RESUMO
BACKGROUND AND PURPOSE: Proteinase-activated receptor 2 (PAR(2)) is a G-protein coupled receptor associated with many pathophysiological functions. To date, the development of PAR(2) antagonists has been limited. Here, we identify a number of novel peptide-mimetic PAR(2) antagonists and demonstrate inhibitory effects on PAR(2)-mediated intracellular signalling pathways and vascular responses. EXPERIMENTAL APPROACH: The peptide-mimetic compound library based on the structures of PAR(2) agonist peptides were screened for inhibition of PAR(2)-induced calcium mobilisation in human keratinocytes. Representative compounds were further evaluated by radioligand binding and inhibition of NFkappaB transcriptional activity and IL-8 production. The vascular effects of the antagonists were assessed using in vitro and in vivo models. KEY RESULTS: Two compounds, K-12940 and K-14585, significantly reduced SLIGKV-induced Ca(2+) mobilisation in primary human keratinocytes. Both K-12940 and K-14585 exhibited competitive inhibition for the binding of a high-affinity radiolabelled PAR(2)-ligand, [(3)H]-2-furoyl-LIGRL-NH(2), to human PAR(2) with K(i) values of 1.94 and 0.627 microM respectively. NFkappaB reporter activity and IL-8 production were also significantly reduced. Furthermore, relaxation of rat-isolated aorta induced by SLIGRL-NH(2) was inhibited competitively by K-14585. K-14585 also significantly lowered plasma extravasation in the dorsal skin of guinea pigs and reduced salivation in mice. CONCLUSIONS AND IMPLICATIONS: K-12940 and K-14585 antagonized PAR(2) competitively, resulting in inhibition of PAR(2)-mediated signalling and physiological responses both in vitro and in vivo. These peptide-mimetic PAR(2) antagonists could be useful in evaluating PAR(2)-mediated biological events and might lead to a new generation of therapeutically useful antagonists.
Assuntos
Permeabilidade Capilar/fisiologia , Queratinócitos/fisiologia , Oligopeptídeos/farmacologia , Peptídeos/antagonistas & inibidores , Peptídeos/fisiologia , Receptor PAR-2/antagonistas & inibidores , Receptor PAR-2/fisiologia , Ureia/análogos & derivados , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Cobaias , Humanos , Técnicas In Vitro , Queratinócitos/efeitos dos fármacos , Queratinócitos/enzimologia , Masculino , Camundongos , Mimetismo Molecular , Peptídeos/agonistas , Ratos , Ratos Wistar , Receptor PAR-2/agonistas , Ureia/farmacologiaRESUMO
The neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and leukemia inhibitory factor (LIF) were investigated in human neuroblastoma SH-SY5Y cells. Effects on differentiation were assessed through monitoring morphological changes and Western blot analysis of the expression of neuronal marker proteins. In contrast to PACAP-38, which induced a 5.5-fold increase in the number of neurite-bearing cells, LIF had no significant effect on cell morphology compared to control cells over the 4-day time course. Cells co-treated with PACAP-38+LIF showed a similar increase in neurite-bearing cells compared to those treated with PACAP-38 alone. Cell morphology was similar for PACAP-38-treated and PACAP-38+LIF-co-treated cells, with the formation of bipolar neuron-like cells with long thin neurites, topped by growth cone-like structures and varicosities. SH-SY5Y cells express tyrosine hydroxylase (TH) but only low levels of the neuronal marker proteins: Bcl-2, GAP-43 and choline acetyltransferase (ChAT). Treatment of cells with PACAP-38 induced the expression of Bcl-2, GAP-43, and ChAT but did not appear to alter the expression of TH. LIF failed to induce the expression of GAP-43 and had little effect on the expression of TH, but did induce the expression of Bcl-2 and upregulated the expression of ChAT. Co-treatment with LIF had no effect on PACAP-38-induced expression of Bcl-2, GAP-43, and ChAT. Cells differentiated for 4 days with PACAP-38 or treated with LIF also displayed increased resistance to hypoxic conditions and to treatment with H2O2 and TNFalpha. The increased resistance to hypoxic conditions for PACAP-differentiated cells was blocked by the p38 MAP kinase inhibitor, SB203580, but not by the MEK1 inhibitor, PD98059. Additionally, cell proliferation assays show that LIF, but not PACAP-38, stimulates proliferation of SH-SY5Y cells, and this observed increase by LIF is not attenuated by co-treatment with PACAP. Further investigation of the intracellular signaling pathways mediating the neurotrophic effects of PACAP on SH-SY5Y cells indicate that neither phospholipase C activation nor Ca2+/calmodulin-dependent kinase II (CAMKII) are involved.
