Serotonin activation of the ERK pathway in Hermissenda: contribution of calcium-dependent protein kinase C

The mitogen‐activated protein kinase (MAPK) cascade is an important contributor to synaptic plasticity and learning in both vertebrates and invertebrates. In the nudibranch mollusk Hermissenda, phosphorylation and activation of the extracellular signal‐regulated protein kinase (ERK), a key member of a MAPK cascade, is produced by one‐trial and multitrial Pavlovian conditioning. Several signal transduction pathways that are activated by 5‐hydroxytryptamine (5‐HT) and may contribute to conditioning have been identified in type B photoreceptors. However, the regulation of ERK activity by ‘upstream’ signaling molecules has not been previously investigated in Hermissenda. In the present study we examined the role of protein kinase C (PKC) in the serotonin (5‐HT) activation of the ERK pathway. The phorbol ester TPA produced an increase in ERK phosphorylation that was blocked by the PKC inhibitors GF109203X or Gö6976. TPA‐dependent ERK phosphorylation was also blocked by the MEK1 inhibitors PD098059 or U0126. The increased phosphorylation of ERK by 5‐HT was reduced but not blocked by pretreatment with the calcium chelator BAPTA‐AM or pretreatment with Gö6976 or GF109203X. These results indicate that Ca²⁺‐dependent PKC activation contributes to ERK phosphorylation, although a PKC‐independent pathway is also involved in 5‐HT‐dependent ERK phosphorylation and activation.     

PKC刺激与抑制对HeLa细胞G_1/S期进程的影响

蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ,ＰＫＣ）是一类分布广泛的丝氨酸／苏氨酸蛋白激酶,在真核细胞的跨膜信号传递中发挥重要的作用［１］．而细胞周期进程是一个复杂的调控过程,受控于多种磷酸酶与磷酸激酶作用下的中心调控体系［２］．大量报道表明ＰＫＣ信号...     

Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity.

Vero cell heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a 20- to 30-kDa membrane-anchored HB-EGF precursor (proHB-EGF). Localization and processing of proHB-EGF, both constitutive and 12-O-tetradecanoylphorbol 13-acetate (TPA)-inducible, was examined in Vero cells overexpressing recombinant HB-EGF (Vero H cells). Flow cytometry and fluorescence immunostaining demonstrated that Vero cell proHB-EGF is cell surface-associated and localized at the interface of cell to cell contact. Cell surface biotinylation and immunoprecipitation detected a 20- to 30-kDa heterogeneous proHB-EGF species. Vero H cell surface proHB-EGF turned over constitutively with a half-life of 1.5 h. Some of the 20- to 30-kDa cell surface-associated proHB-EGF was processed and a 14-kDa species of bioactive HB-EGF was released slowly, but most of the proHB-EGF was internalized, displaying a diffuse immunofluorescent staining pattern and accumulation of proHB-EGF in endosomes. Addition of TPA induced a rapid processing of proHB-EGF at a Pro148-Val149 site with a half-life of 7min. The TPA effect was abrogated by the protein kinase C inhibitors, staurosporine and H7. Kinetic analysis showed that loss of cell surface proHB-EGF is maximal at 30 min after addition of TPA and that proHB-EGF is resynthesized and the initial cell surface levels are regained within 12-24 h. Loss of cell surface proHB-EGF was concomitant with appearance of 14- and 19-kDa soluble HB-EGF species in conditioned medium. Vero H cell-associated proHB-EGF is a juxtacrine growth factor for EP170.7 cells in coculture. Processing of proHB-EGF resulted in loss of juxtacrine activity and a simultaneous increase in soluble HB-EGF paracrine mitogenic activity. It was concluded that processing regulates HB-EGF bioactivity by converting it from a cell-surface juxtacrine growth factor to a processed, released soluble paracrine growth factor.     

