Inflammation can increase the excitability of bronchopulmonary C-fibers leading to excessive sensations and reflexes (e.g. wheeze and cough). We have previously shown modulation of peripheral nerve terminal mitochondria by antimycin A causes hyperexcitability in TRPV1-expressing bronchopulmonary C-fibers through the activation of protein kinase C (PKC). Here, we have investigated the PKC isoform responsible for this signaling. We found PKCβ1, PKCδ and PKCε were expressed by many vagal neurons, with PKCα and PKCβ2 expressed by subsets of vagal neurons. In dissociated vagal neurons, antimycin A caused translocation of PKCα but not the other isoforms, and only in TRPV1-lineage neurons. In bronchopulmonary C-fiber recordings, antimycin A increased the number of action potentials evoked by α,β-methylene ATP. Selective inhibition of PKCα, PKCβ1 and PKCβ2 with 50 nM bisindolylmaleimide I prevented the antimycin-induced bronchopulmonary C-fiber hyperexcitability, whereas selective inhibition of only PKCβ1 and PKCβ2 with 50 nM LY333531 had no effect. We therefore conclude that PKCα is required for antimycin-induced increases in bronchopulmonary C-fiber excitability.

Activation of PKC depends on the availability of DAG, a signaling lipid that is tightly and dynamically regulated. DAG kinase (DGK) terminates DAG signaling by converting it to phosphatidic acid. Here, we demonstrate that DGKzeta inhibits PKCalpha activity and that DGK activity is required for this inhibition. We also show that DGKzeta directly interacts with PKCalpha in a signaling complex and that the binding site in DGKzeta is located within the catalytic domain. Because PKCalpha can phosphorylate the myristoylated alanine-rich C-kinase substrate (MARCKS) motif of DGKzeta, we tested whether this modification could affect their interaction. Phosphorylation of this motif significantly attenuated coimmunoprecipitation of DGKzeta and PKCalpha and abolished their colocalization in cells, indicating that it negatively regulates binding. Expression of a phosphorylation-mimicking DGKzeta mutant that was unable to bind PKCalpha did not inhibit PKCalpha activity. Together, our results suggest that DGKzeta spatially regulates PKCalpha activity by attenuating local accumulation of signaling DAG. This regulation is impaired by PKCalpha-mediated DGKzeta phosphorylation.

1 We have investigated the effects of alpha(1)-adrenoceptor stimulation upon contractility, Ca(2+) influx, inositol phosphate production, and protein kinase C (PKC) translocation in human cultured prostatic stromal cells (HCPSC). 2 The alpha(1)-adrenoceptor selective agonist phenylephrine elicited contractile responses of HCPSC, i.e. a maximal cell shortening of 45+/-6% of initial cell length, with an EC(50) of 1.6+/-0.1 microM. The alpha(1)-adrenoceptor selective antagonists prazosin (1 microM) and terazosin (1 microM) both blocked contractions to phenylephrine (10 microM). The L-type calcium channel blocker nifedipine (10 microM), and the PKC inhibitors Gö 6976 (1 microM) and bisindolylmaleimide (1 microM) also inhibited phenylephrine-induced contractions. 3 Phenylephrine caused a concentration dependent increase in inositol phosphate production (EC(50) 119+/-67 nM). This response was blocked by terazosin (1 microM). 4 Phenylephrine caused the translocation of the PKC alpha isoform, but not the beta, delta, gamma, epsilon or lambda isoforms, from the cytosolic to the particulate fraction of HCPSC, with an EC(50) of 5.7+/-0.5 microM. 5 In FURA-2AM (5 microM) loaded cells, phenylephrine elicited concentration dependent increases in [Ca(2+)](i), with an EC(50) of 3.9+/-0.4 microM. The response to phenylephrine (10 microM) was blocked by prazosin (1 microM), bisindolymaleimide (1 microM), and nifedipine (10 microM). 6 In conclusion, this study has shown that HCPSC express functional alpha(1)-adrenoceptors, and that the intracellular pathways responsible for contractility may be largely dependent upon protein kinase C activation and subsequent opening of L-type calcium channels.

