Stimulation Of Protein Kinase C Research Articles

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37 degrees C. After subtraction of unspecific uptake determined in WT at 37 degrees C or in hOCT2 at 8 degrees C saturable specific uptake of both substrates was measured. Km values of hOCT2-mediated uptake of 95 microM amiloride and 24 microM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC50 (in microM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca2+/CaM complex (-55 +/- 5%, n = 10 and -63 +/- 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (-54 +/- 5%, n = 14, and -31 +/- 6%, n = 26) and PKA (-16 +/- 5%, n = 16, and -18 +/- 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 +/- 6%, n = 8, and +55 +/- 17%, n = 16). Inhibition of Ca2+/CaM complex resulted in a significant decrease of Vmax (160-99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (-22 +/- 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca2+/CaM complex causes changes in transport capacity via hOCT2 trafficking.

Protein kinase C mediates erythrocyte “programmed cell death” following glucose depletion

Glucose depletion of erythrocytes leads to activation of Ca2+-permeable cation channels, Ca2+ entry, activation of a Ca2+-sensitive erythrocyte scramblase, and subsequent exposure of phosphatidylserine at the erythrocyte surface. Ca2+ entry into erythrocytes was previously shown to be stimulated by phorbol esters and to be inhibited by staurosporine and chelerythrine and is thus thought to be regulated by protein phosphorylation/dephosphorylation, presumably via protein kinase C (PKC) and the corresponding phosphoserine/threonine phosphatases. The present experiments explored whether PKC could contribute to effects of energy depletion on erythrocyte phosphatidylserine exposure and cell volume. Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorter analysis. Removal of extracellular glucose led to depletion of cellular ATP, stimulated PKC activity, led to translocation of PKCalpha, enhanced serine phosphorylation of membrane proteins, decreased cell volume, and increased annexin binding, the latter effect being blunted but not abolished in the presence of 1 microM staurosporine or 50 nM calphostin C. The PKC stimulator phorbol-12-myristate-13-acetate (3 microM) and the phosphatase inhibitor okadaic acid (1-10 microM) mimicked the effect of glucose depletion and similarly led to translocation of PKCalpha and enhanced serine phosphorylation, increased annexin binding, and decreased forward scatter, the latter effects being abrogated by PKC inhibitor staurosporine (1 microM). Fluo-3 fluorescence measurements revealed that okadaic acid also enhanced erythrocyte Ca2+ activity. The present observations suggest that protein phosphorylation and dephosphorylation via PKC and the corresponding protein phosphatases contribute to phosphatidylserine exposure and cell shrinkage after energy depletion.

Induction of protein degradation in skeletal muscle by a phorbol ester involves upregulation of the ubiquitin–proteasome proteolytic pathway

Although muscle atrophy is common to a number of disease states there is incomplete knowledge of the cellular mechanisms involved. In this study murine myotubes were treated with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to evaluate the role of protein kinase C (PKC) as an upstream intermediate in protein degradation. TPA showed a parabolic dose–response curve for the induction of total protein degradation, with an optimal effect at a concentration of 25 nM, and an optimal incubation time of 3 h. Protein degradation was attenuated by co-incubation with the proteasome inhibitor lactacystin (5 μM), suggesting that it was mediated through the ubiquitin–proteasome proteolytic pathway. TPA induced an increased expression and activity of the ubiquitin–proteasome pathway, as evidenced by an increased functional activity, and increased expression of the 20S proteasome α-subunits, the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme E2 14k, also with a maximal effect at a concentration of 25 nM and with a 3 h incubation time. There was also a reciprocal decrease in the cellular content of the myofibrillar protein myosin. TPA induced activation of PKC maximally at a concentration of 25 nM and this effect was attenuated by the PKC inhibitor calphostin C (300 nM), as was also total protein degradation. These results suggest that stimulation of PKC in muscle cells initiates protein degradation through the ubiquitin–proteasome pathway. TPA also induced degradation of the inhibitory protein, I-κBα, and increased nuclear accumulation of nuclear factor-κB (NF-κB) at the same time and concentrations as those inducing proteasome expression. In addition inhibition of NF-κB activation by resveratrol (30 μM) attenuated protein degradation induced by TPA. These results suggest that the induction of proteasome expression by TPA may involve the transcription factor NF-κB.