Phorbol ester-potentiated liposomal transfection to monocytic PLB-985 cells

In order to improve transient gene transfer into PLB-985 cells, we treated cells with 12-tetradecanoylphorbol 13-acetate (TPA) 4 h before transfection, and increased by 540-fold the reporter activity of the firefly luciferase gene transfected by TFL-01, a cationic liposome. Dioctanolyglycerol added before TPA addition inhibited the TPA-dependent increase in activity, suggesting it to be a TPA competitor for PKC binding. H7, staurosporine, GO 6976, and H8, but not GF 109203X and forskolin, inhibited the TPA-dependent increase in reporter activity when added 8 h after TFL-01/gene addition. Forskolin and GF 109203X also inhibited the increase when added before TPA. Therefore, for the potentiation of transfection by the TPA/TFL-01 method, conventional PKC activity with significant but low protein kinase A (PKA) activity are first required, and then a novel PKC activity with significant PKA activity. TPA enhanced the uptake of FITC-labeled phosphorothioated oligonucleotides and their prolonged maintenance by cells, suggesting increased TFL-01-assisted plasmid uptake and its stabilization in TPA-treated PLB-985 cells. This method was used successfully for the sensitive analysis of the promoter function of the gp91(phox) gene, implying the method to be generally useful for promoter analyses of various genes expressed in differentiated human monocytic cells.     

The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C

Staurosporine is the most potent inhibitor of protein kinase C (PKC) described in the literature with a half-maximal inhibitory concentration (IC50) of 10 nM. Nevertheless, this natural product is poorly selective when assayed against other protein kinases. In order to obtain specific PKC inhibitors, a series of bisindolylmaleimides has been synthesized. Structure-activity relationship studies allowed the determination of the substructure responsible for conferring high potency and lack of selectivity in the staurosporine molecule. Several aminoalkyl bisindolylmaleimides were found to be potent and selective PKC inhibitors (IC50 values from 5 to 70 nM). Among these compounds GF 109203X has been chosen for further studies aiming at the characterization of this chemical family. GF 109203X was a competitive inhibitor with respect to ATP (Ki = 14 +/- 3 NM) and displayed high selectivity for PKC as compared to five different protein kinases. We further determined the potency and specificity of GF 109203X in two cellular models: human platelets and Swiss 3T3 fibroblasts. GF 109203X efficiently prevented PKC-mediated phosphorylations of an Mr = 47,000 protein in platelets and of an Mr = 80,000 protein in Swiss 3T3 cells. In contrast, in the same models, the PKC inhibitor failed to prevent PKC-independent phosphorylations. GF 109203X inhibited collagen- and alpha-thrombin-induced platelet aggregation as well as collagen-triggered ATP secretion. However, ADP-dependent reversible aggregation was not modified. In Swiss 3T3 fibroblasts, GF 109203X reversed the inhibition of epidermal growth factor binding induced by phorbol 12,13-dibutyrate and prevented [3H] thymidine incorporation into DNA, only when this was elicited by growth promoting agents which activate PKC. Our results illustrate the potential of GF 109203X as a tool for studying the involvement of PKC in signal transduction pathways.     

Different pathways leading to activation of extracellular signal-regulated kinase and p38 MAP kinase by formyl-methionyl-leucyl-phenylalanine or platelet activating factor in human neutrophils

Summary The signaling pathways leading to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activation by N-formyl-Met-Leu-Phe (fMLP) or platelet activating factor (PAF) in human neutrophils were examined. Previously, we found that changes of intracellular Ca²⁺ ([Ca ) stimulated by PAF and fMLP were due to Ca²⁺ influx and internal Ca²⁺ release, respectively. To further determine the mechanism of MAPK activation and its relation with Ca²⁺ influx, blood from healthy human volunteers was taken by venous puncture. Human polymorphonuclear cells (PMNs) were isolated and incubated with protein kinase C (PKC) inhibitor Calphostin C, PKC- isoform inhibitor GF109203X, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002, phospholipase C (PLC) inhibitor U73122, phospholipase A₂ (PLA₂) inhibitor Aristolochic acid, store-operated calcium (SOC) channel inhibitor SKF96365, or extracellular calcium chelator EGTA followed by fMLP or PAF treatment. Phosphorylation of ERK p38 was determined by immunoblotting analysis. Our data indicate that neutrophil MAPK signaling pathways mediated by fMLP and PAF are different. PAF-induced ERK phosphorylation is mediated by PI3K, PKC, PLA₂, PLC, and extracellular calcium, whereas fMLP-induced ERK phosphorylation does not involve the PKC- isoform and extracellular calcium. PAF-induced p38 phosphorylation involves PLA2, whereas fMLP-induced p38 activation is PLC dependent.     

