RESUMO
In our continuing search for novel cancer chemopreventive compounds of natural and synthetic origin, we have evaluated 14 commonly used ultraviolet (UV) sunscreen agents (designated UV-1 to UV-14) for their skin cancer chemoprevention potential. They belong to 8 different chemical categories: aminobenzoate (UV-5, UV-7, UV-8 and UV-14), benzophenone (UV-1, UV-2, UV-3 and UV-13), benzotriazole (UV-10), benzyloxyphenol (UV-9), cinnamate (UV-6), quinolone (UV-4), salicylate (UV-11) and xanthone (UV-12). In the in vitro assay employed, the sunscreens were assessed by their inhibition of the Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in human lymphoblastoid Raji cells. All sunscreens tested were found to exhibit anti-tumour promoting activity: listed in decreasing order, moderate (UV-11, UV-2, UV-7, UV-12, UV-3, UV-9 and UV-14) to weak (UV-1, UV-6, UV-8, UV-16, UV-5, UV-4 and UV-10) with octyl salicylate (UV-11) as the most potent and drometrizole (UV-10) as the least potent among the compounds evaluated. A plausible relationship between the antioxidant property of sunscreens and their ability to promote anti-tumour activity was noted. The results call for a comprehensive analysis of skin cancer chemoprevention potential of currently used UV sunscreen agents around the globe to identify those with the best clinical profile.
Assuntos
Antígenos Virais/imunologia , Neoplasias Cutâneas/prevenção & controle , Protetores Solares/uso terapêutico , Carcinógenos/toxicidade , Humanos , Técnicas In Vitro , Neoplasias Cutâneas/induzido quimicamente , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
Lipoproteins in Escherichia coli are anchored to the periplasmic side of either the inner or the outer membrane by a lipid moiety that is covalently attached to the amino-terminal cysteine residue. Membrane specificity depends on a sorting signal at position 2 of the lipoprotein. Lipoproteins directed to the outer membrane are released from the inner membrane in an ATP-dependent manner through the formation of a complex with LolA, a periplasmic chaperone. However, the ATPase involved in this reaction has not been identified. Here we show, using reconstituted proteoliposomes, that a new complex, LolCDE, belonging to the ATP-binding cassette (ABC) transporter family, catalyses the release of lipoproteins in LolA- and sorting-signal-dependent manners. The LolCDE complex differs mechanistically from all other ABC transporters as it is not involved in the transmembrane transport of substrates. This new mechanism is evolutionarily conserved in other gram-negative bacteria.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Lipoproteínas/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/metabolismo , Expressão Gênica/fisiologia , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Dados de Sequência Molecular , Mutagênese/fisiologia , Plasmídeos , Homologia de Sequência de AminoácidosRESUMO
UNLABELLED: A 60-year-old postmenopausal woman diagnosed as primary osteoporosis began to take raloxifene. The spontaneous microaggregates of platelets induced by shear stress were accelerated after the treatment, concomitant with the significant upregulation of p44/p42 mitogen-activated protein (MAP) kinase induced by adenosine diphosphate (ADP). After the cessation of raloxifene, the spontaneous microaggregates of platelets and the acceleration of ADP-induced p44/p42 MAP kinase phosphorylation was diminished. We concluded that raloxifene caused platelet hyperaggregability to shear stress and p44/p42 MAP kinase was involved in the pathological state. INTRODUCTION: A 60-year-old postmenopausal woman suffering from severe lumbago was diagnosed as primary osteoporosis with combined vertebral fractures. After the acute phase, she began to take 60 mg daily of oral raloxifene. The spontaneous microaggregates of platelets induced by shear stress were accelerated significantly after 8 weeks from the beginning of raloxifene treatment and observed at 12 weeks. RESULTS: The platelet aggregation induced by ADP was little changed; however, low doses (0.3 and 1 microM) of ADP significantly induced the phosphorylation of p44/p42 MAP kinase in the platelets obtained at 12 weeks. Although there were few subjective complaints except for paroxysmal headache, the medication was stopped with her consent to avoid any adverse effects. The spontaneous microaggregates of platelets gradually decreased after the cessation of medication. At 12 weeks after the cessation, the phosphorylation of p44/p42 MAP kinase induced by low doses of ADP was no more observed. CONCLUSION: These results strongly suggest that raloxifene caused platelet hyperaggregability to shear stress and subclinical thrombus formation in this case and that p44/p42 MAP kinase was involved in the pathological state.