Stable knockdown of CFTR establishes a role for the channel in P2Y receptor‐stimulated anion secretion

P2Y receptor regulation of anion secretion was investigated in porcine endometrial gland (PEG) epithelial cells. P2Y2, P2Y4, and P2Y6 receptors were detected in monolayers of PEG cells and immunocytochemistry indicated that P2Y4 receptors were located in the apical membrane. Apical membrane current measurements showed that Ca2+-dependent and PKC-dependent Cl- channels were activated following treatment with uridine triphosphate (UTP) (5 microM). Current-voltage relationships comparing calcium-dependent and PKC-dependent UTP responses under biionic conditions showed significant differences in selectivity between Cl-)and I- for the PKC-dependent conductance (P(I)/P(Cl) = 0.76), but not for Ca2+-dependent conductance (PI/P(Cl) = 1.02). The I-/Cl- permeability ratio for the PKC-dependent conductance was identical to that measured for 8-cpt cAMP. Furthermore, PKC stimulation using phorbol 12-myristate 13-acetate (PMA) activated an apical membrane Cl- conductance that was blocked by the CFTR selective inhibitor, CFTRinh-172. CFTR silencing, accomplished by stable expression of small hairpin RNAs (shRNA), blocked the PKC-activated conductance associated with UTP stimulation and provided definitive evidence of a role for CFTR in anion secretion. CFTR activation increased the initial magnitude of Cl- secretion, and provided a more sustained secretory response compared to conditions where only Ca2+-activated Cl- channels were activated by UTP. Measurements of [cAMP]i following UTP and PMA stimulation were not significantly different than untreated controls. Thus, these results demonstrate that UTP and PMA activation of CFTR occurs independently of increases in intracellular cAMP and extend the findings of earlier studies of CFTR regulation by PKC in Xenopus oocytes to a mammalian anion secreting epithelium.

Protein kinases influence bovine oocyte competence during short-term treatment with recombinant human follicle stimulating hormone

The aim of this study was to investigate the effect of short-term treatment (first 2 or 6 h) with recombinant human follicle-stimulating hormone (r-hFSH) during in vitro maturation (IVM) on the developmental competence of bovine oocytes. The roles of protein kinase A (PKA) and protein kinase C (PKC) (possibly involved in FSH response), were investigated using activators (Sp-cAMPS, PMA) or inhibitors (Rp-cAMPS, sphingosine) of these two protein kinases, respectively. The developmental competence of bovine oocytes was measured by the rate of blastocyst formation after in vitro fertilization (IVF). Our results showed that when cumulus-oocyte complexes (COCs) were cultured with r-hFSH for the first 6 h, a highly significant (P < 0.0001) improvement is seen in blastocyst development rate as a proportion of oocytes in culture compared with those matured with r-hFSH for the first 2 or 24 h. A transient exposure (6 h) to the highest dose (100 microM) of forskolin (an activator of adenylate cyclase) increased (P < 0.05) the rate of blastocyst formation. But the PKA inhibitors (Rp-cAMPS) did not affect the stimulatory effects of r-hFSH on the blastocyst yield. However, stimulation of PKC by low doses of PMA (0.1-0.5 microM) during short-term treatment, enhanced (P < 0.0001) the developmental capacity of oocytes, while sphingosine (a specific inhibitor of PKC) inhibited (P < 0.05) the stimulatory effects of r-hFSH on the rate of blastocyst formation. Our results indicate that although the developmental capacity of bovine oocytes in vitro can be modulated by both the PKA, and the PKC pathways, the activation of PKC during short-term treatment can mimic the effect of r-hFSH on the cytoplasmic maturation in bovine oocytes in vitro.

Neuronal ELAV proteins enhance mRNA stability by a PKCα-dependent pathway

More than 1 in 20 human genes bear in the mRNA 3' UTR a specific motif called the adenine- and uridine-rich element (ARE), which posttranscriptionally determines its expression in response to cell environmental signals. ELAV (embryonic lethal abnormal vision) proteins are the only known ARE-binding factors that are able to stabilize the bound mRNAs, thereby positively controlling gene expression. Here, we show that in human neuroblastoma SH-SY5Y cells, neuron-specific ELAV (nELAV) proteins (HuB, HuC, and HuD) are up-regulated and redistributed by 15 min of treatment with the activators of PKC phorbol esters and bryostatin-1. PKC stimulation also induces nELAV proteins to colocalize with the translocated PKCalpha isozyme preferentially on the cytoskeleton, with a concomitant increase of nELAV threonine phosphorylation. The same treatment promotes stabilization of growth-associated protein 43 (GAP-43) mRNA, a well known nELAV target, and induces an early increase in GAP-43 protein concentration, again only in the cytoskeletal cell fraction. Genetic or pharmacological inactivation of PKCalpha abolishes nELAV protein cytoskeletal up-regulation, GAP-43 mRNA stabilization, and GAP-43 protein increase, demonstrating the primary role of this specific PKC isozyme in the cascade of nELAV recruitment. Finally, in vivo PKC activation is associated with an up-regulation of nELAV proteins in the hippocampal rat brain. These findings suggest a model for gene expression regulation by nELAV proteins through a PKCalpha-dependent pathway that is relevant for the cellular programs in which ARE-mediated control plays a pivotal role.