Assuntos
Fator Inibidor de Leucemia/farmacologia , Fatores de Crescimento Neural/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Animais , Biomarcadores/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular , Humanos , Neuroblastoma , Receptores de OSM-LIF/genética , Receptores de OSM-LIF/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
The intracellular signaling pathways mediating the neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP) were investigated in human neuroblastoma SH-SY5Y cells. Previously, we showed that SH-SY5Y cells express the PAC(1) and VIP/PACAP receptor type 2 (VPAC(2)) receptors, and that the robust cAMP production in response to PACAP and vasoactive intestinal peptide (VIP) was mediated by PAC(1) receptors (Lutz et al. 2006). Here, we investigated the ability of PACAP-38 to differentiate SH-SY5Y cells by measuring morphological changes and the expression of neuronal markers. PACAP-38 caused a concentration-dependent increase in the number of neurite-bearing cells and an up-regulation in the expression of the neuronal proteins Bcl-2, growth-associated protein-43 (GAP-43) and choline acetyltransferase: VIP was less effective than PACAP-38 and the VPAC(2) receptor-specific agonist, Ro 25-1553, had no effect. The effects of PACAP-38 and VIP were blocked by the PAC(1) receptor antagonist, PACAP6-38. As observed with PACAP-38, the adenylyl cyclase activator, forskolin, also induced an increase in the number of neurite-bearing cells and an up-regulation in the expression of Bcl-2 and GAP-43. PACAP-induced differentiation was prevented by the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine (DDA), but not the protein kinase A (PKA) inhibitor, H89, or by siRNA-mediated knock-down of the PKA catalytic subunit. PACAP-38 and forskolin stimulated the activation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAP; p38 MAP kinase) and c-Jun N-terminal kinase (JNK). PACAP-induced neuritogenesis was blocked by the MEK1 inhibitor PD98059 and partially by the p38 MAP kinase inhibitor SB203580. Activation of exchange protein directly activated by cAMP (Epac) partially mimicked the effects of PACAP-38, and led to the phosphorylation of ERK but not p38 MAP kinase. These results provide evidence that the neurotrophic effects of PACAP-38 on human SH-SY5Y neuroblastoma cells are mediated by the PAC(1) receptor through a cAMP-dependent but PKA-independent mechanism, and furthermore suggest that this involves Epac-dependent activation of ERK as well as activation of the p38 MAP kinase signaling pathway.
Assuntos
AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Substâncias de Crescimento/farmacologia , Neurônios/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Tamanho Celular , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
OBJECTIVE: Mast cells are hypothesized to play a role in the pathogenesis of rheumatoid arthritis (RA) by mechanisms requiring elucidation. Tryptase released from these cells can activate protease-activated receptor 2 (PAR-2), which was recently shown to have proinflammatory actions. The purpose of this study was to examine the relationship between synovial mast cells and PAR-2. Mast cell proximity to PAR-2-expressing cells was investigated in RA synovium. In murine studies, we assessed the capacity of mast cell tryptase to mediate synovial proinflammatory responses via PAR-2 and whether degranulating mast cells induced synovial hyperemia by PAR-2 activation. METHODS: RA synovial tissue was examined by immunohistochemistry. PAR-2(+/+) and PAR-2(-/-) C57BL/6J mice were used to investigate the PAR-2 dependence of compound 48/80-induced synovial hyperemia, as measured by laser Doppler imaging, and joint swelling and hyperemic responses to recombinant human beta-tryptase. RESULTS: Mast cells and synovial lining cells staining for PAR-2 were colocalized in RA articular tissue. Compound 48/80 administration resulted in vasodilatation in PAR-2(+/+) mice but not in PAR-2(-/-) mice, which showed a vasoconstrictor response. Eliminating the 5-hydroxytryptamine-mediated component of this response with methysergide unveiled an enhanced PAR-2-mediated vasodilatation to compound 48/80 in PAR-2(+/+) mice and ablated the vasoconstrictor response in PAR-2(-/-) mice. Treatment with beta-tryptase resulted in dose-dependent knee joint swelling and synovial vasodilatation in PAR-2(+/+) mice but not PAR-2(-/-) mice. CONCLUSION: This in vivo study is the first to explore the relationship between synovial mast cells and PAR-2. Our results support the hypothesis that mast cells contribute to the pathogenesis of inflammatory arthritis through PAR-2 activation via release of mast cell tryptase.