Ionizing radiation is a well known human carcinogen. Evidence accumulated over the past decade suggested that extranuclear/extracellular targets and events may also play a critical role in modulating biological responses to ionizing radiation. However, the underlying mechanism(s) of radiation-induced bystander effect is still unclear. In the current study, AL cells were irradiated with alpha particles and responses of bystander cells were investigated. We found out that in bystander AL cells, protein kinase C alpha (PKCα) translocated from cytosol to membrane fraction. Pre-treatment of cells with PKC translocation inhibitor chelerythrine chloride suppressed the induced extracellular signal-regulated kinases (ERK) activity and the increased cyclooxygenase 2 (COX-2) expression as well as the mutagenic effect in bystander cells. Furthermore, tumor necrosis factor alpha (TNFα) was elevated in directly irradiated but not bystander cells; while TNFα receptor 1 (TNFR1) increased in the membrane fraction of bystander cells. Further analysis revealed that PKC activation caused accelerated internalization and recycling of TNFR1. Our data suggested that PKCα translocation may occur as an early event in radiation-induced bystander responses and mediate TNFα-induced signaling pathways that lead to the activation of ERK and up-regulation of COX-2.

Sensitization of purinergic P2X3 receptors (P2X3Rs) is a major mechanism contributing to injury-induced exaggerated pain responses. We showed in a previous study that cyclic adenosine monophosphate (cAMP)-dependent guanine nucleotide exchange factor 1 (Epac1) in rat sensory dorsal root ganglia (DRGs) is upregulated after inflammatory injury, and it plays a critical role in P2X3R sensitization by activating protein kinase C epsilon (PKCε) inside the cells. protein kinase C epsilon has been established as the major PKC isoform mediating injury-induced hyperalgesic responses. On the other hand, the role of PKCα in receptor sensitization was seldom considered. Here, we studied the participation of PKCα in Epac signaling in P2X3R-mediated hyperalgesia. The expression of both Epac1 and Epac2 and the level of cAMP in DRGs are greatly enhanced after complete Freund adjuvant (CFA)-induced inflammation. The expression of phosphorylated PKCα is also upregulated. Complete Freund adjuvant (CFA)-induced P2X3R-mediated hyperalgesia is not only blocked by Epac antagonists but also by the classical PKC isoform inhibitors, Go6976, and PKCα-siRNA. These CFA effects are mimicked by the application of the Epac agonist, 8-(4-chlorophenylthio)-2 -O-methyl-cAMP (CPT), in control rats, further confirming the involvement of Epacs. Because the application of Go6976 prior to CPT still reduces CPT-induced hyperalgesia, PKCα is downstream of Epacs to mediate the enhancement of P2X3R responses in DRGs. The pattern of translocation of PKCα inside DRG neurons in response to CPT or CFA stimulation is distinct from that of PKCε. Thus, in contrast to prevalent view, PKCα also plays an essential role in producing complex inflammation-induced receptor-mediated hyperalgesia.

Striated muscle laminopathies are cardiac and skeletal muscle conditions caused by mutations in the lamin A/C gene (LMNA). LMNA codes for the A-type lamins, which are nuclear intermediate filaments that maintain the nuclear structure and nuclear processes such as gene expression. Protein kinase C alpha (PKC-α) interacts with lamin A/C and with several lamin A/C partners involved in striated muscle laminopathies. To determine PKC-α's involvement in muscular laminopathies, PKC-α's localization, activation, and interactions with the A-type lamins were examined in various cell types expressing pathogenic lamin A/C mutations. The results showed aberrant nuclear PKC-α cellular distribution in mutant cells compared to WT. PKC-α activation (phos-PKC-α) was decreased or unchanged in the studied cells expressing LMNA mutations, and the activation of its downstream targets, ERK 1/2, paralleled PKC-α activation alteration. Furthermore, the phos-PKC-α-lamin A/C proximity was altered. Overall, the data showed that PKC-α localization, activation, and proximity with lamin A/C were affected by certain pathogenic LMNA mutations, suggesting PKC-α involvement in striated muscle laminopathies.

Intracellular trafficking of mycobacteria is comprehensively dependent on the unusual regulation of host proteins. Recently, we have reported that infection of macrophages by Mycobacterium tuberculosis H37Rv (Rv) selectively downregulates the expression of PKCalpha while infection by Mycobacterium smegmatis (MS) does not.