PKC-induced ERK1/2 interactions and downstream effectors in ovine cerebral arteries

Both protein kinase C (PKC) and extracellular signal-regulated kinases (ERK1/2) are involved in mediating vascular smooth muscle contraction. We tested the hypotheses that in addition to PKC activation of ERK1/2, by negative feedback ERKs modulate PKC-induced contraction, and that their interactions modulate both thick and thin myofilament pathways. In ovine middle cerebral arteries (MCA), we measured isometric tension and intracellular free calcium concentration ([Ca(2+)](i)) responses to PKC stimulation [phorbol 12,13-dibutyrate (PDBu), 3 x 10(-6) M] in the absence or presence of ERK1/2 inhibition (U-0126, 10(-5) M). After PDBu +/- ERK1/2 inhibition, we also examined by Western immunoblot the levels of total and phosphorylated ERK1/2, caldesmon(Ser789), myosin light chain(20) (MLC(20)), and CPI-17. PDBu induced significant increase in tension in the absence of increased [Ca(2+)](i). PDBu also increased phosphorylated ERK1/2 levels, a response blocked by U-0126. In turn, U-0126 augmented PDBu-induced contractions. PDBu also was associated with significant increases in phosphorylated caldesmon(Ser789) and MLC(20) levels, each of which peaked at 5 to 10 min. PDBu also increased phosphorylated CPI-17 levels, which peaked at 2 to 3 min. Rho kinase inhibition (Y-27632, 3 x 10(-7) M) did not alter PDBu-induced contraction. These results support the idea that PKC activation can increase CPI-17 phosphorylation to decrease myosin light chain phosphatase activity. In turn, this increases MLC(20) phosphorylation in the thick filament pathway and increases Ca(2+) sensitivity. In addition, ERK1/2-dependent phosphorylation of caldesmon(Ser789) was not necessary for PDBu-induced contraction and appears not to be involved in the reversal of caldesmon's inhibitory effect on actin-myosin ATPase.

Activation of phospholipase A 2 and MAP kinases by oxidized low-density lipoproteins in immortalized GP8.39 endothelial cells

In immortalized rat brain endothelial cells (GP8.39), we have previously shown that oxidized LDL (oxLDL), after 24-h treatment, stimulates arachidonic acid release and phosphatidylcholine hydrolysis by activation of cytosolic phospholipase A 2 (cPLA 2). A putative role for MAPKs in this process has emerged. Here, we studied the contribution of Ca 2+-independent phospholipase A 2 (iPLA 2), and the role of the MAP kinase family as well as both cPLA 2 and iPLA 2 mRNA expression by RT-PCR in oxLDL toxicity to GP8.39 cells in vitro. The activation of extracellular signal-regulated kinases ERK1/2, p38 and c-Jun NH 2-terminal kinase (JNK) was assessed with Western blotting and kinase activity assays. iPLA 2 activity, which was found as a membrane-associated enzyme, was more stimulated by oxLDL compared with native LDL. The phosphorylation of ERK1/2, p38 and JNKs was also significantly enhanced in a dose-dependent manner. PD98059, an ERK inhibitor, SB203580, a p38 inhibitor, and SP600125, an JNK inhibitor, abolished the stimulation of all three members of the MAPK family by oxLDL. Confocal microscopy analysis and subcellular fractionation confirmed either an increase in phosphorylated form of ERKs, p38 and JNKs, or their nuclear translocation upon activation. A strong inhibition of MAPK activation was also observed when endothelial cells were treated with GF109203X, a PKC inhibitor, indicating the important role of both PKC and all three MAPKs in mediating the maximal oxLDL response. Finally, compared with samples untreated or treated with native LDL, treatment with oxLDL (100 μM hydroperoxides) for 24 h significantly increased the levels of constitutively expressed iPLA 2 protein (by 5.1-fold) and mRNA (by 3.1-fold), as well as cPLA 2 protein (by 4.4-fold) and mRNA (by 1.5-fold). Together, these data link the stimulation of PKC–ERK–p38–JNK pathways and PLA 2 activity by oxLDL to the prooxidant mechanism of the lipoprotein complex, which may initially stimulate the endothelial cell reaction against noxious stimuli as well as metabolic repair, such as during inflammation and atherosclerosis.