Activation of protein kinase C inhibits ATP-induced [Ca2+]i elevation in rat osteoblastic cells: Selective effects on P2Y and P2U signaling pathways

Extracellular ATP elicits transient elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in osteoblasts through interaction with more than one subtype of cell surface P₂-purinoceptor. Elevation of [Ca²⁺]_i arises, at least in part, by release of Ca²⁺ from intracellular stores. In the present study, we investigated the possible roles of protein kinase C (PKC) in regulating these signaling pathways. [Ca²⁺]_i of indo-1-loaded UMR-106 osteoblastic cells was monitored by spectrofluorimetry. In the absence of extracellular Ca²⁺, ATP (100 μM) induced transient elevation of [Ca²⁺]_i to a peak 57 ± 7 nM above basal levels (31 ± 2 nM, means ± S. E. M., n = 25). Exposure of cells to the PKC activator 12-O-tetradecanoyl-β-phorbol 13-acetate (TPA, 100 nM) for 2 min significantly reduced the amplitude of the ATP response to 13 ± 4 nM (n = 11), without altering basal [Ca²⁺]_i. Inhibition was half-maximal at approximately 1 nM TPA. The Ca²⁺ response to ATP was also inhibited by the PKC activators 1,2-dioctanoyl-sn-glycerol or 4β-phorbol 12, 13-dibutyrate, but not by the control compounds 4α-phorbol or 4α-phorbol 12, 13-didecanoate. Furthermore, exposure of cells to the protein kinase inhibitors H-7 or staurosporine for 10 min significantly attenuated the inhibitory effect of TPA. However, these protein kinase inhibitors did not prolong the [Ca²⁺]_i response to ATP alone, indicating that activation of PKC does not account for the transient nature of this response. When the effects of other nucleotides were examined, TPA was found to cause significantly greater inhibition of the response to the P_2Y-receptor agonists, ADP and 2-methylthioATP, than the response to the P_2U-receptor agonist, UTP. These data indicate that activation of PKC selectively inhibits the P_2Y signaling pathway in osteoblastic cells. In vivo, endocrine or paracrine factors, acting through PKC, may regulate the responsiveness of osteoblasts to extracellular nucleotides. © 1995 Wiley-Liss, Inc.     

Up-regulation of Na(+)-Ca2+ exchange activity by interferon-gamma in cultured rat microglia

The Na(+)-Ca2+ exchanger (NCX) plays a role in regulating intracellular Ca2+ concentration, but little is known about NCX in microglia. We examined mRNA expression of NCX isoforms in cultured rat microglia and the effect of interferon-gamma (IFN-gamma) on NCX activity. RT-PCR showed that all of the known NCX isoforms, NCX1-3, are expressed in cultured microglia. Ouabain and monensin increased 45Ca2+ uptake and intracellular Ca2+ concentration in microglia, suggesting the presence of NCX activity in the reverse mode. Treatment with IFN-gamma (100 U/mL) caused a biphasic increase in NCX activity. The transient increase in NCX activity by IFN-gamma for 1 h was blocked by the protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and the tyrosine kinase inhibitor, herbimycin A. The delayed increase in NCX activity by IFN-gamma for 24 h was blocked by the protein synthesis inhibitor cycloheximide and actinomycin D. Treatment with IFN-gamma for 24 h increased NCX mRNA and protein levels. The increase in NCX activity and NCX protein by IFN-gamma for 24 h was blocked by staurosporine, GF109203X, herbimycin A and the extracellular signal-regulated kinase inhibitor, PD98059. These findings suggest that NCX is up-regulated by IFN-gamma in a biphasic manner in microglia. Moreover, PKC and tyrosine kinase are involved in the up-regulation of NCX and the extracellular signal-regulated protein kinase is also involved in the delayed increase in NCX activity.     