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/sangue , Proteína Quinase 3 Ativada por Mitógeno/sangue , Agregação Plaquetária/efeitos dos fármacos , Cloridrato de Raloxifeno/farmacologia , Plaquetas/enzimologia , Células Cultivadas , Feminino , Humanos , Pessoa de Meia-Idade , Osteoporose Pós-Menopausa/sangue , Osteoporose Pós-Menopausa/tratamento farmacológico , Agregação Plaquetária/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: The aim of this study is to understand whether the responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis to stress increases excessively with aging in senescence-accelerated mice-prone 10 (SAMP10) and to investigate the role of arachidonic acid (ARA) in this process. MATERIALS AND METHODS: The area under the curve of CORT concentration (CORT-AUC), an index of the HPA axis responsiveness to stress, was assessed in SAMP10 subjected to a 30-minute restraint stress up to 120 minutes after the restraint stress onset. Furthermore, the HPA axis responsiveness was evaluated in aged SAMP10 fed 0.4% ARA-containing diet (ARA group) or control diet (CON group) for 4 weeks. Three weeks later, these mice were divided into a group with a 30-minute restraint stress (CON-S or ARA-S group) and a group without restraint stress (CON-NS or ARA-NS group). Hippocampi were collected after stress release and fatty acid and glucocorticoid receptor (GR) protein levels were evaluated in the nucleus and cytosol. RESULTS: The CORT-AUC of aged SAMP10 was 21% significantly higher than that of young SAMP10. In the ARA group, hippocampal ARA was 0.5% significantly higher than that in the CON group. CORT-AUC in the ARA group was 24% significantly lower than that in the CON group. The ratio of GR protein levels in the nucleus and cytosol in the ARA-S group was 1.72 times significantly higher than that in the ARA-NS group but no difference was observed between the CON-S and CON-NS groups. CONCLUSIONS: Dietary ARA seems to suppress age-related excessive enhancement of the HPA axis responsiveness via attenuation of age-related decline in hippocampal GR translocation into the nucleus after stress loading, which may contribute to an improvement of mental health.
Assuntos
Envelhecimento/efeitos dos fármacos , Ácido Araquidônico/farmacologia , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Ácido Araquidônico/administração & dosagem , Suplementos Nutricionais , Sistema Hipotálamo-Hipofisário/metabolismo , Camundongos , Sistema Hipófise-Suprarrenal/metabolismoRESUMO
Pheochromocytomas are tumors that may produce a variety of substances in addition to catecholamines. To date, among several cases of systemic inflammatory syndrome associated with interleukin-6 (IL-6) secretion, IL-6-producing pheochromocytomas, have been reported. However, the mechanism underlying IL-6 oversecretion in these cases has not yet been clarified. This report describes a patient with pheochromocytoma who exhibited pyrexia and marked inflammatory signs including C-reactive protein elevation. The inflammatory symptoms were easily controlled by the administration of loxoprofen, a nonsteroidal anti-inflammatory drug. The plasma concentration of IL-6 and 11-d-TXB(2), a stable metabolite of thromboxane A(2) (TXA(2)), were significantly elevated in parallel with an elevation of norepinephrine in the samples obtained by selective venous sampling. A left adrenalectomy was performed, and the acute inflammatory symptoms naturally diminished without loxoprofen. Cultured tumor cells obtained from the resected specimen spontaneously released IL-6, and indomethacin inhibited the IL-6 release. According to a cDNA microarray analysis, mRNA of protein kinase C-delta (PKC-delta), prostaglandin D synthase, and arachidonate release-relating enzymes were significantly overexpressed in the tumor tissue in comparison to the adjacent nontumor tissue. The constitutive phosphorylation of PKC-delta was observed in the tumor tissue. These results strongly suggest that the systemic inflammatory syndrome in IL-6-producing pheochromocytoma, at least in part, is caused by the overexpression of PKC-delta, resulting in an excess of arachidonate derivatives such as prostaglandins.