The Interaction of Isoflurane and Protein Kinase C-Activators on Sarcolemmal KATP Channels

Protein kinase C (PKC)-dependent signaling pathways may be involved in the "memory" effect of anesthetic and ischemic preconditioning, which facilitates activation of cardioprotective adenosine triphosphate (ATP)-sensitive potassium channels during later ischemic challenge and ATP depletion. Using patch-clamp techniques, we found that exposure of isolated guinea pig cardiomyocytes to 1 mM of isoflurane after phorbol ester stimulation of PKC facilitates the induction of larger (P < or = 0.05) sarcolemmal K(ATP) channel currents (IKATP) during cell dialysis with 0.5, compared to 1.0, mM of ATP in the pipette (10 +/- 5 versus 2 +/- 1 pA/pF in five and six cells, respectively). A PKC inhibitor, bisindolylmaleimide, abolished the induction of IKATP by a second brief isoflurane exposure under these conditions. A diacylglycerol PKC activator applied via the pipette elicited concentration-related activation of IKATP. The diacylglycerol alone (0.5 microM) elicited I(KATP), averaging 5 +/- 3 pA/pF in nine cells. Briefly treating myocytes on the microscope stage with isoflurane, followed by washout and patching with the same diacylglycerol solution, elicited larger (P < or = 0.01) IKATP, averaging 40 +/- 9 pA/pF (10 cells), with an onset 48 +/- 2 min after anesthetic pretreatment. Facilitation of IKATP by isoflurane during the reduction of intracellular ATP is dependent on PKC, whereas "preconditioning" myocytes with isoflurane causes persistent changes in sarcolemmal KATP channel function, which enhance the induction of IKATP by a diacylglycerol.

Long‐lasting change in brain dynamics induced by methamphetamine: enhancement of protein kinase C‐dependent astrocytic response and behavioral sensitization

It is well known that long-term exposure to psychostimulants induces neuronal plasticity. Recently, accumulating evidence suggests that astrocytes may actively participate in synaptic plasticity. In this study, we found that in vitro treatment of cortical neuron/glia co-cultures with either methamphetamine (METH) or morphine (MRP) caused the activation of astrocytes via protein kinase C (PKC). Purified astrocytes were markedly activated by METH, whereas MRP had no such effect. METH, but not MRP, caused a long-lasting astrocytic activation in cortical neuron/glia co-cultures. Furthermore, MRP-induced behavioral sensitization to hyper-locomotion was reversed by 2 months of withdrawal following intermitted MRP administration, whereas behavioral sensitization to METH-induced hyper-locomotion was maintained even after 2 months of withdrawal. Consistent with this cell culture study, in vivo treatment with METH, which was associated with behavioral sensitization, caused a PKC-dependent astrocytic activation in the cingulate cortex and nucleus accumbens of mice. These findings provide direct evidence that METH induces a long-lasting astrocytic activation and behavioral sensitization through the stimulation of PKC in the rodent brain. In contrast, MRP produced a reversible activation of astrocytes via neuronal PKC and a reversibility of behavioral sensitization. This information can break through the definition of drugs of abuse and the misleading of concept that morphine produces a long-lasting neurotoxicity.

OX1 Orexin Receptors Couple to Adenylyl Cyclase Regulation via Multiple Mechanisms

In this study, the mechanism of OX(1) orexin receptors to regulate adenylyl cyclase activity when recombinantly expressed in Chinese hamster ovary cells was investigated. In intact cells, stimulation with orexin-A led to two responses, a weak (21%), high potency (EC(50) approximately 1 nm) inhibition and a strong (4-fold), low potency (EC(50) = approximately 300 nm) stimulation. The inhibition was reversed by pertussis toxin, suggesting the involvement of G(i/o) proteins. Orexin-B was, surprisingly, almost equally as potent as orexin-A in elevating cAMP (pEC(50) = approximately 500 nm). cAMP elevation was not caused by Ca(2+) elevation or by Gbetagamma. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKCdelta, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of G(s) activity, orexins still elevated cAMP; however, the potencies were greatly increased (EC(50) of orexin-A = approximately 10 nm and EC(50) of orexin-B = approximately 100 nm), and the response was fully dependent on PKCdelta. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-Galpha(s) antibodies. We conclude that OX(1) receptors stimulate adenylyl cyclase via a low potency G(s) coupling and a high potency phospholipase C --> PKC coupling. The former or some exogenous G activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX(1) receptors couple to G(s) almost as efficiently as the orexin-A-activated receptors, in contrast to Ca(2+) elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.