(1) The phorbolester 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C (PKC), inhibits cholinergic stimulation of gastric acid secretion. We observed that this effect strongly correlated with the inhibition of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity in rabbit parietal cells. (2) The aim of this study was to specify the function of PKC-alpha in cholinergically stimulated H(+) secretion. PKC-alpha represents the only calcium-dependent PKC isoenzyme that has been detected in rabbit parietal cells. (3) Gö 6976, an inhibitor of calcium-dependent PKC, concentration-dependently antagonized the inhibitory effect of TPA, and, therefore, revealed the action of PKC-alpha on carbachol-induced acid secretion in rabbit parietal cells. (4) TPA exerted no additive inhibition of carbachol-stimulated acid secretion if acid secretion was partially inhibited by the potent CaMKII inhibitor 1-[N,O-bis(5-isoquinolinsulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62). (5) Since both kinase modulators, TPA and KN-62, affected no divergent signal transduction pathways in the parietal cell, an in vitro model has been used to study if PKC directly targets CaMKII. CaMKII purified from parietal cell-containing gastric mucosa of pig, was transphosphorylated by purified cPKC containing PKC-alpha up to 1.8 mol P(i) per mol CaMKII in vitro. The autonomy site of CaMKII was not transphosphorylated by PKC. (6) The phosphotransferase activity of the purified CaMKII was in vitro inhibited after transphosphorylation by PKC if calmodulin was absent during transphosphorylation. Attenuation of CaMKII activity by PKC showed strong similarity to the downregulation of CaMKII by basal autophosphorylation. (7) Our results suggest that PKC-alpha and CaMKII are closely functionally linked in a cholinergically induced signalling pathway in rabbit parietal cells. We assume that in cholinergically stimulated parietal cells PKC-alpha transinhibits CaMKII activity, resulting in an attenuation of acid secretion.

Cardioplegic arrest (CP) and cardiopulmonary bypass (CPB) can lead to dysfunction in the coronary and skeletal microcirculation leading to impaired tissue perfusion. alpha-Adrenergic signaling pathways acting on these microcirculatory beds are thought to involve protein kinase C (PKC). We investigate here the role of the conventional PKCs in microvascular function in the setting of CP/CPB.

Prolonged exposure to organic barn dusts can lead to chronic inflammation and a broad range of lung problems over time, mediated by innate immune mechanisms. The immune surfactant or collectin surfactant protein D (SP-D) is a crucial multifunctional innate immune receptor. Little work to date has examined the effect of such collectins in response to organic dusts. We provide evidence here that agricultural organic dusts can inhibit mRNA and protein expression of SP-D in a human alveolar epithelial cell line, and an in vivo mouse model. This inhibition was not a result of lipopolysaccharide (LPS) or peptidoglycans, the two most commonly cited immune active components of these dusts. We further show that inhibition of the signaling molecule protein kinase C alpha (PKCα) can reverse this inhibition implicating it as a mechanism of SP-D inhibition. Examination of the SP-D regulatory receptor GPR116 showed that its mRNA expression was increased in response to dust and inhibited by blocking PKCα, implicating it as a means of inhibiting SP-D in the lungs in response to organic dusts. This reduction shows that organic barn dust can reduce lung SP-D, thus leaving workers potentially at risk for a host of pathogens.

Nephronectin (Npnt) is an extracellular matrix protein and ligand of integrin α8β1 known to promote differentiation of osteoblasts. A search for factors that regulate Npnt gene expression in osteoblasts revealed that phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), had a strong effect to suppress that expression. Research was then conducted to elucidate the signaling pathway responsible for regulation of Npnt gene expression by PMA in osteoblasts. Treatment of MC3T3-E1 cells with PMA suppressed cell differentiation and Npnt gene expression. Effects were noted at a low concentration of PMA, and were time- and dose-dependent. Furthermore, treatment with the PKC signal inhibitor Gö6983 inhibited down-regulation of Npnt expression, while transfection with small interfering RNA (siRNA) of PKCα, c-Jun, and c-Fos suppressed that down-regulation. The present results suggest regulation of Npnt gene expression via the PKCα and c-Jun/c-Fos pathway.

