Basic fibroblast growth factor stimulates fibronectin expression through phospholipase C gamma, protein kinase C alpha, c-Src, NF-kappaB, and p300 pathway in osteoblasts

Fibronectin (Fn) is involved in early stages of bone formation and basic fibroblast growth factor (bFGF) is an important factor regulating osteogenesis. bFGF increased Fn expression, which was attenuated by phosphatidylinositol phospholipase inhibitor (U73122), protein kinase C inhibitor (GF109203X), Src inhibitor (PP2), NF-kappaB inhibitor (PDTC), IkappaBalpha phosphorylation inhibitor (Bay 117082), or IkappaB protease inhibitor (TPCK). bFGF-induced increase of Fn-luciferase activity was antagonized by cells transfected with Fn construct without NF-kappaB regulatory site. Stimulation of osteoblasts with bFGF activated IkappaB kinase alpha/beta (IKK alpha/beta) and increased IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-kappaB-specific DNA-protein complex and kappaB-luciferase activity. bFGF-mediated an increase of IKKalpha/beta activity and DNA-binding activity was inhibited by U73122, GF109203X, or PP2. The binding of p65 to the NF-kappaB element, as well as the recruitment of p300 and the enhancement of p50 acetylation on the Fn promoter was enhanced by bFGF. Overexpression of constitutively active FGF receptor 2 (FGFR2) increased Fn-luciferase activity, which was inhibited by co-transfection with dominant negative (DN) mutants of PLCgamma2, PKCalpha, c-Src, IKKalpha, or IKKbeta. Our results suggest that bFGF increased Fn expression in rat osteoblasts via the FGFR2/PLCgamma2/PKCalpha/c-Src/NF-kappaB signaling pathway.     

Regulation and function of COX-2 gene expression in isolated gastric parietal cells

We examined expression, function, and regulation of the cyclooxygenase (COX)-2 gene in gastric parietal cells. COX-2-specific mRNA was isolated from purified (>95%) canine gastric parietal cells in primary culture and measured by Northern blots using a human COX-2 cDNA probe. Carbachol was the most potent inducer of COX-2 gene expression. Gastrin and histamine exhibited minor stimulatory effects. Carbachol-stimulated expression was inhibited by intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (90%), protein kinase C (PKC) inhibitor GF-109203X (48%), and p38 kinase inhibitor SB-203580 (48%). Nuclear factor (NF)-kappaB inhibitor 1-pyrrolidinecarbodithioic acid inhibited carbachol-stimulated expression by 80%. Similar results were observed in the presence of adenoviral vector Ad.dom.neg.IkappaB, which expresses a repressor of NF-kappaB. Addition of SB-203580 with Ad.dom.neg.IkappaB almost completely blocked carbachol stimulation of COX-2 gene expression. We examined the effect of carbachol on PGE(2) release by enzyme-linked immunoassay. Carbachol induced PGE(2) release. Ad.dom.neg.IkappaB, alone or with SB-203580, produced, respectively, partial (70%) and almost complete (>80%) inhibition of carbachol-stimulated PGE(2) production. Selective COX-2 inhibitor NS-398 blocked carbachol-stimulated PGE(2) release without affecting basal PGE(2) production. In contrast, indomethacin inhibited both basal and carbachol-stimulated PGE(2) release. Carbachol induces COX-2 gene expression in the parietal cells through signaling pathways that involve intracellular Ca(2+), PKC, p38 kinase, and activation of NF-kappaB. The functional significance of these effects seems to be stimulation of PGE(2) release.     

Autophosphorylation suppresses whereas kinase inhibition augments the translocation of protein kinase Calpha in response to diacylglycerol

We have seen that protein kinase Calpha (PKCalpha) is transiently translocated to the plasma membrane by carbachol stimulation of neuroblastoma cells. This is induced by the Ca2+ increase, and PKCalpha does not respond to diacylglycerol (DAG). The unresponsiveness is dependent on structures in the catalytic domain of PKCalpha. This study was designed to investigate if and how the kinase activity and autophosphorylation are involved in regulating the translocation. PKCalpha enhanced green fluorescent protein translocation was studied in living neuroblastoma cells by confocal microscopy. Carbachol stimulation induced a transient translocation of PKCalpha to the plasma membrane and a sustained translocation of kinase-dead PKCalpha. In cells treated with the PKC inhibitor GF109203X, wild-type PKCalpha also showed a sustained translocation. The same effects were seen with PKCbetaI, PKCbetaII, and PKCdelta. Only kinase-dead and not wild-type PKCalpha translocated in response to 1,2-dioctanoylglycerol. To examine whether autophosphorylation regulates relocation to the cytosol, the autophosphorylation sites in PKCalpha were mutated to glutamate, to mimic phosphorylation, or alanine, to mimic the non-phosphorylated protein. After stimulation with carbachol, glutamate mutants behaved like wild-type PKCalpha, whereas alanine mutants behaved like kinase-dead PKCalpha. When the alanine mutants were treated with 1,2-dioctanoylglycerol, all cells showed a sustained translocation of the protein. However, neither carbachol nor GF109203X had any major effects on the level of autophosphorylation, and GF109203X potentiated the translocation of the glutamate mutants. We, therefore, hypothesize that 1) autophosphorylation of PKCalpha limits its sensitivity to DAG and 2) that kinase inhibitors augment the DAG sensitivity of PKCalpha, perhaps by destabilizing the closed conformation.     