Roles for Protein Kinase C and Mitogen-Activated Protein Kinase in Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Abstract: Both the Ca²⁺/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca²⁺ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca²⁺ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC₅₀ 1–5 µM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC₅₀～10 µM) than that induced by nicotine (IC₅₀～30 µM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca²⁺ ionophore and the participation of both PKC and MEK/MAPK in optimal secretion induced by nicotine.     

Involvement of protein kinase C in translocation of desmoplakins from cytosol to plasma membrane during desmosome formation in human squamous cell carcinoma cells grown in low to normal calcium concentration

The intracellular signal transduction mechanism leading to desmosome formation in low-calcium-grown keratinocytes after addition of calcium to the medium was studied by immunofluorescence using antibodies to desmoplakins I and II (cytoplasmic desmosomal proteins) and by electron microscopy before and after addition of calcium; protein kinase C (PKC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoylglycerol (DOG); calcium ionophore A23187; selective PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine; and a Ca2+/calmodulin-dependent kinase inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). In previous studies using a low-calcium-grown human epidermal squamous cell carcinoma, we have shown that an increase in extracellular Ca2+ caused a four-fold increase in PKC activity and addition of TPA (10 ng/ml) induced a transient increase in membrane-bound PKC activity in association with cell-cell contact formation. The present study showed that TPA (10 ng/ml). PDBu (10 ng/ml), and DOG (1 mg/ml) induced a rapid cell-cell contact and redistribution of desmoplakins from cytoplasm to the plasma membrane with desmosome formation within 60-120 min, which was similar, although less marked, to the effect of increased Ca2+. The TPA-induced desmosome formation was inhibited by selective PKC inhibitors, H-7 (20 microM) or staurosporine (100 nM). On the other hand, calcium ionophore A23187 induced only a temporary increase in the number of desmoplakin-containing fluorescent spots in the cytoplasm and a temporary cell-cell attachment without desmosome formation. The calcium-induced desmosome formation was partially inhibited by 20-100 microM H-7 or 100 nM staurosporine; however, it was not inhibited by W-7 at a concentration of 25 microM, at which this agent selectively inhibits calmodulin-dependent protein kinase. These results suggest that PKC activation plays an important role in desmoplakin translocation from the cytoplasm to the plasma membrane as one of the processes of calcium-induced desmosome formation.     

Gangliosides inhibit PDGF-induced signal transduction events in U-1242 MG human glioma cells

In this study we investigated the responses of intracellular calcium ([Ca²⁺]_i) and protein kinase C (PKC) to PDGF in U-1242 MG cells. PDGF-BB stimulated [³H]PDBu binding approximately 2–3 fold. This response was inhibited by preincubating the cells with an inhibitor of phospholipase C (PLC), U73122, suggesting that PLC mediates the induction of PKC translocation by PDGF. PDGF also increased the concentration of [Ca²⁺]_i that was attenuated in a calcium-free medium. This indicates that PDGF-induced elevation of [Ca²⁺]_i is mainly due to influx of extracellular calcium. PDGF-stimulated translocation of PKC was inhibited by the intracellular calcium buffer BAPTA/AM. All gangliosides studied except GM3 inhibited these responses with similar efficacy. Collectively, these results indicate that the signal transduction pathway initiated by PDGF leading to PKC translocation in U-1242 MG cells is intact, and this pathway is inhibited by several gangliosides.Special issue dedicated to Dr. Leon S. Wolfe.     

FGF-2 and TPA induce matrix metalloproteinase-9 secretion in MCF-7 cells through PKC activation of the Ras/ERK pathway