Assuntos
Artrite Reumatoide/imunologia , Mastócitos/imunologia , Receptor PAR-2/imunologia , Receptor PAR-2/metabolismo , Membrana Sinovial/imunologia , Animais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Degranulação Celular/imunologia , Edema/imunologia , Edema/metabolismo , Edema/patologia , Hiperemia/imunologia , Hiperemia/metabolismo , Hiperemia/patologia , Imuno-Histoquímica , Articulação do Joelho/irrigação sanguínea , Articulação do Joelho/imunologia , Articulação do Joelho/patologia , Fluxometria por Laser-Doppler , Mastócitos/enzimologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptor PAR-2/genética , Membrana Sinovial/patologia , Triptases/metabolismoRESUMO
BACKGROUND AND PURPOSE: Up-regulation of proteinase-activated receptor-2 (PAR2) is a factor in a number of disease states and we have therefore examined the signalling pathways involved in the expression of the receptor. EXPERIMENTAL APPROACH: We investigated the effects of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), trypsin and the PAR2 activating peptide, 2-furoyl(2f)-LIGKV-OH on both mRNA and functional expression of PAR2 in human umbilical vein endothelial cells (HUVECs). The effect of specific chemical inhibitors and dominant negative adenovirus constructs of the mitogen-activated protein kinase (MAPK) cascade and the nuclear factor kappa B (NF-kappaB) signalling pathway was assessed. Methods included semi-quantitative and quantitative RT-PCR, [(3)H]inositol phosphate (IP) accumulation and Ca(2+)-dependent fluorescence. KEY RESULTS: The above agonists induced both mRNA and functional expression of PAR2; PAR4 mRNA, but not that for PAR1 or PAR-3, also increased following TNFalpha treatment. Inhibition of p38 MAP kinase reduced PAR2 and PAR4 expression, whilst inhibition of MEK1/ERK/JNK was without effect. A similar dependency upon p38 MAP kinase was observed for the expression of PAR4. TNFalpha -induced enhancement of PAR2 stimulated [(3)H]-inositol phosphate accumulation (IP) and Ca(2+) signalling was abolished following SB203580 pre-treatment. Infection with adenovirus encoding dominant-negative IKKbeta (Ad.IKKbeta(+/-)) and to a lesser extent dominant-negative IKKalpha (Ad.IKKalpha(+/-)), substantially reduced both control and IL-1beta- induced expression of both PAR2 and PAR4 mRNA and enhancement of PAR2-stimulated IP accumulation and Ca(2+) mobilisation. CONCLUSIONS AND IMPLICATIONS: These data reveal for the first time the signalling events involved in the upregulation of both PAR2 and PAR4 during pro-inflammatory challenge.
Assuntos
Citocinas/fisiologia , Células Endoteliais/metabolismo , Quinase I-kappa B/metabolismo , Sistema de Sinalização das MAP Quinases , Receptor PAR-2/biossíntese , Receptores de Trombina/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adenoviridae/genética , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Genes Dominantes , Humanos , Quinase I-kappa B/genética , Imidazóis/farmacologia , Fosfatos de Inositol/metabolismo , Interleucina-1beta/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Oligopeptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA Mensageiro/biossíntese , Receptor PAR-2/genética , Receptores de Trombina/genética , Fatores de Tempo , Tripsina/farmacologia , Fator de Necrose Tumoral alfa/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: In this study we examined the effect of the natural product cardamonin, upon lipopolysaccharide (LPS)-induced inflammatory gene expression in order to attempt to pinpoint the mechanism of action. EXPERIMENTAL APPROACHES: Cardamonin was isolated from the Greek plant A. absinthium L. Its effects were assessed on LPS-induced nitrite release and iNOS and COX-2 protein expression in two macrophage cell lines. Western blotting was used to investigate its effects on phosphorylation of the mitogen activated protein (MAP) kinases, ERK, JNK and p38 MAP kinase, and activation of the NFkappaB pathway, at the level of IkappaBalpha degradation and phosphorylation of NFkappaB. Also its effects on NFkappaB and GAS/GAF-DNA binding were assessed by EMSA. KEY RESULTS: Cardamonin concentration-dependently inhibited both NO release and iNOS expression but had no effect on COX-2 expression. It did not affect phosphorylation of the MAP kinases, degradation of IkappaBalpha or phosphorylation of NFkappaB. However, it inhibited NFkappaB DNA-binding in both LPS-stimulated cells and nuclear extracts of the cells (in vitro). It also inhibited IFNgamma-stimulated iNOS induction and GAS/GAF-DNA binding. CONCLUSIONS AND IMPLICATIONS: These results show that the inhibitory effect of cardamonin on LPS-induced iNOS induction is not mediated via effects on the initial activation of the NFkappaB or MAP kinase pathways but is due to a direct effect on transcription factor binding to DNA. However, although some selectivity in cardamonin's action is implicated by its inability to affect COX-2 expression, its exact mechanism(s) of action has yet to be identified.