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.     

Sphingosine and phorbol ester modulate protein kinase C activity and modify ATP-evoked calcium mobilization in glioma C6 cells.

The effect of sphingosine and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on ATP-evoked Ca(2+) mobilization in glioma C6 cells was studied with the Fura-2 video-imaging technique. Treatment of the cells with TPA, an activator of protein kinase C, reduced the ATP-evoked release of Ca(2+) from the intracellular stores, whereas sphingosine, known from in vitro studies as a protein kinase C inhibitor, potentiated Ca(2+) release synergistically with ATP. ATP-induced Ca(2+) mobilization was also enhanced by a specific protein kinase C inhibitor, GF 109203X. Pretreatment of the cells with GF 109203X prevented TPA action, whereas TPA diminished the stimulatory effect of sphingosine. However, this sphingosine effect was only observed after a short (1 min) treatment, whereas a longer treatment (5 min) reduced ATP-evoked Ca(2+) release. It is therefore concluded that sphingosine has two apparent actions: it inhibits protein kinase C providing a positive feedback regulation of receptor signals and it releases Ca(2+) from intracellular stores by an unknown mechanism, possibly independent of protein kinase C.     

Adreno-muscarinic synergy in the bladder trigone: calcium-dependent and -independent mechanisms

We have recently demonstrated a strong synergy between both excitatory pathways in the trigone which--as part of the bladder base--is believed to play a key role in outflow control. The aim of this study was to reveal whether modulation of intracellular Ca2+, [Ca2+]i, is mainly responsible for this synergistic effect. Intact muscle strips from the superficial trigone of male guinea-pigs were used for tension experiments. In isolated trigonal cells, [Ca2+]i was measured by epifluorescence microscopy using the fluorescent Ca2+-indicator Fura-2. Phenylephrine (PE, 10 microM) augmented contractions induced by carbachol (1 microM) to 4.0+/-0.8-fold of control, while corresponding [Ca2+]i levels did not exceed 1.3+/-0.2-fold of control. Furthermore, PE generated significantly greater contractions for a given rise of [Ca2+]i, compared to depolarising KCl solutions. The protein kinase C inhibitor GF 109203X (5 microM) and the Rho-kinase inhibitor Y-27632 (5microM) reduced the PE contracture to 37.3+/-9.4 and 60.1+/-12.4% of control, respectively, without significantly altering the [Ca2+]i transients. GF 109203X reduced the augmentation of 1microM carbachol by PE to 1.5+/-0.1-fold. Muscarinic and adrenergic receptor activation exerts a strong synergistic effect in the bladder trigone without similar changes to the [Ca2+]i transient. Ca2+-sensitisation of contractile proteins is likely to play a key role in this synergism, particularly for adrenergic activation.     

Regulation and function of p38 protein kinase in isolated canine gastric parietal cells

We examined the regulation and functional role of p38 kinase in gastric acid secretion. p38 kinase was immunoprecipitated from cell lysates of highly purified gastric parietal cells in primary culture, and its activity was quantitated by in vitro kinase assay. Carbachol effects were dose- and time-dependent, with a maximal 10-fold stimulatory effect detected after 30 min of incubation. SB-203580, a highly selective inhibitor of p38 kinase, blocked carbachol induction of p38 kinase activity, with maximal inhibition at 10 microM. Stimulation by carbachol was unaffected by preincubation of parietal cells with the intracellular Ca(2+) chelator BAPTA-AM, but incubation of cells in Ca(2+)-free medium led to a 50% inhibition of carbachol induction of p38 kinase activity. Because some of the effects of carbachol are mediated by the small GTP-binding protein Rho, we examined the role of Rho in carbachol induction of p38 kinase activity. We tested the effect of exoenzyme C3 from Clostridium botulinum (C3), a toxin known to ADP-ribosylate and specifically inactivate Rho. C3 led to complete ADP-ribosylation of Rho, and it inhibited carbachol induction of p38 kinase by 50%. We then tested the effect of SB-203580 and C3 on carbachol-stimulated uptake of [(14)C]aminopyrine (AP). Inhibition of p38 kinase by SB-203580 led to a dose-dependent increase in AP uptake induced by carbachol, with maximal (threefold) effect at 10 microM SB-203580. Similarly, preincubation of parietal cells with C3 led to a twofold increase in AP uptake induced by carbachol. Thus carbachol induces a cascade of events in parietal cells that results in activation of p38 kinase through signaling pathways that are at least in part dependent on Rho activation and on the presence of extracellular Ca(2+). p38 kinase appears to inhibit gastric acid secretion.     