Dual Phospholipase C/Diacylglycerol Requirement for Protein Kinase D1 Activation in Lymphocytes

The serine/threonine kinase protein kinase D1 (PKD1) is a protein kinase C (PKC) substrate that mediates antigen receptor signal transduction in lymphocytes. PKC phosphorylates serines 744/748 within the PKD1 catalytic domain, and this is proposed to be necessary and sufficient for enzyme activation. Hence, a PKD1 mutant with alanine substituted at positions 744 and 748 (PKD-S744A/S748A) is catalytically inactive. Conversely, a PKD1 mutant with glutamic residues substituted at positions 744 and 748 as phospho-mimics (PKD-S744E/S748E) is constitutively active when expressed in Cos7 or HeLa cells. The present study reveals that Ser-744/Ser-748 phosphorylation is required for PKD1 activation in lymphocytes. However, PKD-S744E/S748E is not constitutively active but, like the wild type enzyme, requires antigen receptor triggering or phorbol ester stimulation. Antigen receptor activation of wild type PKD is dependent on phospholipase C (PLC)/diacylglycerol (DAG) and PKC, whereas PKD-S744E/S748E is only dependent on PLC/DAG but no longer requires PKC. Hence, substitution of serines 744 and 748 with glutamic residues as phospho-mimics bypasses the PKC requirement for PKD1 activation but does not bypass the need for antigen receptors, PLC, or DAG. In lymphocytes, PKD1 is, thus, not regulated by PLC and PKC in a linear pathway; rather, PKD1 activation has more stringent requirements for integration of dual PLC signals, one mediated by PKCs and one that is PKC-independent.

Establishment of an immortalized porcine granulosa cell line (PGV) and the study on the potential mechanisms of PGV cell proliferation

In order to establish an immortalized granulosa cell line and to investigate the potential mechanisms of immortalized cell proliferation, simian virus (SV) 40 was used to infect porcine granulosa cells from small follicles (1-2 mm in diameter), and one colony was selected after four weeks of culture. The colony was digested with trypsin and the cells were cultured for more than 300 days (named PGV). The SV40 large T antigen gene and its products were confirmed in immortalized cells by Southern blotting and immunohistochemistry. Progesterone production was not detected in the conditioned culture media with follicle-stimulating hormone (FSH) and forskolin, possibly due to the lack of P450scc gene transcription as examined by Northern blotting. PGV cells responded significantly to the stimulation of sera (fetal bovine and horse sera) and protein kinase C (PKC) stimulators (PMA and OAG), while PKC inhibitors (staurosporine and calphostin C) blocked both sera and PKC stimulation. Phospholipase C (PLC) and phosphatidic acid phosphatase (PAP) inhibitors (U73122 and propranolol) significantly reduced PGV cell proliferation, while PMA restored PLC and PAP inhibition. These data suggest that diacylglycerol (DAG) is produced in PGV cells by PLD as well as by PLC, and that DAG then activates PKC stimulating the PGV cell cycle through yet unknown mechanisms. Thus, an immortalized granulosa cell line is very useful to study granulosa cells in vitro, as the cells are homogeneous and are a functionally defined population.

Identification of Signaling-Induced Transcription Pathway Determinants that Influence Erythro-Megakaryocytic Lineage Commitment.