Assuntos
Chalconas/fisiologia , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Humanos , Inflamação/metabolismo , Macrófagos/enzimologia , Macrófagos/imunologia , Camundongos , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossínteseRESUMO
1. In this study we examined the signalling events that regulate lipopolysaccharide (LPS)-stimulated induction of interferon regulatory factor (IRF)-1 in human umbilical vein endothelial cells (HUVECs). 2. LPS stimulated a time- and concentration-dependent increase in IRF-1 protein expression, an effect that was mimicked by the cytokine, tumour necrosis factor (TNF)-alpha. 3. LPS stimulated a rapid increase in nuclear factor kappa B (NFkappaB) DNA-binding activity. Pre-incubation with the NFkappaB pathway inhibitors, N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK) or pyrrolidine dithiocarbamate (PDTC), or infection with adenovirus encoding IkappaBalpha, blocked both IRF-1 induction and NFkappaB DNA-binding activity. 4. LPS and TNFalpha also stimulated a rapid activation of gamma interferon activation site/gamma interferon activation factor (GAS/GAF) DNA-binding in HUVECs. Preincubation with the Janus kinase (JAK)-2 inhibitor, AG490 blocked LPS-stimulated IRF-1 induction but did not affect GAS/GAF DNA-binding. 5. Preincubation with TLCK, PDTC or infection with IkappaBalpha adenovirus abolished LPS-stimulated GAS/GAF DNA-binding. 6. Incubation of nuclear extracts with antibodies to RelA/p50 supershifted GAS/GAF DNA-binding demonstrating the involvement of NFkappaB isoforms in the formation of the GAS/GAF complex. 7. These studies show that NFkappaB plays an important role in the regulation of IRF-1 induction in HUVECs. This is in part due to the interaction of NFkappaB isoforms with the GAS/GAF complex either directly or via an intermediate protein.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/metabolismo , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Fosfoproteínas/biossíntese , Prolina/análogos & derivados , Fatores de Transcrição/metabolismo , Adenoviridae/genética , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Vetores Genéticos , Humanos , Recém-Nascido , Fator Regulador 1 de Interferon , Fator Gênico 3 Estimulado por Interferon , Interferon gama/metabolismo , NF-kappa B/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Prolina/farmacologia , Isoformas de Proteínas/metabolismo , Tiocarbamatos/farmacologia , Fatores de Tempo , Tosilina Clorometil Cetona/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Tirfostinas/farmacologia , Veias Umbilicais/citologiaRESUMO
1. In rat aortic smooth muscle cells (RASMC), exposure to lipopolysaccharide (LPS) resulted in NF-kappaB-DNA binding, degradation of IkappaB-alpha, -beta and -epsilon and increased activity of both alpha and beta isoforms of inhibitory kappa B kinase (IKK). 2. Expression of dominant-negative (DN)-IKK-alpha, IKK-beta and NF-kappaB-inducing kinase (NIK) abolished LPS-stimulated NF-kappaB reporter activity, suggesting that activation of a NIK/IKK-dependent pathway is indispensable for NF-kappaB activation by LPS in this cell type. 3. The tyrosine phosphatase inhibitor, pervanadate, abolished LPS-stimulated NF-kappaB-DNA-binding activity. However, the effect of pervanadate was shown to be mediated by excess hydrogen peroxide (H(2)O(2)) present in the reaction mix. Preincubation of RASMC with H(2)O(2) inhibited LPS-stimulated IKK kinase activity and downstream NF-kappaB-DNA binding activity. 4. H(2)O(2) also strongly stimulated p38 MAP kinase activity in RASMCs. Effective inhibition of this pathway using SB203580 did not reverse the effects of H(2)O(2) on LPS-stimulated IKK/NF-kappaB signalling. 5. These studies show that hydrogen peroxide-mediated inhibition of LPS-stimulated NF-kappaB activation in RASMC occurs upstream of IKK. The inhibitory effect of H(2)O(2) is not due to tyrosine phosphatase inhibition, it is mediated by H(2)O(2) through a mechanism which is independent of any cross-talk involving MAP kinase homologues.
