of PKC in the spinal cord of ALS patients was previously reported, therefore, the present results indicate that PKC activation might be involved in neuroprotection.

Nuclear localization of regucalcin is enhanced in culture with protein kinase C activation in cloned normal rat kidney proximal tubular epithelial NRK52E cells

In this study we investigated whether the nuclear localization of regucalcin in cloned normal rat kidney tubular epithelial NRK52E cells is regulated after culture with hormonal signaling factors. Stable regucalcin/pCXN2 transfectants with subconfluent monolayers were further cultured for 24 or 48 h in a serum-free medium containing either vehicle, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1), parathyroid hormone (PTH), phorbol 12-myristate 13-acetate (PMA), or other factors. Culture with TNF-alpha (1.0 ng/ml of medium) or TGF-beta1 (5.0 ng/ml) for 48 h caused a significant decrease in regucalcin mRNA levels in NRK52E cells (wild-type), while regucalcin mRNA levels were markedly increased in the presence of PMA (10(-6) M), an activator of protein kinase C, in wild-type cells. Immunocytochemical observation showed that HA-regucalcin was markedly localized in the nucleus of HA-regucalcin/ phCMV2-transfected cells. The nuclear localization was enhanced in culture with BS (5%), PTH (10(-7) M), Bay K 8644 (2.5x10(-6) M), or PMA (10(-6) M) for 24 or 48 h. Culture with staurosporine, an inhibitor of protein kinase C, caused a remarkable decrease in the localization of HA-regucalcin in the nucleus of HA-RGPR-p117/phCMV2-transfected cells with PMA. Culture with PMA (10(-6) M) for 24 or 48 h caused a remarkable increase in nuclear regucalcin protein levels. The effect of PMA in increasing nuclear regucalcin levels was completely absent in culture with staurosporine (10(-8) M). The nuclear localization of regucalcin in the stable regucalcin/pCXN2-transfected cells (transfectant) increased markedly as compared with that of wild-type cells, whereas the increase was less evident in the transfectants cultured with staurosporine. This study demonstrated that regucalcin localizes in the nucleus of cloned normal rat kidney proximal tubular epithelial NRK52E cells, and that its nuclear localization is enhanced through an intracellular signaling process which involves protein kinase C.

Protein kinase CβI interacts with the β1-adrenergic signaling pathway to attenuate lipolysis in rat adipocytes

We have shown previously that insulin attenuates β1-adrenergic receptor (β1-AR)-mediated lipolysis via activation of protein kinase C (PKC) in rat adipocytes. This antilipolysis persists after removal of insulin and is independent of the phosphodiesterase 3B activity, and phorbol 12-myristate 13-acetate (PMA) could substitute for insulin to produce the same effect. Here, we attempted to identify the PKC isoform responsible for antilipolysis. Isolated adipocytes were treated with high and low concentrations of PMA for up to 6 h to degrade specific PKC isoforms. In the PMA-treated cells, the downregulation profiles of PKC isoforms α and βI, but not βII, δ, ɛ, or ζ, correlated well with a decrease of lipolysis-attenuating effect of PMA. After rats fasted for 24 h, adipocyte expression of PKC isoform α increased, while expression of PKCδ decreased. Fasting did not change the potency of PMA to attenuate lipolysis, however. The lipolysis-attenuating effect of PMA was blocked by the PKCβI/βII inhibitor LY 333531, but not by the PKCβII inhibitor CGP 53353 or the PKCδ inhibitor rottlerin. These data suggest that PKCβI interacts with β1-AR signaling and attenuates lipolysis in rat adipocytes.

The angiotensin receptor type 1–Gq protein–phosphatidyl inositol phospholipase Cβ–protein kinase C pathway is involved in activation of proximal tubule Na+‐ATPase activity by angiotensin(1–7) in pig kidneys

In a previous study, we observed that angiotensin(1-7) (Ang(1-7)) stimulates proximal tubule Na+-ATPase activity through the angiotensin receptor type 1 (AT1R). Here we aimed to study the signalling pathways involved. Our results show that the stimulatory effect of Ang(1-7) on Na+-ATPase activity through AT1R involves a Gq protein-phosphatidyl inositol-phospholipase Cbeta (PI-PLCbeta) pathway because: (1) the effect was reversed by GDPbetaS, a non-hydrolysable GDP analogue, and by a monoclonal Gq protein antibody; (2) the effect was similar and not additive to that of GTPgammaS, a non-hydrolysable GTP analogue; (3) Ang(1-7) induced a rapid decrease (30 s) in phosphatidylinositol 4,5-bisphosphate levels, a PI-PLCbeta substrate; and (4) U73122, a specific inhibitor of PI-PLCbeta, abolished Ang(1-7)-induced stimulation of Na+-ATPase activity. Angiotensin(1-7) increased the protein kinase C (PKC) activity similarly to phorbol-12-myristate-13-acetate (PMA), an activator of PKC. This effect was reversed by losartan, a specific antagonist of AT1R. The stimulatory effects of Ang(1-7) and PMA on Na+-ATPase activity are similar, non-additive and reversed by calphostin C, a specific inhibitor of PKC. A catalytic subunit of PKC (PKC-M) increased the Na+-ATPase activity. These data show that Ang(1-7) stimulates Na+-ATPase activity through the AT1R-Gq protein-PI-PLCbeta-PKC pathway. This effect is similar to that described for angiotensin II, showing for the first time that these compounds could have similar effects in the renal system.

S-Phase-specific Activation of PKCα Induces Senescence in Non-small Cell Lung Cancer Cells

Protein kinase C (PKC) has been widely implicated in positive and negative control of cell proliferation. We have recently shown that treatment of non-small cell lung cancer (NSCLC) cells with phorbol 12-myristate 13-acetate (PMA) during G1 phase inhibits the progression into S phase, an effect mediated by PKC delta-induced up-regulation of the cell cycle inhibitor p21 Cip1. However, PMA treatment in asynchronously growing NSCLC cells leads to accumulation of cells in G2/M. Studies in post-G1 phases revealed that PMA induced an irreversible G2/M cell cycle arrest in NSCLC cells and conferred morphological and biochemical features of senescence, including elevated SA-beta-Gal activity and reduced telomerase activity. Remarkably, this effect was phase-specific, as it occurred only when PKC was activated in S, but not in G1, phase. Mechanistic analysis revealed a crucial role for the classical PKC alpha isozyme as mediator of the G2/M arrest and senescence, as well as for inducing p21(Cip1) an obligatory event for conferring the senescence phenotype. In addition to the unappreciated role of PKC isozymes, and specifically PKC alpha, in senescence, our data introduce the paradigm that discrete PKCs trigger distinctive responses when activated in different phases of the cell cycle via a common mechanism that involves p21 Cip1 up-regulation.