Erythroid and megakaryocytic cells are derived from a common precursor and share many of the same transcriptional regulators. We and others have demonstrated a critical role for sustained activation of extracellular-related (ERK) kinase in PKC-induced megakaryocytic differentiation of K562 cells. However, the transcriptional mechanisms underlying this process are largely unknown. Our prior studies have focused on late stage megakaryocyte specific genes such as glycoprotein IIb/CD41. In order to understand the early transcriptional events regulating this process, we have begun to examine the mechanisms regulating the expression of CD9, a tetraspanin molecule that is currently the earliest identified marker of megakaryocyte differentiation. The PKC agonist ingenol 3,20 dibenzoate (IDB) rapidly induced CD9 expression in both K562 cells as well as in primary human CD34+ progenitor cells, much earlier than the kinetics for CD41 induction. CD9 induction by PKC was dependent on ERK activation. Recently, it has been shown that immediate early gene expression, including the transcription factor Egr-1, is highly sensitive to the duration and amplitude of ERK activity. Egr-1 has been previously implicated in the regulation of hematopoietic lineage commitment. PKC/ERK activation rapidly and strongly induced Egr-1 expression, which remained elevated for up to 96 hrs after IDB treatment of K562 cells. Egr-1 induction was closely followed by CD9 induction, suggesting a causal relationship. This was supported by chromatin immunoprecipitation (ChIP) analysis, which showed strong recruitment of Egr-1 to the CD9 promoter during PKC/ERK-induced differentiation. KLF5, a known downstream target of Egr-1, was identified as an additional target of PKC/ERK signaling during megakaryocytic differentiation. KLF5, a novel GC binding factor, was found to be expressed in primary megakaryocytes and not in erythroid cells. KLF5 expression was induced by IDB treatment, and ChIP analysis showed that Egr-1 was recruited to the KLF5 promoter during PKC/ERK-induced differentiation. KLF5 has been shown previously to regulate known megakaryocyte-expressed genes PDGF-A and TGF-β expression in vascular smooth muscle, but a role in megakaryocyte gene expression has not been previously suggested. To better understand the regulation of Egr-1 in erythromegakaryocytic differentiation, Nab2, a natural co-repressor of Egr-1 was also evaluated. Control K562 cells strongly expressed c-myc and Nab2. ChIP studies show that c-myc binds to the Nab2 promoter in control K562 cells and both c-myc and Nab2 expression strongly declined during the first 24 hours after PKC stimulation. Enforced expression of Nab2 not only blocked CD9 induction by IDB, but also induced marked hemoglobinization of K562 cells. Taken together, these findings suggest a novel and critical role for the balance in Egr-1/Nab2 activity as a regulatory mechanism in erythroid versus megakaryocytic differentiation pathways.

17beta-estradiol enhances the production of granulocyte-macrophage colony-stimulating factor in human keratinocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is effective for impaired wound repair. Estrogen is known to enhance wound repair. We examined if 17beta-estradiol (E2) may in vitro enhance GM-CSF production in human keratinocytes. E2 and membrane-impermeable bovine serum albumin-conjugated E2 increased GM-CSF secretion, mRNA stability, and promoter activity. The element homologous to activator protein-1 (AP-1) on the promoter was responsible for the activation. E2 enhanced transcriptional activity and DNA binding of AP-1. E2 transiently generated c-Fos protein, and shifted AP-1 composition from c-Jun homodimers to c-Fos/c-Jun heterodimers in keratinocytes. E2-induced enhancement of GM-CSF secretion, mRNA stability, and promoter activity were not suppressed by estrogen receptor antagonist ICI 182,780, however, suppressed by conventional protein kinase C inhibitor Gö6976 and PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK). Gö6976 and PD98059 suppressed E2-induced c-Fos expression and enhancement of DNA-binding and transcriptional activity at AP-1. E2 induced membrane translocation of protein kinase Calpha, which was suppressed by phosphatidylinositol (PI)-specific phospholipase C (PLC) inhibitor U73122. E2 stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), which was suppressed by PD98059, Gö6976, and U73122. E2 transiently generated inositol 1,4,5-triphosphate in keratinocytes, which was suppressed by U73122 and guanine nucleotide-binding protein inhibitor. These results suggest that E2 may enhance GM-CSF production via guanine nucleotide-binding protein-coupled membrane receptors and signaling cascade of PI-specific PLC/protein kinase Calpha/MEK/ERK.

Isoenzyme-specific Translocation of Protein Kinase C (PKC)βII and not PKCβI to a Juxtanuclear Subset of Recycling Endosomes: INVOLVEMENT OF PHOSPHOLIPASE D

Elucidation of isoenzyme-specific functions of individual protein kinase C (PKC) isoenzymes has emerged as an important goal in the study of this family of kinases, but this task has been complicated by modest substrate specificity and high homology among the individual members of each PKC subfamily. The classical PKCbetaI and PKCbetaII isoenzymes provide a unique opportunity because they are the alternatively spliced products of the beta gene and are 100% identical except for the last 50 of 52 amino acids. In this study, it is shown that green fluorescent protein-tagged PKCbetaII and not PKCbetaI translocates to a recently described juxtanuclear site of localization for PKCalpha and PKCbetaII isoenzymes that arises with sustained stimulation of PKC. Mechanistically, translocation of PKCbetaII to the juxtanuclear region required kinase activity. PKCbetaII, but not PKCbetaI, was found to activate phospholipase D within this time frame. Inhibitors of phospholipase D (1-butanol and a dominant negative construct) prevented the translocation of PKCbetaII to the juxtanuclear region but not to the plasma membrane, thus demonstrating a role for phospholipase D in the juxtanuclear translocation of PKCbetaII. Taken together, these results define specific biochemical and cellular actions of PKCbetaII when compared with PKCbetaI.