Matrix metalloproteinases (MMPs) play an important role in cancer metastasis. Here, we investigated the effect of fibroblast growth factor-2 (FGF-2) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the secretion of type IV collagenases (MMP-2, MMP-9) in breast cancer MCF-7 cells. As shown by gelatin zymography, both FGF-2 and TPA stimulated the secretion of MMP-9 in MCF-7 cells while they did not change the level of MMP-2 secretion. Signaling cascade studies indicated that both FGF-2 and TPA induced Ras activation, c-Raf phosphorylation, mitogen-activated protein kinase/ERK kinase (MEK(1/2)) phosphorylation, and extracellular signal-regulated kinase (ERK(1/2)) phosphorylation. The FGF-2- and TPA-induced MMP-9 secretion was significantly inhibited by transient transfection of MCF-7 cells with dominant negative Ras (Ras-N17) and by treatment with MEK(1/2) inhibitor PD98059. A pan-protein kinase C (PKC) inhibitor, GF109203X, was found to totally abolish the FGF-2- and TPA-induced MMP-9 secretion and ERK(1/2) phosphorylation. Use of isoform-specific PKC inhibitors such as Rotllerin and G?6976 suggested, moreover, that the PKC-delta isoform is a likely component of FGF-2 and TPA trophic signaling. These results demonstrated that FGF-2 and TPA induce MMP-9 secretion in MCF-7 cells mainly through PKC-dependent activation of the Ras/ERK(1/2) signaling pathway.     

Involvement of different protein kinases and phospholipases A2 in phorbol ester (TPA)-induced arachidonic acid liberation in bovine platelets

The effect of various phospholipase A2 and protein kinase inhibitors on the arachidonic acid liberation in bovine platelets induced by the protein kinase activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. TPA stimulates arachidonic acid release mainly by activating group IV cytosolic PLA2 (cPLA2), since inhibitors of this enzyme markedly inhibited arachidonic acid formation. However, group VI Ca2+-independent PLA2 (iPLA2) seems to contribute to the arachidonic acid liberation too, since the relatively specific iPLA2 inhibitor bromoenol lactone (BEL) decreased arachidonic acid generation in part. The pronounced inhibition of the TPA-induced arachidonic acid release by the protein kinase C (PKC) inhibitors GF 109203X and Ro 31-82220, respectively, and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA2s by TPA is mediated via PKC and p38 MAP kinase.     

Mechanosensitive synoviocytes: a Ca2+ -PKCalpha-MAP kinase pathway contributes to stretch-induced hyaluronan synthesis in vitro.

Hyaluronan (HA) is central to joint function, contributing to synovial fluid retention, lubrication, matrix organisation and joint embryogenesis. HA synthesis by intimal synoviocytes is stimulated by stretch (SSHA), linking HA production to joint usage; but the signal transduction paths are unknown. Low passage rabbit synoviocytes (RS), cultured from micro dissected synovial intima, were subjected to 10 min of 10% static stretch followed by 170-min relaxation, or to sustained stretch for 180 min in a Flexcell 2000 apparatus. Medium HA content was analysed by a HA-binding assay. The roles of protein kinase C (PKC) isoforms, extracellular signal-regulated kinases (ERK1/2) and Ca²⁺ signalling in SSHA were tested using kinase inhibitors, Ca²⁺ chelators and Ca²⁺ channel activators combined with Western blots for activated kinases. Stretch increased HA secretion by 57%, independently of stretch duration. PKCα translocated from cytosol to membrane and triggered the phosphorylation of ERK1/2. The PKC inhibitor bisindolylmaleimide (BIM) blocked both SSHA and ERK phosphorylation, as did Gö 6976, a specific inhibitor of Ca²⁺-dependent PKC. The Ca²⁺ channel activator Bay K stimulated HA secretion and ERK phosphorylation. Extra- and intra-cellular Ca²⁺ chelation by EGTA and BAPTA-AM (respectively) inhibited SSHA. SSHA was also blocked by the partially selective protein kinase A inhibitor, H-89. Connective tissue growth factor, CTGF, was not involved in SSHA. Thus, stimulation of synoviocyte HA secretion by static stretch is due at least in part the o activation of a Ca²⁺ influx-dependent activation of the PKCα–MEK–ERK1/2 cascade. This is functionally important because it links joint lubrication to joint use.     