Aberrant expression of protein kinase C (PKC) isozymes is a hallmark of cancer. The different members of the PKC family control cellular events associated with cancer development and progression. Whereas the classical/conventional PKCα isozyme has been linked to tumor suppression in most cancer types, here we demonstrate that this kinase is required for the mitogenic activity of aggressive human prostate cancer cells displaying aberrantly high PKCα expression. Immunohistochemical analysis showed abnormal up-regulation of PKCα in human primary prostate tumors. Interestingly, silencing PKCα expression from aggressive prostate cancer cells impairs cell cycle progression, proliferation and invasion, as well as their tumorigenic activity in a mouse xenograft model. Mechanistic analysis revealed that PKCα exerts a profound control of gene expression, particularly over genes and transcriptional networks associated with cell cycle progression and E2F transcription factors. PKCα RNAi depletion from PC3 prostate cancer cells led to a reduction in the expression of pro-inflammatory cytokine and epithelial-to-mesenchymal transition (EMT) genes, as well as a prominent down-regulation of the immune checkpoint ligand PD-L1. This PKCα-dependent gene expression profile was corroborated in silico using human prostate cancer databases. Our studies established PKCα as a multifunctional kinase that plays pleiotropic roles in prostate cancer, particularly by controlling genetic networks associated with tumor growth and progression. The identification of PKCα as a pro-tumorigenic kinase in human prostate cancer provides strong rationale for the development of therapeutic approaches towards targeting PKCα or its effectors.

Protein kinase C (PKC) isoenzymes require membrane translocation for physiological activation. We have recently shown that the growth factors such as epidermal growth factor and hepatocyte growth factor (HGF), but not keratinocyte growth factor (KGF), regulate PKCalpha activation to promote epithelial wound healing [Sharma, G.D., Ottino, P., Bazan, H.E.P., 2005. Epidermal and hepatocyte growth factors, but not keratinocyte growth factor, modulate protein kinase C alpha translocation to the plasma membrane through 15(S)-hydroxyeicosatetraenoic acid synthesis. J. Biol. Chem. 280, 7917--924]. Protein kinase C alpha (PKCalpha) and protein kinase C epsilon (PKCvarepsilon) are two differentially regulated isoenzymes. While PKCalpha requires Ca(2+) for its activation, PKEvarepsilon is Ca(2+) independent. However, growth factor-induced activation of these enzymes and their specific regulation of epithelial migration and proliferation have not been explored. In the present study, we overexpressed PKCvarepsilon fused to green fluorescent protein to examine its translocation in real-time to the plasma membrane in living human corneal epithelial cells. Stimulation with HGF and KGF demonstrated translocation of PKCvarepsilon to the plasma membrane. Because HGF activates both PKCs, this growth factor was used to stimulate wound healing. PKCalpha or PKCvarepsilon-genes were knocked down individually without affecting the basal expression of the other PKC isoforms. Gene knockdown of PKCalpha significantly inhibited HGF-stimulated proliferation of human corneal epithelial cells. In contrast, PKCvarepsilon-gene-silencing severely impaired the HGF-stimulated migratory ability of human corneal epithelial cells. When migrating epithelial cells in the cornea wound bed after injury were transfected with specific PKCalpha- or PKCvarepsilon-siRNA, there was a significant delay in wound healing. Corneal wound healing stimulated with HGF in similar conditions was also inhibited. On the other hand, overexpression of PKCalpha or PKCvarepsilon-genes fused with green fluorescent protein in migrating corneal epithelium accelerated repair of the epithelial defect. Our findings demonstrate that PKCalpha and PKCvarepsilon modulate different stages of wound healing stimulated by HGF and contribute to epithelial repair by playing selective regulatory roles in epithelial proliferation and migration, both crucial to corneal wound healing.

A principal challenge in treating acute myeloid leukemia (AML) is chemotherapy refractory disease. As such, there remains a critical need to identify key regulators of chemotherapy resistance in AML. In this study, we demonstrate that the membrane scaffold, CD82, contributes to the chemoresistant phenotype of AML. Using an RNA-seq approach, we identified the increased expression of the tetraspanin family member, CD82, in response to the chemotherapeutic, daunorubicin. Analysis of the TARGET and BEAT AML databases identifies a correlation between CD82 expression and overall survival of AML patients. Moreover, using a combination of cell lines and patient samples, we find that CD82 overexpression results in significantly reduced cell death in response to chemotherapy. Investigation of the mechanism by which CD82 promotes AML survival in response to chemotherapy identified a crucial role for enhanced protein kinase c alpha (PKCα) signaling and downstream activation of the β1 integrin. In addition, analysis of β1 integrin clustering by super-resolution imaging demonstrates that CD82 expression promotes the formation of dense β1 integrin membrane clusters. Lastly, evaluation of survival signaling following daunorubicin treatment identified robust activation of p38 mitogen-activated protein kinase (MAPK) downstream of PKCα and β1 integrin signaling when CD82 is overexpressed. Together, these data propose a mechanism where CD82 promotes chemoresistance by increasing PKCα activation and downstream activation/clustering of β1 integrin, leading to AML cell survival via activation of p38 MAPK. These observations suggest that the CD82-PKCα signaling axis may be a potential therapeutic target for attenuating chemoresistance signaling in AML.

