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Victor M. Elner - One of the best experts on this subject based on the ideXlab platform.
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dual involvement of caspase 4 in inflammatory and er stress induced apoptotic responses in human retinal pigment epithelial cells
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Zong Mei Bian, Susan G Elner, Victor M. ElnerAbstract:PURPOSE. To investigate the functional involvement of caspase-4 in human retinal pigment epithelial (hRPE) cells. METHODS. Expression and activation of caspase-4 in hRPE cells were measured after stimulation with proinflammatory Agents IL-I(3 (2 ng/mL), TNF-α (20 ng/mL), lipopolysaccharide (1000 ng/mL), interferon-γ (500 U/mL), or monocyte coculture in the absence or presence of Immunomodulating Agent cyclosporine (3 or 30 ng/mL), dexamethasone (10 μM), or IL-10 (100 U/mL) and endoplasmic reticulum (ER) stress inducer thapsigargin (25 nM) or tunicamycin (3 or 10 μM). The onset of ER stress was determined by expression of GRP78. The involvement of caspase-4 in inflammation and apoptosis was further examined by treating the cells with caspase-4 inhibitor Z-LEVD-fmk, caspase-1 and -4 inhibitor Z-YVAD-fmk, and pan-caspase inhibitor Z-VAD-fmk. RESULTS. Caspase-4 mRNA expression and protein activation were induced by all the proinflammatory Agents and ER stress inducers tested in this study. Caspase-4 activation was blocked or reduced by dexamethasone and IL-10. Elevated ER stress by proinflammatory Agents and ER stress inducers was shown by increased expression of the ER stress marker GRP78. The induced caspase-4 and caspase-3 activities by tunicamycin and the stimulated IL-8 protein expression by IL-1β were markedly reduced by caspase-4 inhibitor Z-LEVD-fmk. Although caspase-4 inhibitor Z-LEVD-fmk and caspase-1 and -4 inhibitor Z-YVAD-fmk reduced tunicamycin-induced hRPE apoptotic cell death by 59% and 86%, respectively, pan-caspase inhibitor Z-VAD-fmk completely abolished the induced apoptosis. CONCLUSIONS. Caspase-4 is dually involved in hRPE proinflammatory and proapoptotic responses. Various proinflammatory stimuli and ER stress induce hRPE caspase-4 mRNA synthesis and protein activation. ER stress-induced hRPE cell death is caspase and, in part, caspase-4 dependent.
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regulated expression of caspase 12 gene in human retinal pigment epithelial cells suggests its Immunomodulating role
Investigative Ophthalmology & Visual Science, 2008Co-Authors: Zong Mei Bian, Susan G Elner, Victor M. ElnerAbstract:Purpose To investigate the expression and regulation of the short form of caspase-12, caspase-12S, in human retinal pigment epithelial (hRPE) cells. Methods hRPE cells were stimulated by the proinflammatory Agents IL-1beta (2 ng/mL) and TNF-alpha (20 ng/mL); LPS (1000 ng/mL); coculture with monocytes; the Immunomodulating Agent cyclosporine (Cys; 30 ng/mL); the anti-inflammatory cytokine IL-10 (100 U/mL); and the endoplasmic reticulum (ER) stress inducers tunicamycin (3 or 10 muM) and thapsigargin (25 or 100 nM) for 6 hours or longer. The total RNAs were isolated and subjected to semiquantitative and quantitative real-time RT-PCR analysis. Effects of tunicamycin and thapsigargin on IL-1beta- and TNF-alpha-stimulated MCP-1 mRNA expression and protein production were further examined by RT-PCR and ELISA, respectively. Results RT-PCR results showed that caspase-12S is the predominant form of caspase-12 in the examined hRPE cells of this study, with expression at levels as high as those in many other human tissues such as pancreas, prostate, small intestine, lung, spleen, and kidney. Treatment with IL-1beta and TNF-alpha, as well as LPS and coculture with monocytes reduced hRPE caspase-12S mRNA expression within 6 hours. In contrast, hRPE exposure to cyclosporine (Cys) and the cytokine IL-10 for 6 hours increased caspase-12S mRNA expression. Compared to Cys and IL-10, the ER stress activators tunicamycin and thapsigargin were even more potent enhancers of hRPE caspase-12S gene expression. They also caused corresponding reductions in IL-1beta- and TNF-alpha-induced MCP-1 mRNA expression and protein production. Conclusions hRPE cells express a high level of caspase-12S. The regulated expression of caspase-12S suggests that this caspase recruitment domain (CARD)-only protein may be an endogenous dominant negative regulator that modulates inflammatory responses in hRPE cells.
Ribeiro, Elton Brito - One of the best experts on this subject based on the ideXlab platform.