Assuntos
Expressão Gênica , Interleucina-6/sangue , Feocromocitoma/genética , Feocromocitoma/imunologia , Proteína Quinase C-delta/genética , Idoso , Feminino , Humanos , Interleucina-6/imunologia , Feocromocitoma/sangue , Feocromocitoma/cirurgia , Proteína Quinase C-delta/imunologia , Células Tumorais CultivadasRESUMO
We previously showed that tumor necrosis factor-alpha (TNF-alpha) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt) is involved in TNF-alpha-stimulated IL-6 synthesis in MC3T3-E1 cells. TNF-alpha induced the phosphorylation of Akt depending upon time. Akt inhibitor, 1L-6-hydroxymethyl-CHIRO-inositol 2-( R)-2- O-methyl-3-O-octadecylcarbonate, significantly suppressed the TNF-alpha-stimulated IL-6 synthesis, but the inhibitory effect was partial. The phosphorylation of Akt induced by TNF-alpha was markedly attenuated by LY294002 and wortmannin, inhibitors of PI3-kinase. Wortmannin and LY294002 significantly reduce the TNF-alpha-induced IL-6 synthesis. On the contrary, the suppressive effects of Akt inhibitor, wortmannin or LY294002 on TNF-alpha-induced phosphorylation of p44/p42 MAP kinase were minor. PD98059, a specific inhibitor of MEK, had little effect on the TNF-alpha-induced phosphorylation of Akt. A combination of Akt inhibitor and PD98059 suppressed the TNF-alpha-induced IL-6 synthesis in an additive manner. These results strongly suggest that PI3-kinase/Akt plays a role in the TNF-alpha-stimulated IL-6 synthesis mainly independent of p44/p42 MAP kinase in osteoblasts.
Assuntos
Interleucina-6/biossíntese , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Androstadienos/farmacologia , Animais , Células Cultivadas , Cromonas/farmacologia , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Morfolinas/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sirolimo/farmacologia , WortmaninaRESUMO
Cellular retinoic acid-binding protein (cRABP) was detected in the cytosol of virus-induced papilloma (Shope) of rabbit skin. The Shope papilloma cRABP showed the same ligand specificity and sedimentation value (2S) as was found in other animal species. The level of cRABP in the papillomatous tissue was significantly higher than that in the normal rabbit skin and increased in accordance with the growth and development of the tumor, reaching a peak about 40 days after the inoculation of the Shope papilloma virus. Although this binding capacity was about 15 times greater than in the normal skin, the level of cRABP in the transplantable carcinomas Vx2 and Vx7, both originating from the virus-induced papillomas over 20 years ago, was much the same as in normal rabbit skin.