Although protein kinase C (PKC) has been implicated as an effector of erythropoietin (EPO) production, its exact role is still uncertain. Hep3B human hepatocellular carcinoma cells were used for this study and were depleted of PKC in three different ways: long-term treatment with phorbol 12-myristate 13-acetate (PMA), selective inhibition with calphostin C, and treatment with PKCalpha antisense oligonucleotides. When EPO-producing Hep3B cells were incubated in 1% O2 (hypoxia) for 24 h, PMA treatment resulted in significant decreases in medium levels of EPO in Hep3B cell cultures at concentrations higher than 10 nM. The specific PKC inhibitor, calphostin C, significantly inhibited medium levels of EPO and EPO mRNA levels in Hep3B cells exposed to 1% O2. Western blot analysis revealed that Hep3B cells express the classical PKCalpha and gamma isoforms, as well as novel PKCepsilon and delta and the atypical zeta isoform. Preincubation with PMA for 6 h specifically down-regulated PKCalpha protein expression. Phosphorothioate modified antisense oligonucleotides specific for PKCalpha also decreased EPO production in Hep3B cells exposed to hypoxia for 20 h when compared to PKCalpha sense treatment. The translocation of PKCalpha from the soluble to particulate fractions was increased in Hep3B cells incubated under hypoxia compared with normoxia (21% O2) controls. These results suggest that the PKCalpha isoform plays an important role in sustaining hypoxia-regulated EPO production.

We previously identified a novel Rab small GTPase protein, Rab37, which plays a critical role in regulating exocytosis of secreted glycoproteins, tissue inhibitor of metalloproteinases 1 (TIMP1) to suppress lung cancer metastasis. Patients with preserved Rab37 protein expression were associated with better prognosis. However, a significant number of the patients with preserved Rab37 expression showed poor survival. In addition, the molecular mechanism for the regulation of Rab37-mediated exocytosis remained to be further identified. Therefore, we investigated the molecular mechanism underlying the dysregulation of Rab37-mediated exocytosis and metastasis suppression. Here, we report a novel mechanism for Rab37 inactivation by phosphorylation. Lung cancer patients with preserved Rab37, low TIMP1, and high PKCα expression profile correlate with worse progression-free survival examined by Kaplan-Meier survival, suggesting that PKCα overexpression leads to dysfunction of Rab37. This PKCα-Rab37-TIMP1 expression profile predicts the poor outcome by multivariate Cox regression analysis. We also show that Rab37 is phosphorylated by protein kinase Cα (PKCα) at threonine 172 (T172), leading to attenuation of its GTP-bound state, and impairment of the Rab37-mediated exocytosis of TIMP1, and thus reduces its suppression activity on lung cancer cell motility. We further demonstrate that PKCα reduces vesicle colocalization of Rab37 and TIMP1, and therefore inhibits Rab37-mediated TIMP1 trafficking. Moreover, Phospho-mimetic aspartate substitution mutant T172D of Rab37 significantly promotes tumor metastasis in vivo. Our findings reveal a novel regulation of Rab37 activity by PKCα-mediated phosphorylation which inhibits exocytic transport of TIMP1 and thereby enhances lung tumor metastasis.

Gene deletion-induced autophagy deficiency leads to neural tube defects (NTDs), similar to those in diabetic pregnancy. Here we report the key autophagy regulators modulated by diabetes in the murine developing neuroepithelium. Diabetes predominantly leads to exencephaly, induces neuroepithelial cell apoptosis and suppresses autophagy in the forebrain and midbrain of NTD embryos. Deleting the Prkca gene, which encodes PKCα, reverses diabetes-induced autophagy impairment, cellular organelle stress and apoptosis, leading to an NTD reduction. PKCα increases the expression of miR-129-2, which is a negative regulator of autophagy. miR-129-2 represses autophagy by directly targeting PGC-1α, a positive regulator for mitochondrial function, which is disturbed by maternal diabetes. PGC-1α supports neurulation by stimulating autophagy in neuroepithelial cells. These findings identify two negative autophagy regulators, PKCα and miR-129-2, which mediate the teratogenicity of hyperglycaemia leading to NTDs. We also reveal a function for PGC-1α in embryonic development through promoting autophagy and ameliorating hyperglycaemia-induced NTDs.