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Desenvolvimento de nanocarreadores formados por micelas de pluronic F127 ou nanoemulsões para veiculação de interferon gamma e avaliação do potencial imunomodulador
'Biblioteca Central da UNB', 2016Co-Authors: Ribeiro, Elton BritoAbstract:Tese (Doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Nanociência e Nanobiotecnologia, 2016.O objetivo deste estudo foi desenvolver novas formulações contendo IFN-γ, visando sua utilização como Agente imunomodulador. As formulações foram produzidas a partir de diferentes sistemas coloidais formados por nanoemulsões ou micelas nanoestruturadas. As nanoemulsões à base de água destilada, triglicerídeo de ácido cáprico/caprílico, oleoato de sorbitano (SP), polissorbato 80 (TW) e propilenoglicol (PG) ou 1-butanol (BT) foram preparadas por ultra-homogeneização. As dispersões, formadas pelo copolímero Pluronic F127 em solução tampão fosfato (pH 7.4), foram preparadas nas concentrações de 10 mg.mL-1, 50 mg.mL-1 e 250 mg.mL-1. As nanoemulsões e dispersões desenvolvidas foram caracterizadas para obter o tamanho hidrodinâmico, índice de polidispersão, carga superficial, estabilidade física preliminar e acelerada e propriedades reológicas. As nanoemulsões e dispersões estáveis foram selecionadas para incorporar nano doses de IFN-γ (100 ng.mL-1). A influência do IFN-γ incorporado nas formulações desenvolvidas na atividade funcional das células mononucleares para Escherichia coli enteropatogênica foi analisada por meio da liberação de ânion superóxido, fagocitose e atividade microbicida. Em adição, a influência das formulações sobre a atividade funcional das células mononucleares, das células de câncer de mama humano (MCF-7) e da co-cultura de ambas, foi estudada por meio do teste de permeabilidade celular ao iodeto de propídeo e da liberação de cálcio intracelular. As formulações otimizadas permaneceram estáveis em condições extremas durante 90 dias, com valores de pH biocompatíveis, sem alteração significativa dos perfis reológicos, diâmetro hidrodinâmico com dimensões nanométricas, potencial zeta negativo, e sem variações significativas da dose do IFN-γ (100 ng.mL-1). Os ensaios biológicos mostraram que as formulações desenvolvidas não alteram a taxa de viabilidade das células mononucleares e aumentaram a liberação de superóxido, o índice de fagocitose e a liberação de cálcio intracelular das células mononucleares do sangue humano, quando se comparou ao grupo espontâneo. Além disso, as nanoemulsões são capazes de promover a morte das células de câncer de mama humano MCF-7 ou em co-cultura. Os resultados indicam que as formulações produzidas podem melhorar a atividade biológica do IFN-γ.The aim of this study was to design and develop different formulations containing IFN-γ to probe their use as an Immunomodulating Agent. Produced formulations were based on two different nanoemulsion systems or nanostructured micelles. The nanoemulsions comprising distilled water, triglycerides of capric/caprylic acid, sobitan-oleate (SP), polysorbate 80 (TW) and propylene glycol (PG) or 1-butanol (BT) were prepared through ultra-homogenization. The pluronic 127 dispersions in phosphate buffer solution (pH 7.4) were prepared at concentrations of 10 mg.mL-1, 50 mg.mL-1 and 250 mg.mL-1. The developed nanoemulsions and dispersions were characterized regarding droplet size, polydispersity, surface charge, preliminary and accelerated physical stability, and rheological properties. Stable products were selected to incorporate nano doses of IFN-γ (100 ng.mL-1) and characterized under the same parameters. The influence of IFN-γ incorporated formulations on functional activity of mononuclear cell for Escherichia coli enteropathogenic was analyzed through superoxide release, phagocytosis and microbicidal activity. By means of cell permeability tests to propidium iodide and intracellular calcium release were employed to study the formulations influence on functional activity of mononuclear cell, human breast cancer (MCF-7) and co-culturing both. The optimized formulations remained stable in extreme conditions during 90 days, displaying biocompatible pH value, significant maintenance of its rheological profile, nanometric hydrodynamic diameter, negative zeta potential and no significant variations of IFN-γ dose (100 ng.mL-1). Additionaly, developed formulations did not alter the MN cell viability rates and increased the superoxide release, phagocytosis index and intracellular calcium release of mononuclear cells of human blood compared to the spontaneous group. Furthermore, the nanoemulsions were able to promote the death of MCF-7 human breast cancer or co-culturing. Our findings indicate that the produced formulations can improve the biological activity of IFN-γ.The aim of this study was to design and develop different formulations containing IFN-γ to probe their use as an Immunomodulating Agent. Produced formulations were based on two different nanoemulsion systems or nanostructured micelles. The nanoemulsions comprising distilled water, triglycerides of capric/caprylic acid, sobitan-oleate (SP), polysorbate 80 (TW) and propylene glycol (PG) or 1-butanol (BT) were prepared through ultra-homogenization. The pluronic 127 dispersions in phosphate buffer solution (pH 7.4) were prepared at concentrations of 10 mg.mL-1, 50 mg.mL-1 