Assuntos
Proteínas de Transporte/metabolismo , Neoplasias Cutâneas/metabolismo , Tretinoína/metabolismo , Infecções Tumorais por Vírus/metabolismo , Animais , Linhagem Celular , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , Coelhos , Neoplasias Cutâneas/patologia , Fatores de Tempo , Infecções Tumorais por Vírus/patologiaRESUMO
Although beta-carotene has been considered to be a key cancer preventive agent in green and yellow vegetables, other types of carotenoids, such as alpha-carotene, may also contribute to anticarcinogenic action, since these carotenoids usually coexist with beta-carotene and are detectable in human blood and tissues. In this study, we compared the inhibitory effect of natural alpha-carotene, obtained from palm oil, with that of beta-carotene on spontaneous liver carcinogenesis in C3H/He male mice. The mean number of hepatomas per mouse was significantly decreased by alpha-carotene supplementation (per os administration in drinking water at a concentration of 0.05%, ad libitum) as compared with that in the control group (P < 0.001, Student's t test). On the other hand, beta-carotene, at the same dose as alpha-carotene, did not show any such significant difference from the control group. Furthermore, we also compared the antitumor-promoting activity of alpha-carotene with that of beta-carotene against two-stage mouse lung carcinogenesis (initiator, 4-nitroquinoline 1-oxide; promoter, glycerol). alpha-Carotene, but not beta-carotene, reduced the number of lung tumors per mouse to about 30% of that in the control group (P < 0.001, Student's t test). The higher potency of the antitumor-promoting action of alpha-carotene compared to beta-carotene was confirmed in other experimental systems; e.g., alpha-carotene was also found to have a stronger effect than beta-carotene in suppressing the promoting activity of 12-O-tetradecanoylphorbol-13-acetate on skin carcinogenesis in 7,12-dimethylbenz[a]anthracene-initiated mice. These results suggest that not only beta-carotene, but also other types of carotenoids, such as alpha-carotene, may play an important role in cancer prevention.
Assuntos
Carotenoides/uso terapêutico , Neoplasias Hepáticas/prevenção & controle , Neoplasias Pulmonares/prevenção & controle , Neoplasias Cutâneas/prevenção & controle , 4-Nitroquinolina-1-Óxido , 9,10-Dimetil-1,2-benzantraceno , Administração Oral , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Pulmonares/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C3H , Ornitina Descarboxilase/análise , Papiloma/induzido quimicamente , Papiloma/prevenção & controle , Neoplasias Cutâneas/induzido quimicamente , Organismos Livres de Patógenos Específicos , Acetato de Tetradecanoilforbol , beta CarotenoRESUMO
The intake of citrus fruits has been suggested as a way to prevent the development of some types of human cancer. Nitric oxide (NO) is closely associated with the processes of epithelial carcinogenesis. We attempted a search for NO generation inhibitors in Citrus unshiu. The active constituent was traced by an activity-guiding separation. NO and superoxide (O2-) generation was induced by a combination of lipopolysaccharide and IFN-gamma in mouse macrophage RAW 264.7 cells, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated human promyelocyte HL-60, respectively. Expression of inducible NO synthase and cyclooxygenase 2 proteins were detected by Western blotting. The in vivo anti-inflammatory and antitumor promoting activities were evaluated by topical TPA application to ICR mouse skin with measurement of edema formation, epidermal thickness, leukocyte infiltration, hydrogen peroxide production, and the rate of proliferating cell nuclear antigen-stained cells. As a result, nobiletin, a polymethoxyflavonoid, was identified as an inhibitor of both NO and O2- generation. Nobiletin significantly inhibited two distinct stages of skin inflammation induced by double TPA application [first stage priming (leukocyte infiltration) and second stage activation (oxidative insult by leukocytes)] by decreasing the inflammatory parameters. It also suppressed the expression of cyclooxygenase-2 and inducible NO synthase proteins and prostaglandin E2 release. Nobiletin inhibited dimethylbenz[a]anthracene (0.19 micromol)/TPA (1.6 nmol)-induced skin tumor formation at doses of 160 and 320 nmol by reducing the number of tumors per mouse by 61.2% (P < 0.001) and 75.7% (P < 0.001), respectively. The present study suggests that nobiletin is a functionally novel and possible chemopreventive agent in inflammation-associated tumorigenesis.