Assuntos
Peróxido de Hidrogênio/farmacologia , Lipopolissacarídeos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Oxidantes/farmacologia , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/enzimologia , Células Cultivadas , DNA/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Quinase I-kappa B , Proteínas Quinases JNK Ativadas por Mitógeno , Luciferases/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , NF-kappa B/efeitos dos fármacos , NF-kappa B/genética , NF-kappa B/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Vanadatos/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
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 Gö6983, 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, Gö6983 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.
Assuntos
Queratinócitos/enzimologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Trombina/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA , Ativação Enzimática , Humanos , Quinase I-kappa B , Receptor PAR-2 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Proteinase-activated receptors are a recently described, novel family of seven-transmembrane G-protein-coupled receptors. Rather then being stimulated through ligand receptor occupancy, activation is initiated by cleavage of the N terminus of the receptor by a serine protease resulting in the generation of a new tethered ligand that interacts with the receptor within extracellular loop-2. To date, four proteinase-activated receptors (PARs) have been identified, with distinct N-terminal cleavage sites and tethered ligand pharmacology. In addition to the progress in the generation of PAR-1 antagonists, we describe the role of thrombin in such processes as wound healing and the evidence implicating PAR-1 in vascular disorders and cancer. We also identify advances in the understanding of PAR-1-mediated intracellular signaling and receptor desensitization. The cellular functions, signaling events, and desensitization processes involved in PAR-2 activation are also assessed. However, other major aspects of PAR-2 are highlighted, in particular the ability of several serine protease enzymes, in addition to trypsin, to function as activators of PAR-2. The likely physiological and pathophysiological roles for PAR-2 in skin, intestine, blood vessels, and the peripheral nervous system are considered in the context of PAR-2 activation by multiple serine proteases. The recent discovery of PAR-3 and PAR-4 as additional thrombin-sensitive PARs further highlights the complexity in assessing the effects of thrombin in several different systems, an issue that remains to be fully addressed. These discoveries have also highlighted possible PAR-PAR interactions at both functional and molecular levels. The future identification of other PARs and their modes of activation are an important future direction for this expanding field of study.
Assuntos
Sistema Cardiovascular/metabolismo , Receptores de Trombina , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Humanos , Fosforilação , Fosfotransferases/metabolismo , Agregação Plaquetária , Receptor PAR-1 , Receptores de Trombina/metabolismo , Receptores de Trombina/fisiologia , Transdução de SinaisRESUMO
We have investigated the termination of agonist-stimulated mitogen-activated protein (MAP) kinase activity in EAhy926 cells by MAP kinase phosphatase-2 (MKP-2). In cells expressing either wild-type (WT) or catalytically inactive (CI)-MKP-2, there was no significant differences in TNFalpha-stimulated JNK or p38 MAP kinase activity, however hydrogen peroxide (H2O2)-stimulated JNK activity was substantially reduced in WT-MKP-2 expressing clones and enhanced in cells expressing CI-MKP-2. Consistent with these findings, we observed substantial nuclear translocation of JNK occurred in response to H2O2 but not TNFalpha. Using a phosphospecific anti-JNK antibody, we found that TNFalpha-stimulated JNK activity was associated principally with the cytosol while in response to H2O2, JNK activity was found within the nucleus. These results show that the role of MKP-2 in terminating JNK activity is determined by the translocation of JNK to the nucleus, which is under agonist-specific regulation and not a universal cellular response to stimulation.
Assuntos
Núcleo Celular/enzimologia , Endotélio/citologia , Endotélio/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Anticorpos Anti-Idiotípicos/imunologia , Fosfatases de Especificidade Dupla , Humanos , Peróxido de Hidrogênio/agonistas , Peróxido de Hidrogênio/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Fosfatases da Proteína Quinase Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/química , Fosforilação , Proteína Fosfatase 2 , Transporte Proteico/fisiologia , Especificidade por Substrato , Fator de Necrose Tumoral alfa/agonistas , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF-kappaB (kidney), expression of iNOS and COX-2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF-kappaB, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF-kappaB to DNA and the degradation of IkappaBalpha, IkappaBbeta, and IkappaBvarepsilon. Selective inhibition of iNOS activity with L-NIL reduced the circulatory failure and liver injury, while selective inhibition of COX-2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF-kappaB and thus prevention of the expression of NFkappaB-dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX-2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.