Proteinase-activated receptor-2-mediated activation of stress-activated protein kinases and inhibitory kappa B kinases in NCTC 2544 keratinocytes.

In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory kappa B kinases (IKKs) through stimulation of the novel G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity. Trypsin also stimulated NF kappa B-DNA binding and the activation of the upstream kinases IKK alpha and -beta. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and G?6983, or transfection of dominant negative (DN)-PKC alpha, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, G?6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKC alpha or PKC zeta had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKK alpha activity but abolished that of IKK beta, whereas DN-PKC alpha but not DN-PKC zeta substantially reduced trypsin-stimulated Flag-IKK beta activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.     

Complete reconstitution of human IkappaB kinase (IKK) complex in yeast. Assessment of its stoichiometry and the role of IKKgamma on the complex activity in the absence of stimulation

The IkappaB kinase (IKK) complex, composed of two catalytic subunits (IKKalpha and IKKbeta) and a regulatory subunit (IKKgamma), is the key enzyme in activation of nuclear factor kappaB (NF-kappaB). To study the mechanism and structure of the complex, we wanted to recombinantly express IKK in a model organism that lacks IKK. For this purpose, we have recombinantly reconstituted all three subunits together in yeast and have found that it is biochemically similar to IKK isolated from human cells. We show that there is one regulatory subunit per kinase subunit. Thus, the core subunit composition of IKKalpha.beta.gamma complex is alpha(1)beta(1)gamma(2), and the core subunit composition of IKKbeta.gamma is beta(2)gamma(2). The activity of the IKK complex (alpha+beta+gamma or beta+gamma) expressed in yeast (which lack NF-kappaB and IKK) is 4-5-fold higher than an equivalent amount of IKK from nonstimulated HeLa cells. In the absence of IKKgamma, IKKbeta shows a level of activity similar to that of IKK from nonstimulated HeLa cells. Thus, IKKgamma activates IKK complex in the absence of upstream stimuli. Deleting the gamma binding domain of IKKbeta or IKKalpha prevented IKKgamma induced activation of IKK complex in yeast, but it did not prevent the incorporation of IKKgamma into IKK and large complex formation. The possibility of IKK complex being under negative control in mammalian cells is discussed.     

Interleukin-1-induced NF-kappaB activation is NEMO-dependent but does not require IKKbeta

Activation of NF-kappaB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IkappaB kinase (IKK) complex, which contains two kinases named IKKalpha and IKKbeta and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKbeta is required for TNF-induced NF-kappaB activation. To determine whether NEMO and IKKalpha can form a functional IKK complex capable of activating the classical NF-kappaB pathway in the absence of IKKbeta, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IkappaBalpha degradation absolutely requires NEMO and IKKbeta. In contrast, we consistently observed intact IkappaBalpha degradation and NF-kappaB activation in response to IL-1 in two separate cell lines lacking IKKbeta. Furthermore, exogenously expressed, catalytically inactive IKKbeta blocked TNF- but not IL-1-induced IkappaBalpha degradation in wild-type MEFs, and reconstitution of IKKalpha/beta double knockout cells with IKKalpha rescued IL-1- but not TNF-induced NF-kappaB activation. Finally, we have shown that incubation of IKKbeta-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-kappaB activation. Our results therefore demonstrate that NEMO and IKKalpha can form a functional IKK complex that activates the classical NF-kappaB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways.     

Interleukin-1beta-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKS,and NF-kappaB pathways in canine tracheal smooth muscle cells

Interleukin-beta (IL-1beta) was found to induce inflammatory responses in the airways, which exerted a potent stimulus for PG synthesis. This study was to determine the mechanisms of IL-1beta-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). IL-1beta markedly increased COX-2 expression and PGE(2) formation in a time- and concentration-dependent manner in TSMCs. Both COX-2 expression and PGE(2) formation in response to IL-1beta were attenuated by a tyrosine kinase inhibitor, genistein, a phosphatidylcholine-phospholipase C inhibitor, D609, a phosphatidylinositol-phospholipase C inhibitor, U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. IL-1beta-induced activation of NF-kappaB correlated with the degradation of IkappaB-alpha in TSMCs. IL-1beta-induced NF-kappaB activation, COX-2 expression, and PGE(2) synthesis were inhibited by the dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. IL-1beta-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 inhibitor), but these two inhibitors had no effect on IL-1beta-induced NF-kappaB activation, indicating that activation of p42/44 and p38 MAPK and NF-kappaB signalling pathways were independently required for these responses. These findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from IL-1beta-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways in canine TSMCs. IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.     