Assuntos
Anticarcinógenos/uso terapêutico , Citrus/química , Toxidermias/prevenção & controle , Flavonas , Flavonoides/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Neoplasias Cutâneas/prevenção & controle , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Antioxidantes/farmacologia , Carcinógenos , Linhagem Celular , Ciclo-Oxigenase 2 , Dinoprostona/biossíntese , Toxidermias/metabolismo , Feminino , Flavonoides/isolamento & purificação , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Humanos , Interferon gama/farmacologia , Isoenzimas/biossíntese , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos ICR , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Prostaglandina-Endoperóxido Sintases/biossíntese , Pele/efeitos dos fármacos , Pele/metabolismo , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/metabolismo , Superóxidos/antagonistas & inibidores , Superóxidos/metabolismo , Acetato de TetradecanoilforbolRESUMO
The marine bacterium, Vibrio alginolyticus, regulates the cytoplasmic pH at about 7.8 over the pH range 6.0-9.0. By the addition of diethanolamine (a membrane-permeable amine) at pH 9.0, the internal pH was alkalized and simultaneously the cellular K+ was released. Following the K+ exit, the internal pH was acidified until 7.8, where the K+ exit leveled off. The K+ exit was mediated by a K+/H+ antiporter that is driven by the outwardly directed K+ gradient and ceases to function at the internal pH of 7.8 and below. The Na+-loaded cells assayed in the absence of KCl generated inside acidic delta pH at alkaline pH due to the function of an Na+/H+ antiporter, but the internal pH was not maintained at a constant value. At acidic pH range, the addition of KCl to the external medium was necessary for the alkalization of cell interior. These results suggested that in cooperation with the K+ uptake system and H+ pumps, the K+/H+ antiporter functions as a regulator of cytoplasmic pH to maintain a constant value of 7.8 over the pH range 6.0-9.0.
Assuntos
Proteínas de Transporte/fisiologia , Concentração de Íons de Hidrogênio , Hidrogênio/metabolismo , Potássio/metabolismo , Vibrio/fisiologia , Potenciais da Membrana , Antiportadores de Potássio-HidrogênioRESUMO
Using a reconstitution system for protein translocation, the involvement of SecY in the translocation of secretory proteins across the cytoplasmic membrane of Escherichia coli was studied. Anti-SecY antibodies raised against the N- and C-terminal sequences prevented the functional reconstitution of the translocation system. Depletion of SecY from the solubilized membrane preparation was performed by treatment with anti-SecY IgG, followed by removal of IgG with protein A-agarose. The SecY-depleted preparation was inactive as to functional reconstitution. However, reconstitution with it was demonstrated in the presence of a protein fraction, which was released from the anti-SecY immunoprecipitate upon addition of the SecY fragment used to raise the antibody. Reconstitution with the SecY-depleted membrane fraction was also demonstrated in the presence of a purified SecY preparation. OmpT proteinase specifically cleaved SecY in the solubilized membrane preparation. The cleavage was accompanied by a decrease in the reconstituted activity. Based on these findings we conclude that SecY is an indispensable component of the secretory machinery.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Sequência de Aminoácidos , Anticorpos , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Transporte Biológico , Membrana Celular/metabolismo , Cinética , Peso Molecular , Canais de Translocação SEC , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismoRESUMO
Superoxide dismutase (SOD) was modified into cationized form (Cat-SOD) in order to enhance its pharmacological efficacy based on an electrostatic interaction. The inhibitory effect of Cat-SOD on superoxide anion release from inflammatory macrophages and its cellular interaction were studied in vitro. Cat-SOD exhibited an excellent inhibitory effect on superoxide anion release from the macrophages, and this effect surpassed those of native SOD and SOD modified with mannose (Man-SOD) which is taken up via mannose receptor-mediated endocytosis by macrophages. In the presence of colchicine, a microtubule-disruptive agent, the inhibitory effect of Cat-SOD was slightly impaired, whereas the effect of Man-SOD completely disappeared. The intracellular localization of fluorescein isothiocyanate-labeled SOD, Cat-SOD and Man-SOD observed by confocal laser microscopy supported the difference in their abilities to eliminate superoxide anions. The different sensitivities of Cat-SOD and Man-SOD to colchicine were also confirmed by the confocal laser microscopic images, suggesting their distinct intracellular trafficking pathways in the macrophages. In conclusion, Cat-SOD is desirable for its pharmacological activity, which is probably the result of its ability to be delivered to the vicinity of NADPH-oxidase which locates in the cell membrane and generates superoxide anions.