Protein kinase C theta and epsilon promote T-cell survival by a rsk-dependent phosphorylation and inactivation of BAD

Both MAPK and protein kinase C (PKC) signaling pathways promote cell survival and protect against cell death. Here, we show that 12-O-tetradecanoylphorbol-13-acetate (TPA) prevents Fas-induced apoptosis in T lymphocytes. The effect of TPA was specifically abolished by the PKC inhibitor GF109203X and by dominant negative PKCtheta, PKCepsilon, and PKCalpha, suggesting that novel and conventional PKC isoforms mediate phorbol ester action. Moreover, TPA stimulated phosphorylation of BAD at serine 112, an effect abrogated by GF109203X but not by the MEK inhibitor PD98059. Expression of constitutively active PKC increased the phosphorylation of BAD at serine 112 but not at serine 136. Additionally, Fas-mediated cell death was enhanced by overexpression of a catalytically inactive form of p90Rsk (Rsk2-KN). Finally, Rsk2-KN abolished the protective effect of constitutively active PKC and totally blocked phosphorylation of BAD on serine 112. Thus, novel PKCtheta and PKCepsilon rescue T lymphocytes from Fas-mediated apoptosis via a p90Rsk-dependent phosphorylation and inactivation of BAD.     

Induction of cytosolic phospholipase A2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of MAPKs and NF-kappaB pathways

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandins (PG) synthesis induced by bacterial lipopolysaccharide (LPS) and cytokines. However, the intracellular signaling pathways mediating LPS-induced cPLA2 expression and PGE2 synthesis in canine tracheal smooth muscle cells (TSMCs) remains unknown. LPS-induced expression of cPLA2 and release of PGE2 was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (D609), phosphatidylinositol-phospholipase C (U73122), PKC (GF109203X and staurosporine), removal of Ca2+ by BAPTA/AM plus EDTA, MEK1/2 (PD98059), p38 (SB202190), JNK (SP600125), and phosphatidylinositol 3-kinase (PI3-K; LY294002 and wortmannin). The involvement of MPAKs in LPS-induced responses was further confirmed by transfection of TSMCs with dominant negative mutants of ERK2 and p38. LPS-induced cPLA2 expression and PGE2 synthesis was inhibited by a selective NF-kappaB inhibitor (helenalin) and transfection with dominant negative mutants of NF-kappaB inducing kinase (NIK), IkappaB kinase (IKK)-alpha, and IKK-beta, consistent with that LPS-stimulated both IkappaB-alpha degradation and NF-kappaB translocation into nucleus in these cells. LPS-stimulated cPLA2 phosphorylation was inhibited by PD98059, GF109203X, and staurosporine, indicating the regulation by p42/p44 MAPK and PKC. Moreover, LPS-induced up-regulation of cPLA2 and COX-2 linked to PGE2 synthesis was inhibited by AACOCF3 (a selective cPLA2 inhibitor), implying the involvement of cPLA2 in these responses. These findings suggest that phosphorylation and expression of cPLA2 correlates with the release of PGE2 from LPS-challenged TSMCs, at least in part, mediated through MAPKs and NF-kappaB signaling pathways. LPS-mediated responses were modulated by PLC, Ca2+, PKC, tyrosine kinase, and PI3-K in TSMCs.     

Thrombin Mediates Migration of Rat Brain Astrocytes via PLC, Ca2+, CaMKII, PKCα, and AP-1-Dependent Matrix Metalloproteinase-9 Expression

Matrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) remain unclear. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 and migration of RBA-1 cells, which were inhibited by pretreatment with the inhibitor of Gq-coupled receptor (GPAnt2A), Gi/o-coupled receptor (GPAnt2), PC-PLC (D609), PI-PLC (U73122), Ca²⁺-ATPase (thapsigargin, TG), calmodulin (CaMI), CaMKII (KN62), PKC (Gö6976 or GF109203X), MEK1/2 (PD98059), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) or the intracellular calcium chelator (BAPTA/AM) and transfection with siRNA of PKCα, Erk2, JNK1, p38 MAPK, c-Jun, or c-Fos. In addition, thrombin-induced elevation of intracellular Ca²⁺ concentration was attenuated by PPACK (a thrombin inhibitor). Thrombin further induced CaMKII phosphorylation and PKCα translocation, which were inhibited by U73122, D609, KN62, TG, or BAPTA/AM. Thrombin also induced PKCα-dependent p42/p44 MAPK and JNK1/2, but not p38 MAPK activation. Finally, we showed that thrombin enhanced c-Fos expression and c-Jun phosphorylation. c-Fos mRNA levels induced by thrombin were reduced by PD98059, SP600125, and Gö6976, but not SB202190. Thrombin stimulated in vivo binding of c-Fos to the MMP-9 promoter, which was reduced by pretreatment with SP600125 or PD98059, but not SB202190. These results concluded that thrombin activated a PLC/Ca²⁺/CaMKII/PKCα/p42/p44 MAPK and JNK1/2 pathway, which in turn triggered AP-1 activation and ultimately induced MMP-9 expression in RBA-1 cells.     