Assuntos
Glicoproteínas/farmacologia , NF-kappa B/metabolismo , Choque Hemorrágico/metabolismo , Choque Hemorrágico/patologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Ciclo-Oxigenase 2 , DNA/genética , DNA/metabolismo , Glicoproteínas/uso terapêutico , Frequência Cardíaca/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/patologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Rim/enzimologia , Rim/metabolismo , Rim/patologia , Lipase/sangue , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Fígado/patologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/metabolismo , Miocárdio/enzimologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Pâncreas/metabolismo , Pâncreas/patologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Ligação Proteica/efeitos dos fármacos , Ratos , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Ressuscitação , Choque Hemorrágico/enzimologia , Choque Hemorrágico/prevenção & controle , SíndromeRESUMO
In the EAhy926 endothelial cell line, UTP, ATP, and forskolin, but not UDP and epidermal growth factor, inhibited tumor necrosis factor alpha (TNFalpha)- and sorbitol stimulation of the stress-activated protein kinases, JNK, and p38 mitogen-activated protein (MAP) kinase, and MAPKAP kinase-2, the downstream target of p38 MAP kinase. In NCT2544 keratinocytes, UTP and a proteinase-activated receptor-2 agonist caused similar inhibition, but in 13121N1 cells, transfected with the human P2Y(2) or P2Y(4) receptor, UTP stimulated JNK and p38 MAP kinase activities. This suggests that the effects mediated by P2Y receptors are cell-specific. The inhibitory effects of UTP were not due to induction of MAP kinase phosphatase-1, but were manifest upstream in the pathway at the level of MEK-4. The inhibitory effect of UTP was insensitive to the MEK-1 inhibitor PD 098059, changes in intracellular Ca(2+) levels, or pertussis toxin. Acute phorbol 12-myristate 13-acetate pretreatment also inhibited TNFalpha-stimulated SAP kinase activity, while chronic pretreatment reversed the effects of UTP. Furthermore, the protein kinase C inhibitors Ro318220 and Go6983 reversed the inhibitory action of UTP, but GF109203X was ineffective. These results indicate a novel mechanism of cross-talk regulation between P2Y receptors and TNFalpha-stimulated SAP kinase pathways in endothelial cells, mediated by Ca(2+)-independent isoforms of protein kinase C.
Assuntos
Endotélio Vascular/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Receptores Purinérgicos P2/fisiologia , Transdução de Sinais/fisiologia , Linhagem Celular , Humanos , Sistema de Sinalização das MAP Quinases , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Lipocortin 1 (annexin 1) is a calcium- and phospholipid-binding protein that modulates anti-inflammatory responses including those induced by lipopolysaccharide. To investigate the precise role of lipocortin 1 in regulating the lipopolysaccharide-induced signal transduction pathways, we generated stable RAW 264.7 macrophage cell lines expressing decreased and increased lipocortin 1 protein. Several RAW 264.7 clones with increased lipocortin 1 protein levels showed constitutive activation of the mitogen-activated protein kinase extracellular signal-regulated kinase, which was down-regulated following lipopolysaccharide treatment. Conversely, clones with decreased lipocortin 1 protein expression showed prolonged extracellular signal-regulated kinase activity, following lipopolysaccharide activation. Lipocortin 1 specifically regulates the components of the extracellular signal-regulated kinase pathway, since changes in lipocortin 1 protein expression had no affect on the related mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Lipocortin 1 modulated upstream components of the extracellular signal-regulated kinase pathway and associated with the adaptor protein growth factor binding protein. The downstream consequences of altered extracellular signal-regulated kinase activity were independent of the proinflammatory transcription factor nuclear factor kappa B. These data indicate that lipocortin 1 specifically regulates proximal signaling components of the extracellular signal-regulated kinase signal transduction pathway, resulting in the modulation of biochemical functions in RAW macrophages.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Anexina A1/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Sequência de Bases , Transporte Biológico , Linhagem Celular , Primers do DNA , Ativação Enzimática , Proteína Adaptadora GRB2 , Lipopolissacarídeos/farmacologia , Camundongos , NF-kappa B/metabolismo , Fosforilação , Proteínas/metabolismoRESUMO