Assuntos
Macrófagos Peritoneais/metabolismo , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Animais , Cátions/metabolismo , Membrana Celular/metabolismo , Diaminas/metabolismo , Fluoresceína-5-Isotiocianato , Radioisótopos de Índio , Macrófagos Peritoneais/enzimologia , CamundongosRESUMO
Prostaglandin F2alpha (PGF2alpha) significantly induced p42/p44 mitogen-activated protein (MAP) kinase activity in osteoblast-like MC3T3-E1 cells. PD98059, a selective inhibitor of MAP kinase kinase, inhibited PGF2alpha-induced interleukin-6 (IL-6) synthesis as well as PGF2alpha-induced p42/p44 MAP kinase activation. PD98059 suppressed the IL-6 synthesis induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, or NaF, an activator of heterotrimeric GTP-binding protein, as well as the p42/p44 MAP kinase activation by TPA or NaF. Calphostin C, a highly potent and specific inhibitor of PKC, inhibited the PGF2alpha-induced p42/p44 MAP kinase activity. These results strongly suggest that PKC-dependent p42/p44 MAP kinase activatioin is involved in PGF2alpha-induced IL-6 synthesis in osteoblasts.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Dinoprosta/farmacologia , Interleucina-6/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Osteoblastos/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Linhagem Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno , Mitógenos/farmacologia , Osteoblastos/efeitos dos fármacos , Fluoreto de Sódio/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
In cloned osteoblast-like MC3T3-E1 cells, prostaglandin E2 (PGE2) stimulated 45Ca2+ influx even in the presence of nifedipine, a Ca2+ antagonist that inhibits voltage-dependent Ca2+ channel, in a dose-dependent manner, attaining a maximum at 0.5 microM. Dose of PGE2 above 0.5 microM caused less than maximal stimulation. While PGE2 stimulated the formation of inositol trisphosphate dose dependently in the range between 1 nM and 10 microM. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, which by itself had little effect on 45Ca2+ influx, significantly suppressed the 45Ca2+ influx induced by PGE2 in a dose-dependent manner between 1 nM and 1 microM. 4 alpha-Phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect in this capacity. Staurosporine, a PKC inhibitor, enhanced the PGE2-induced 45Ca2+ influx. On the other hand, dibutyryl cAMP had little effect on the 45Ca2+ influx induced by PGE2. Our data suggest that PGE2 regulates Ca2+ influx through self-induced activation of PKC. These results indicate that there is an autoregulatory mechanism in signal transduction by PGE2, and PGE2 modulates osteoblast functions through the interaction between Ca2+ influx and phosphoinositide hydrolysis in osteoblast-like cells.
Assuntos
Cálcio/metabolismo , Dinoprostona/farmacologia , Osteoblastos/efeitos dos fármacos , Proteína Quinase C/fisiologia , Transdução de Sinais , Animais , Diferenciação Celular , Linhagem Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , Camundongos , Osteoblastos/metabolismo , Fosfatidilinositóis/metabolismoRESUMO
We previously showed that prostaglandin (PG) E1 stimulates the synthesis of interleukin-6 (IL-6) through activation of protein kinase (PK) A in osteoblast-like MC3T3-E1 cells and that PGF2alpha induces IL-6 synthesis through PKC activation. In other studies, we demonstrated that thrombin stimulates IL-6 synthesis, which depends on intracellular Ca2+ mobilisation in these cells and that tumour necrosis factor-alpha (TNF) induces IL-6 synthesis through sphingosine 1-phosphate, a product of sphingomyelin turnover. In the present study, among sphingomyelin metabolites, we examined the effect of ceramide on the IL-6 synthesis induced by various agonists in MC3T3-E1 cells. C2-ceramide, a cell-permeable ceramide analogue, suppressed the PGE1-induced IL-6 synthesis. C2-ceramide inhibited the IL-6 synthesis induced by PGF2alpha or 12-O-tetradecanoylphorbol-13-acetate, an activator of PKC. C2-ceramide reduced the IL-6 synthesis induced by cholera toxin, forskolin or dibutyryl cAMP. C2-ceramide inhibited the IL-6 synthesis induced by thrombin. The IL-6 synthesis stimulated by thapsigargin, which is known to stimulate Ca2+ mobilisation from intracellular Ca2+ stores, or A23187, a Ca-ionophore, was also inhibited by C2-ceramide. C2-ceramide did not affect the IL-6 synthesis induced by interleukin-1. On the contrary, C2-ceramide enhanced the TNF-induced IL-6 synthesis. D,L-threo-dihydrosphingosine, an inhibitor of sphingosine kinase, inhibited the enhancement by C2-ceramide as well as the TNF-effect. These results strongly suggest that ceramide modulates the IL-6 synthesis stimulated by various agonists in osteoblasts.