Translocation of protein kinase Cepsilon and protein kinase Cdelta to membrane is required for ultraviolet B-induced activation of mitogen-activated protein kinases and apoptosis.

UV-induced signal transduction may be involved in tumor promotion and induction of apoptosis. The role of protein kinase C (PKC) in UVB-induced signal transduction is not well understood. This study showed that UVB markedly induced translocation of membrane-associated PKCepsilon and PKCdelta, but not PKCalpha, from cytosol to membrane. Dominant negative mutant (DNM) PKCepsilon or PKCdelta inhibited UVB-induced translocation of PKCepsilon and PKCdelta, respectively. UVB-induced activation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs) was strongly inhibited by DNM PKCepsilon and PKCdelta, whereas the DNM of PKCalpha was less effective on the UVB-induced phosphorylation of Erks and JNKs. Among the PKC inhibitors used only rottlerin, a selective inhibitor of PKCdelta, markedly inhibited the UVB-induced activation of Erks and JNKs, but not p38 kinases. Safingol, a selective inhibitor for PKCalpha, did not show any inhibitory effect on UVB-induced mitogen-activated protein kinase activation. GF109203X is a stronger inhibitor of classical PKC than novel PKC. Lower concentrations of GF109203X (<10 microM) had no effect on UVB-induced activation of Erks or JNKs. However, at higher concentrations (over 20 microM), GF109203X inhibited UVB-induced activation of JNKs, Erks, and even p38 kinases. Meanwhile, rottlerin and GF109203X markedly inhibited UVB-induced apoptosis of JB6 cells, whereas safingol had little inhibitory effect. DNM-Erk2 cells and PD98059, a selective inhibitor for mitogen-activated protein kinase/extracellular signal-regulated kinase 1 that directly activates Erks, inhibited UVB-induced apoptosis. DNM-JNK1 cells also blocked UVB-induced apoptosis, whereas SB202190, a specific inhibitor for p38 kinases, did not produce the inhibitory effect. These data demonstrate that PKCdelta and PKCepsilon, but not PKCalpha, mediate UVB-induced signal transduction and apoptosis in JB6 cells through activation of Erks and JNKs.     

Activation of the PI3K/Akt signaling pathway through P2Y2 receptors by extracellular ATP is involved in osteoblastic cell proliferation

We studied the PI3K/Akt signaling pathway modulation and its involvement in the stimulation of ROS 17/2.8 osteoblast-like cell proliferation by extracellular ATP. A dose- and time-dependent increase in Akt-Ser 473 phosphorylation (p-Akt) was observed. p-Akt was increased by ATPγS and UTP, but not by ADPβS. Akt activation was abolished by PI3K inhibitors and reduced by inhibitors of PI-PLC, Src, calmodulin (CaM) but not of CaMK. p-Akt was diminished by cell incubation in a Ca²⁺-free medium but not by the use of L-type calcium channel blockers. The rise in intracellular Ca²⁺ induced by ATP was potentiated in the presence of Ro318220, a PKC inhibitor, and attenuated by the TPA, a known activator of PKC. ATP-dependent p-Akt was diminished by TPA and augmented by Ro318220 treatment in a Ca²⁺-containing but not in a Ca²⁺-free medium. ATP stimulated the proliferation of both ROS 17/2.8 cells and rat osteoblasts through PI3K/Akt. In the primary osteoblasts, ATP induces alkaline phosphatase activity via PI3K, suggesting that the nucleotide promotes osteoblast differentiation. These results suggest that ATP stimulates osteoblast proliferation through PI-PLC linked-P2Y₂ receptors and PI3K/Akt pathway activation involving Ca²⁺, CaM and Src. PKC seems to regulate Akt activation through Src and the Ca²⁺ influx/CaM pathway.