In RAW 264.7 macrophages lipopolysaccharide (LPS) stimulated the activation of p42 and p44 MAP kinases and their upstream activator mitogen-activated protein (MAP) kinase kinase (MAPKK), and induced the 69-kDa isoform of cyclo-oxygenase-2 (COX-2) and the 130-kDa isoform of nitric oxide synthase (iNOS). PD 098059, a specific inhibitor of the activation of MAPKK, prevented LPS-mediated activation of MAPKK (IC50 = 3.0 +/- 0.1 microM, n = 3) and p42/44 MAP kinases and substantially reduced the induction of COX-2 by approximately 40%-70%, but was without effect upon the induction of iNOS. In parallel, LPS also stimulated the activation of p38 MAP kinase and the MAPKAP kinase-2, a downstream target of p38 MAP kinase. SB 203580, a specific inhibitor of p38 MAP kinase prevented the activation of p38 MAP kinase (IC50 = 3.3 +/- 1.4 microM, n = 3) and MAPKAP kinase-2 by LPS and reduced the induction of COX-2 by approximately 50-90%, with no significant effect upon iNOS expression. These studies indicate the involvement of both the classical p42/44 MAP kinases and p38 MAP kinase in the regulation of COX-2 but not iNOS induction following exposure to LPS.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Isoenzimas/biossíntese , Macrófagos/enzimologia , Proteínas Quinases Ativadas por Mitógeno , Óxido Nítrico Sintase/biossíntese , Prostaglandina-Endoperóxido Sintases/biossíntese , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Linhagem Celular , Ciclo-Oxigenase 2 , Ativação Enzimática , Indução Enzimática , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno , Óxido Nítrico Sintase Tipo II , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Hypertrophy and hyperplasia of airway smooth muscle (ASM) are important pathological features that contribute to airflow obstruction in chronic severe asthma. Despite considerable research effort, the cellular mechanisms that modulate ASM growth remain unknown. Recent evidence suggests that mitogen-induced activation of phosphoinositide (PI)-specific phospholipase C (PLC) and PI-dependent calcium mobilization are neither sufficient nor necessary to stimulate human ASM proliferation. In this study, we identify phosphatidylinositol (PtdIns) 3-kinase as a key regulator of human ASM proliferation. Pretreatment of human ASM with the PtdIns 3-kinase inhibitors wortmannin and LY-294002 significantly reduced thrombin- and epidermal growth factor (EGF)-induced DNA synthesis (IC(50) approximately 10 nM and approximately 3 microM, respectively). In separate experiments, wortmannin and LY-294002 markedly inhibited PtdIns 3-kinase and 70-kDa S6 protein kinase (pp70(S6k)) activation induced by stimulation of human ASM cells with EGF and thrombin but had no effect on EGF- and thrombin-induced p42/p44 mitogen-activated protein kinase (MAPK) activation. The specificity of wortmannin and LY-294002 was further suggested by the demonstrated inability of these compounds to alter thrombin-induced calcium transients, total PI hydrolysis, or basal cAMP levels. Transient expression of constitutively active PtdIns 3-kinase (p110*) activated pp70(S6k), whereas a dominant-negative PtdIns 3-kinase (Deltap85) blocked EGF- and thrombin-stimulated pp70(S6k) activity. Collectively, these data suggest that activation of PtdIns 3-kinase is required for the mitogenic effect of EGF and thrombin in human ASM cells. Further investigation of the role of PtdIns 3-kinase may offer new therapeutic approaches in the treatment of diseases characterized by smooth muscle cell hyperplasia such as asthma and chronic bronchitis.