Assuntos
Interleucina-6/biossíntese , Osteoblastos/efeitos dos fármacos , Esfingosina/análogos & derivados , Alprostadil/farmacologia , Animais , Bucladesina/farmacologia , Calcimicina/farmacologia , Linhagem Celular , Toxina da Cólera/farmacologia , Colforsina/farmacologia , Dinoprosta/farmacologia , Interleucina-1/farmacologia , Camundongos , Osteoblastos/metabolismo , Esfingosina/metabolismo , Esfingosina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tapsigargina/farmacologia , Trombina/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We investigated the effects of retinoic acid (RA) on the signalling pathways by prostaglandin E2 (PGE2) in osteoblast-like MC3T3-E1 cells. The pretreatment with RA significantly inhibited the formation of inositol phosphates induced by 10 microM PGE2 in a dose-dependent manner in the range between 0.1 nM and 0.1 microM, without affecting protein contents in the cultured cells. This effect of RA was dependent on the time of pretreatment up to 8 h. However, RA had little effect on the formation of inositol phosphates induced by NaF, a GTP-binding protein activator. On the other hand, RA significantly inhibited PGE2-induced cAMP accumulation in a dose-dependent manner between 0.1 nM and 0.1 microM. This effect of RA was dependent on the time of pretreatment up to 8 h. RA also inhibited the cAMP accumulation induced by NaF or forskolin which directly activates adenylate cyclase. These results strongly suggest that RA modulates the signalling by PGE2 in osteoblast-like cells as follows: the inhibitory effect on the phosphoinositide hydrolysis is exerted at the point between PGE2 receptor and GTP-binding protein, and the inhibitory effect on the cAMP production is exerted at a point downstream from adenylate cyclase.
Assuntos
Dinoprostona/farmacologia , Fosfatos de Inositol/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tretinoína/farmacologia , Células 3T3 , Animais , Células Clonais , Colforsina/farmacologia , AMP Cíclico/metabolismo , Cinética , Camundongos , Osteoblastos/efeitos dos fármacos , Fluoreto de Sódio/farmacologiaRESUMO
We previously reported that endothelin-1 (ET)-1 stimulates phospholipase D (PLD) independently of phosphoinositide hydrolysis in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of ET-1 on the secretion of interleukin-6 (IL-6) and the involvement of protein kinase C (PKC) activation in the IL-6 secretion in these cells. ET-1 significantly stimulated IL-6 secretion time-dependently up to 72 h. The stimulative effect was dose-dependent in the range between 1 nM and 1 microM. BQ123, a selective antagonist of endothelinA (ETA) receptor, inhibited the ET-1-induced IL-6 secretion. On the contrary, BQ788, a selective antagonist of endothelinB (ETB) receptor, had no effect. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, significantly stimulated IL-6 secretion. However, 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, did not affect IL-6 secretion. The effect of a combination of ET-1 and TPA on IL-6 secretion was not additive. Calphostin C, a specific PKC inhibitor, significantly inhibited the ET-1-induced IL-6 secretion. Both ET-1- and TPA-induced IL-6 secretion were reduced in PKC downregulated MC3T3-E1 cells. These results strongly suggest that ET-1 stimulates IL-6 secretion via ETA receptor in osteoblast-like cells and that PKC activation is involved in the ET-1-induced IL-6 secretion.