Albuminuria in diabetic nephropathy is due to endothelial dysfunction, a loss of negative charges in the basement membrane, and changes a of the slit-membrane diaphragm composition. We have recently shown that protein kinase C alpha (PKCalpha)-deficient mice are protected against the development of albuminuria under diabetic conditions. We here tested the hypothesis that PKCalpha mediates the hyperglycemia-induced downregulation of the slit-diaphragm protein nephrin. After 8 weeks of streptozotocin (STZ)-induced hyperglycemia the expression of glomerular nephrin was significantly reduced. In contrast, other slit-diaphragm proteins such as podocin and CD2AP were unaltered in diabetic state. In PKCalpha-/- mice, hyperglycemia-induced downregulation of nephrin was prevented. Podocin and CD2AP remained unchanged. In addition, the nephrin messenger RNA expression was also reduced in hyperglycemic wild-type mice but remained unaltered in PKCalpha-/- mice. We postulate that the underlying mechanism of the hyperglycemia-induced regulation of various proteins of the glomerular filtration barrier is a PKCalpha-dependent regulation of the Wilms' Tumor Suppressor (WT1) which previously has been shown to act as a direct transcription factor on the nephrin promoter. Our data suggest that PKCalpha activation may be an important intracellular signaling pathway in the regulation of nephrin expression and glomerular albumin permeability in the diabetic state.

The subcellular location of phospholipase D1 (PLD1) and its activation by protein kinase C alpha (PKC alpha) were examined by subcellular fractionation and by microscopic observation of green fluorescent protein-fused PLD1 (GFP-PLD1) or PKC alpha (GFP-PKC alpha) in fibroblastic 3Y1 cells. Major PLD1 immunoreactivity and PKC alpha-stimulated PLD activity segregated with a plasma membrane marker, even though a significant amount was co-fractionated with markers for endoplasmic reticulum (ER) and Golgi. Upon treatment with phorbol myristate acetate (PMA), PKC alpha translocated from the cytosolic fraction to the membrane fraction to which PLD1 also localized. GFP-PLD1 was found in the plasma membrane as well as a in a perinuclear compartment consistent with ER and Golgi and in other dispersed vesicular structures in the cytoplasm. However, most of GFP-PKC alpha was translocated from the cytosol to the plasma membrane after treatment with PMA. From these results, we concluded that the plasma membrane is the major site of PLD1 activation by PKC alpha in 3Y1 cells.

Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis and proinflammatory processes. Abnormalities in these processes are primary features of rheumatoid arthritis (RA) in synovial tissues. We investigated the signaling pathway involved in IL-6 production caused by thrombin in synovial fibroblasts. Thrombin caused concentration- and time-dependent increases in IL-6 production. By using pharmacological inhibitors or activators or genetic inhibition by the protease activated receptor (PAR), siRNA revealed that the PAR1 receptor but not other PAR receptors is involved in thrombin-mediated up-regulation of IL-6. Thrombin-mediated IL-6 production was attenuated by thrombin inhibitor (PPACK), phospholipase C inhibitor (U73122), protein kinase C alpha inhibitor (Ro320432), Src inhibitor (PP2), NF-kappaB inhibitor (PDTC), I kappa B protease inhibitor (TPCK), or NF-kappaB inhibitor peptide. Stimulation of synovial fibroblasts with thrombin activated I kappa B kinase alpha/beta (IKK alpha/beta), I kappa B alpha phosphorylation, I kappa B alpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Thrombin-mediated an increase of IKK alpha/beta activity, kappaB-luciferase activity and p65 and p50 binding to the NF-kappaB element was inhibited by PPACK, U73122, Ro320432 and PP2. The binding of p65 and p50 to the NF-kappaB elements, as well as the recruitment of p300 and the enhancement of p50 acetylation on the IL-6 promoter was enhanced by thrombin. Our results suggest that thrombin increased IL-6 production in synovial fibroblasts via the PAR1 receptor/PI-PLC/PKC alpha/c-Src/NF-kappaB and p300 signaling pathway.