Assuntos
Mitógenos/farmacologia , Músculo Liso/citologia , Fosfatidilinositol 3-Quinases/fisiologia , Traqueia/citologia , Androstadienos/farmacologia , Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Cromonas/farmacologia , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Humanos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Quinases S6 Ribossômicas/metabolismo , Trombina/farmacologia , WortmaninaRESUMO
When pulmonary hypertension occurs in the face of hypoxia there is remodeling of all three layers of the pulmonary vessels, but in particular, there is an increase in number of adventitial fibroblasts. Hypoxia causes vasoconstriction in the pulmonary circulation and vasodilation in the systemic circulation. We hypothesized that there are fundamental differences in oxygen sensing and cell signaling between systemic and pulmonary artery cells in response to hypoxia. Here, we determined the effect of hypoxia either alone or in combination with known growth factors such as serum, endothelin-1 (ET-1), and platelet-derived growth factor (PDGF) on the proliferative responses of bovine pulmonary artery and mesenteric artery fibroblasts. Fibroblasts were obtained from primary cultures. Growth was assessed by [3H]thymidine incorporation. Inositol 1,4,5-triphosphate (IP3) generation was measured using a competitive binding assay. Hypoxia alone increased proliferation of pulmonary artery fibroblasts (611 +/- 24%), but not in those from the mesentery. Furthermore, hypoxia had the effect of increasing the replicative response of pulmonary fibroblasts to serum and PDGF, but no change was observed in the mesenteric cells. ET-1 had no effect on growth of either cell type. PDGF gave rise to a significant elevation in IP3 production under hypoxic conditions in the pulmonary artery cells (234%), but not in the mesenteric cells. ET-1 caused no change in IP3 production in any cell type. These data suggest that hypoxia sensitizes pulmonary artery fibroblasts to the proliferative effect of mitogens through a pathway that is not present, or is present but repressed, in the mesenteric cells.
Assuntos
Fibroblastos/patologia , Hipóxia/patologia , Inositol 1,4,5-Trifosfato/biossíntese , Artérias Mesentéricas/patologia , Artéria Pulmonar/patologia , Análise de Variância , Animais , Sangue , Bovinos , Comunicação Celular , Divisão Celular , Células Cultivadas , Tecido Elástico/metabolismo , Tecido Elástico/patologia , Endotelina-1/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Mitógenos/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Compostos Radiofarmacêuticos , Timidina/metabolismo , Trítio , Vasoconstrição/fisiologia , Vasodilatação/fisiologiaRESUMO
In this study, we examined the ability of tumour necrosis factor-alpha (TNF) to stimulate the mitogen-activated protein (MAP) kinase homologues p42/44 MAP kinase, c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase and its effect upon DNA synthesis in primary cultures of bovine aortic endothelial cells (BAECs). TNF strongly stimulated p38 MAP kinase and JNK activity in both a time- and concentration-dependent manner. By contrast, TNF was a very poor activator of p42/44 MAP kinase relative to the known activator of p42/44 MAP kinase in endothelial cells, adenosine triphosphate (ATP). TNF-stimulated activation of p38 MAP kinase, and MAPKAP kinase-2, a known downstream target of p38 MAP kinase, was strongly inhibited by pre-incubation with the p38 MAP kinase inhibitor SB203580, whereas the minor activation of p42/44 MAP kinase was abolished by pre-incubation of the cell with the novel MAP kinase kinase 1 inhibitor PD098059. Addition of TNF resulted in a 50-60% decrease in DNA synthesis in BAECs. Pre-incubation with PD098059 or co-incubation with ATP failed to modify the inhibitory effect of TNF upon DNA synthesis. SB203580 reduced basal DNA synthesis by approximately 50%; however, if failed to modify the inhibition mediated by TNF. These results indicate that TNF strongly activates both p38 MAP kinase, JNK and, to a minor extent, p42/p44 MAP kinase. It is likely that only one of these kinases, JNK, plays a role in the regulation of DNA synthesis in these cells.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Replicação do DNA/efeitos dos fármacos , Endotélio Vascular/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Fator de Necrose Tumoral alfa/farmacologia , Animais , Aorta , Bovinos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Pulmonary hypertension in response to chronic hypoxia is invariably accompanied by remodeling of the pulmonary vessels but the mechanism by which hypoxia increases the replication of vascular cells is unknown. To investigate the hypothesis that hypoxia stimulates intracellular kinase cascades we measured the activity of "classic" mitogen-activated protein (MAP) kinase pathways and "stress- activated" MAP kinase pathways in bovine pulmonary artery fibroblasts subjected to hypoxia for up to 30 h. Hypoxia (1% O2) stimulated strongly the stress-activated protein kinases, c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase. Two peaks of p38 MAP kinase activity at 6 and 24 h were associated with an increase in the activity of mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2, the immediate downstream target of p38 MAP kinase. Furthermore, the second phase of p38 MAP kinase activity could be reversed if cells were reoxygenated after 12 h. These data suggest that hypoxic stimulation of pulmonary artery cells is mediated by activation of the stress-activated protein kinases.