Assuntos
Endotelina-1/fisiologia , Interleucina-6/metabolismo , Osteoblastos/enzimologia , Osteoblastos/metabolismo , Proteína Quinase C/metabolismo , Animais , Linhagem Celular , Regulação para Baixo , Antagonistas dos Receptores de Endotelina , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Camundongos , Naftalenos/farmacologia , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Piperidinas/farmacologia , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/metabolismoRESUMO
We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O-aminophinoxy)-ethane-N,N,N,N-tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Linfocinas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoblastos/metabolismo , Animais , Calcimicina/farmacologia , Cálcio/metabolismo , Inibidores Enzimáticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Flavonoides/farmacologia , Imidazóis/farmacologia , Técnicas In Vitro , Ionóforos/farmacologia , Linfocinas/efeitos dos fármacos , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Tapsigargina/farmacologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
We previously showed that sphingosine 1-phosphate acts as a second messenger for tumor necrosis factor alpha-induced interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells and that the synthesis by sphingosine 1-phosphate is dependent on p42/p44 mitogen-activated protein (MAP) kinase activation. In the present study, we investigated the effect of sphingosine 1-phosphate on the induction of heat shock protein 27 (HSP27) in MC3T3-E1 cells. Not C2-ceramide, but sphingosine and sphingosine 1-phosphate significantly induced HSP27 accumulation dose dependently in the range between 1microM and 30 microM. DL-threo-dihydrosphingosine, an inhibitor of sphingosine kinase, markedly inhibited the sphingosine-induced HSP27 accumulation. Sphingosine 1-phosphate induced increase in the levels of the mRNA for HSP27. Sphingosine 1-phosphate stimulated the phosphorylation of p38 MAP kinase. The sphingosine 1-phosphate-induced HSP27 accumulation was dose dependently suppressed by SB203580, an inhibitor of p38 MAP kinase, but not PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase. SB203580 reduced the sphingosine 1-phosphate-induced increase of mRNA for HSP27. These results strongly suggest that sphingosine 1-phosphate-stimulated HSP27 induction is mediated via p38 MAP kinase activation in osteoblasts.
Assuntos
Ativadores de Enzimas/farmacologia , Proteínas de Choque Térmico/biossíntese , Lisofosfolipídeos , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Esfingosina/análogos & derivados , Animais , Northern Blotting , Linhagem Celular , Flavonoides/farmacologia , Proteínas de Choque Térmico/genética , Camundongos , Osteoblastos/metabolismo , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Esfingosina/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
To elucidate the precise molecular mechanisms underlying stratum corneum (SC) elasticity, we investigated the molecular dynamics of chemical residues within keratin fibers of human plantar SC under various conditions by cross polarization/magic angle spinning 13C-nuclear magnetic resonance. The intensities of nuclear magnetic resonance spectra responsible for amide carbonyl, C alpha methine, and side-chain aliphatic carbons in the intact SC decreased markedly with increasing water content of up to 30% in dry SC, and then remained constant at greater than 30%. Lipid extraction of intact SC with acetone/ether (1:1) did not induce any significant change in the nuclear magnetic resonance spectrum, whereas additional treatment with water, which released natural moisturizing factors (mainly amino acids), caused the SC to lose elasticity. The observed decrease in elasticity of the SC recovered after treatment with basic and neutral amino acids, but not after treatment with acidic amino acid. With the latter treatment, movement of amino acid molecules was significantly disturbed, suggesting a strong interaction with keratin fibers. Parallel studies of the complex elastic modulus of a pig SC sheet with a rheovibron also demonstrated that removal of natural moisturizing factor reduced the elasticity of the SC; this effect was also reversed by the application of basic and neutral amino acids, but not by the application of acidic amino acid. These findings suggest that structural keratin proteins, mainly consisting of 10-nm filaments, acquire their elasticity with the help of hydrated natural moisturizing factor via the reduction of intermolecular forces, probably through nonhelical regions between keratin fibers.