Nerve growth factor-induced protein kinase C stimulation contributes to TrkA-dependent inhibition of p75 neurotrophin receptor sphingolipid signaling.

Previous studies have established that reciprocal interactions between the low-affinity p75 nerve growth factor (NGF) receptor (p75(NTR)) and the high-affinity TrkA NGF receptor can dictate the cellular response to NGF. As the most important interaction, TrkA signaling was found to inhibit p75(NTR)-mediated sphingomyelinase (SMase) stimulation, ceramide production, and apoptosis. However, the mechanism by which TrkA counteracts p75(NTR)-coupled sphingolipid signaling is still unclear. Considering the stimulatory effect of NGF on protein kinase C (PKC) activity, we investigated the role of PKC in TrkA/p75(NTR) signaling interaction. In this study, we found that, in SK-N-BE cells, which selectively express p75(NTR), phorbol ester-induced PKC stimulation resulted in the abrogation of SMase stimulation and ceramide production induced by NGF. Moreover, in SK-N-BE neuroblastoma cells, which selectively express TrkA, NGF stimulated global PKC activity through two independent pathways involving phospholipase Cgamma (PLCgamma) and phosphoinositide-3 kinase (PI3K). In SH-SY5Y, another neuroblastoma cell line, which coexpresses TrkA and p75(NTR), NGF induced PKC stimulation through a TrkA/PI3K signaling pathway, whereas there was no ceramide production. However, in these cells, the inhibition of TrkA, PI3K, and PKC resulted in the restoration of NGF-induced ceramide production. Thus, our study demonstrates for the first time that TrkA interferes with p75(NTR) signaling through a PI3K/PKC-dependent mechanism.

JNK interacting protein 1 (JIP-1) protects LNCaP prostate cancer cells from growth arrest and apoptosis mediated by 12-0-tetradecanoylphorbol-13-acetate (TPA).

12-0-tetradecanoylphorbol-13-acetate (TPA) stimulates protein kinase C (PKC) which mediates apoptosis in androgen-sensitive LNCaP human prostate cancer cells. The downstream signals of PKC that mediate TPA-induced apoptosis in LNCaP cells are unclear. In this study, we found that TPA activates the c-Jun NH2-terminal kinase (JNK)/c-Jun/AP-1 pathway. To explore the possible role that the JNK/c-Jun/AP-1 signal pathway has on TPA-induced apoptosis in LNCaP cells, we stably transfected the scaffold protein, JNK interacting protein 1 (JIP-1), which binds to JNK inhibiting its ability to phosphorylate c-Jun. TPA (10−9–10−7 mol l−1) caused phosphorylation of JNK in both wild-type and JIP-1-transfected (LNCaP-JIP-1) cells. It resulted in phosphorylation and upregulation of expression of c-Jun protein in the wild-type LNCaP cells, but not in the JIP-1-transfected LNCaP cells. In addition, upregulation of AP-1 reporter activity by TPA (10−9 mol l−1) occurred in LNCaP cells but was abrogated in LNCaP-JIP-1 cells. Thus, TPA stimulated c-Jun through JNK, and JIP-1 effectively blocked JNK. TPA (10−12–10−8 mol l−1) treatment of LNCaP cells caused their growth inhibition, cell cycle arrest, upregulation of p53 and p21waf1, and induction of apoptosis. All of these effects were significantly attenuated when LNCaP-JIP-1 cells were similarly treated with TPA. A previous study showed that c-Jun/AP-1 blocked androgen receptor (AR) signaling by inhibiting AR binding to AR response elements (AREs) of target genes including prostate-specific antigen (PSA). Therefore, we hypothesised that TPA would not be able to disrupt the AR signal pathway in LNCaP-JIP-1 cells. Contrary to expectation, TPA (10−9–10−8 mol l−1) inhibited DHT-induced AREs reporter activity and decreased levels of PSA in the LNCaP-JIP-1 cells. Taken together, TPA, probably by stimulation of PKC, phosphorylates JNK, which phosphorylates and increases expression of c-Jun leading to AP-1 activity. Growth control of prostate cancer cells can be mediated through the JNK/c-Jun pathway, but androgen responsiveness of these cells can be independent of this pathway, suggesting that androgen independence in progressive prostate cancer may not occur through activation of this pathway.