Functional interactions of transforming growth factor beta-activated kinase 1 with IkappaB kinases to stimulate NF-kappaB activation

Several mitogen-activated protein kinase kinase kinases play critical roles in nuclear factor-kappaB (NF-kappaB) activation. We recently reported that the overexpression of transforming growth factor-beta-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, together with its activator TAK1-binding protein 1 (TAB1) stimulates NF-kappaB activation. Here we investigated the molecular mechanism of TAK1-induced NF-kappaB activation. Dominant negative mutants of IkappaB kinase (IKK) alpha and IKKbeta inhibited TAK1-induced NF-kappaB activation. TAK1 activated IKKalpha and IKKbeta in the presence of TAB1. IKKalpha and IKKbeta were coimmunoprecipitated with TAK1 in the absence of TAB1. TAB1-induced TAK1 activation promoted the dissociation of active forms of IKKalpha and IKKbeta from active TAK1, whereas the IKK mutants remained to interact with active TAK1. Furthermore, tumor necrosis factor-alpha activated endogenous TAK1, and the kinase-negative TAK1 acted as a dominant negative inhibitor against tumor necrosis factor-alpha-induced NF-kappaB activation. These results demonstrated a novel signaling pathway to NF-kappaB activation through TAK1 in which TAK1 may act as a regulatory kinase of IKKs.     

Stress-induced inhibition of the NF-kappaB signaling pathway results from the insolubilization of the IkappaB kinase complex following its dissociation from heat shock protein 90

Activation of the stress response attenuates proinflammatory responses by suppressing cytokine-stimulated activation of the NF-kappaB signaling pathway. In this study, we show that the activation of the cellular stress response, either by heat shock treatment or after exposure to sodium arsenite, leads to a transient inhibition of IkappaBalpha phosphorylation. Inhibition of IkappaBalpha phosphorylation after stress was associated with the detergent insolubilization of the upstream kinases, IkappaB kinase alpha (IKKalpha) and IkappaB kinase beta, components involved in IkappaBalpha phosphorylation. Pretreatment of cells with glycerol, a chemical chaperone that reduces the extent of stress-induced protein denaturation, reduced the stress-dependent detergent insolubility of the IKK complex and restored the cytokine-stimulated phosphorylation of IkappaB. The stress-dependent insolubility of the IKK complex appeared reversible; as the cells recovered from the heat shock treatment, the IKK complex reappeared within the soluble fraction of cells and was again capable of mediating the phosphorylation of IkappaBalpha in response to added cytokines. Treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) function, also resulted in IKK detergent insolubility and proteasome-mediated degradation of the IKK complex. Furthermore, while IKKalpha coprecipitated with Hsp90 in control cells, coprecipitation of the two proteins was greatly reduced in those cells early after stress or following exposure to geldanamycin. Stress-induced transient insolubilization of the IkappaB kinase complex following its dissociation from Hsp90 represents a novel mechanism by which the activation of the stress response inhibits the NF-kappaB signaling pathway in response to proinflammatory stimuli.     

The P2X(7) receptor-mediated phospholipase D activation is regulated by both PKC-dependent and PKC-independent pathways in a rat brain-derived Type-2 astrocyte cell line, RBA-2.

The aim of this study was to characterize the regulatory mechanisms of the P2X(7) receptor (P2X(7)R)-mediated phospholipase D (PLD) activation in a rat brain-derived Type-2 astrocyte cell line, RBA-2. A time course study revealed that activation of P2X(7)R resulted in a choline and not phosphorylcholine formation, suggesting that activation of P2X(7)R is associated with the phosphatidylcholine-PLD (PC-PLD) in these cells. GF 109203X, a selective protein kinase C (PKC) inhibitor, partially inhibited the P2X(7)R-mediated PLD activation, while blocking the phorbol 12-myristate 13-acetate (PMA)-stimulated PLD activity. In addition, PMA synergistically activated the P2X(7)R-mediated PLD activity. Furthermore, genistein, a tyrosine kinase inhibitor, blocked the P2X(7)R-activated PLD, while KN62, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, was less effective, whereas the mitogen-activated protein kinase (MAPK) inhibitor PD98059 was ineffective. No additive inhibitory effects were found by simultaneous treatment of GF 109203X and KN62 on P2X(7)R-activated PLD. Taken together, these results demonstrate that both PKC-dependent and PKC-independent signaling pathways are involved in the regulation of P2X(7)R-mediated PLD activation. Additionally, CaMKII may participate in the PKC-dependent pathway, and tyrosine kinase may play a pivotal role on both PKC-dependent and PKC-independent pathways in the P2X(7)R-mediated PLD activation in RBA-2 cells.