Conventional protein kinase C and atypical protein kinase Czeta differentially regulate macrophage production of tumour necrosis factor-alpha and interleukin-10.

In chronic inflammatory diseases such as rheumatoid arthritis, joint macrophages/monocytes are the major source of pro- and anti-inflammatory cytokines. Little is understood regarding the signalling pathways which determine the production of the pro-inflammatory cytokine, tumour necrosis factor-alpha (TNF-alpha) and the anti-inflammatory cytokine, interleukin-10 (IL-10). Two pathways integral to macrophage function are the protein kinase C (PKC)- and the cAMP-dependent pathways. In this report, we have investigated the involvement of PKC and cAMP in the production of TNF-alpha and IL-10 by peripheral blood monocyte-derived macrophages. The utilization of the PKC inhibitors Go6983, Go6976 and RO-32-0432 demonstrated a role for conventional PKCs (alpha and beta) in the production of TNF-alpha in response to stimulation by lipopolysaccharide and phorbol 12-myristate 13-acetate (PMA)/ionomycin. PKC stimulation resulted in the downstream activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway which differentially regulates TNF-alpha and IL-10. The addition of cAMP however, suppressed activation of this MAPK and TNF-alpha production. Cyclic-AMP augmented IL-10 production and cAMP response element binding protein activation upon stimulation by PMA/ionomycin. In addition, cAMP activated PKCzeta; inhibition of which, by a dominant negative adenovirus construct, selectively suppressed IL-10 production. These observations suggest that pro-inflammatory and anti-inflammatory cytokines are differentially regulated by PKC isoforms; TNF-alpha being dependent on conventional PKCs (alpha and beta) whereas IL-10 is regulated by the cAMP-regulated atypical PKCzeta.

Regulation of restitution after superficial injury in isolated guinea pig gastric mucosa.

The immediate response of the gastrointestinal epithelium to superficial (i.e. microscopic) injury is primarily directed towards restoring the disturbed epithelial continuity. Both structural (i.e. cytoskeleton) and humoral (i.e. growth factors and cytokines) involvement in the process has recently been documented. Yet it is unclear whether humoral signaling regulating mucosal recovery after superficial injury is associated with tyrosine phosphorylation, and whether there are other signs of downstream activation of the signaling pathway. To evaluate the effects of exogenous genistein and phorbol-myristate acetate in the assessment of the role of tyrosine receptor-mediated signaling in the immediate repair of gastric mucosa after superficial injury. Guinea pig gastric mucosa was mounted in a Ussing chamber, injured with 1.25 M NaCl, and perfused for 4 h. Simultaneously, potential difference and tissue resistance were recorded. In some sets of experiments the tissue was exposed bilaterally either to genistein in order to inhibit tyrosine receptor-mediated signaling or to 4-phorbol-myristate 13-acetate (PMA) in order to enhance PKC signaling during the 4 h recovery. Phosphotyrosine (PTYR) and protein kinase C (PKC) immunoreactivity were assessed by immunoblotting and by immunohistochemistry. Proliferative activity was determined morphometrically after staining of the tissue for Ki-67 nuclear antigen and expressed as proliferative index (PI). The inhibition of tyrosine kinases with exogenous genistein resulted in a significant decrease of the PTYR and the stimulation of PKC with PMA increased the PTYR. Nevertheless, no change in the PTYR was observed by immunoblotting after superficial injury alone. Several PKC isoenzymes were found in the guinea pig gastric mucosa, including PKC-alpha, -epsilon, -zeta and -iota. They were unaffected either by the injury or the PMA treatment. The mean PI of tissues subjected to NaCl-injury was higher than that of uninjured control tissues (p<0.05) (n=7). Exposure of tissue to genistein during recovery decreased the PI, while stimulation with PMA increased it (p<0.05 for both) (n=6). Both electrophysiologic and morphologic restitution were sensitive to genistein, but not to PMA. Superficial injury alone does not influence tyrosine phosphorylation to a degree which could be assessed by immunoblotting. Nevertheless, exogenous modulation of tyrosine receptor-mediated signaling results in downstream signaling effects. The injury-associated induction of proliferation is sensitive to modulation of tyrosine phosphorylation and PKC, suggesting that superficial epithelial injury results in endogenous activation of the epithelium, presumably after paracrine stimulation of the neighboring cells.