and 250 mg.mL-1. The developed nanoemulsions and dispersions were characterized regarding droplet size, polydispersity, surface charge, preliminary and accelerated physical stability, and rheological properties. Stable products were selected to incorporate nano doses of IFN-γ (100 ng.mL-1) and characterized under the same parameters. The influence of IFN-γ incorporated formulations on functional activity of mononuclear cell for Escherichia coli enteropathogenic was analyzed through superoxide release, phagocytosis and microbicidal activity. By means of cell permeability tests to propidium iodide and intracellular calcium release were employed to study the formulations influence on functional activity of mononuclear cell, human breast cancer (MCF-7) and co-culturing both. The optimized formulations remained stable in extreme conditions during 90 days, displaying biocompatible pH value, significant maintenance of its rheological profile, nanometric hydrodynamic diameter, negative zeta potential and no significant variations of IFN-γ dose (100 ng.mL-1). Additionaly, developed formulations did not alter the MN cell viability rates and increased the superoxide release, phagocytosis index and intracellular calcium release of mononuclear cells of human blood compared to the spontaneous group. Furthermore, the nanoemulsions were able to promote the death of MCF-7 human breast cancer or co-culturing. Our findings indicate that the produced formulations can improve the biological activity of IFN-γ
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Desenvolvimento de nanocarreadores formados por micelas de pluronic F127 ou nanoemulsões para veiculação de interferon gamma e avaliação do potencial imunomodulador
2016Co-Authors: Ribeiro, Elton BritoAbstract:O objetivo deste estudo foi desenvolver novas formulações contendo IFN-γ, visando sua utilização como Agente imunomodulador. As formulações foram produzidas a partir de diferentes sistemas coloidais formados por nanoemulsões ou micelas nanoestruturadas. As nanoemulsões à base de água destilada, triglicerídeo de ácido cáprico/caprílico, oleoato de sorbitano (SP), polissorbato 80 (TW) e propilenoglicol (PG) ou 1-butanol (BT) foram preparadas por ultra-homogeneização. As dispersões, formadas pelo copolímero Pluronic F127 em solução tampão fosfato (pH 7.4), foram preparadas nas concentrações de 10 mg.mL-1, 50 mg.mL-1 e 250 mg.mL-1. As nanoemulsões e dispersões desenvolvidas foram caracterizadas para obter o tamanho hidrodinâmico, índice de polidispersão, carga superficial, estabilidade física preliminar e acelerada e propriedades reológicas. As nanoemulsões e dispersões estáveis foram selecionadas para incorporar nano doses de IFN-γ (100 ng.mL-1). A influência do IFN-γ incorporado nas formulações desenvolvidas na atividade funcional das células mononucleares para Escherichia coli enteropatogênica foi analisada por meio da liberação de ânion superóxido, fagocitose e atividade microbicida. Em adição, a influência das formulações sobre a atividade funcional das células mononucleares, das células de câncer de mama humano (MCF-7) e da co-cultura de ambas, foi estudada por meio do teste de permeabilidade celular ao iodeto de propídeo e da liberação de cálcio intracelular. As formulações otimizadas permaneceram estáveis em condições extremas durante 90 dias, com valores de pH biocompatíveis, sem alteração significativa dos perfis reológicos, diâmetro hidrodinâmico com dimensões nanométricas, potencial zeta negativo, e sem variações significativas da dose do IFN-γ (100 ng.mL-1). Os ensaios biológicos mostraram que as formulações desenvolvidas não alteram a taxa de viabilidade das células mononucleares e aumentaram a liberação de superóxido, o índice de fagocitose e a liberação de cálcio intracelular das células mononucleares do sangue humano, quando se comparou ao grupo espontâneo. Além disso, as nanoemulsões são capazes de promover a morte das células de câncer de mama humano MCF-7 ou em co-cultura. Os resultados indicam que as formulações produzidas podem melhorar a atividade biológica do IFN-γ. _________________________________________________________________________________________________ ABSTRACTThe aim of this study was to design and develop different formulations containing IFN-γ to probe their use as an Immunomodulating Agent. Produced formulations were based on two different nanoemulsion systems or nanostructured micelles. The nanoemulsions comprising distilled water, triglycerides of capric/caprylic acid, sobitan-oleate (SP), polysorbate 80 (TW) and propylene glycol (PG) or 1-butanol (BT) were prepared through ultra-homogenization. The pluronic 127 dispersions in phosphate buffer solution (pH 7.4) were prepared at concentrations of 10 mg.mL-1, 50 mg.mL-1 and 250 mg.mL-1. The developed nanoemulsions and dispersions were characterized regarding droplet size, polydispersity, surface charge, preliminary and accelerated physical stability, and rheological properties. Stable products were selected to incorporate nano doses of IFN-γ (100 ng.mL-1) and characterized under the same parameters. The influence of IFN-γ incorporated formulations on functional activity of mononuclear cell for Escherichia coli enteropathogenic was analyzed through superoxide release, phagocytosis and microbicidal activity. By means of cell permeability tests to propidium iodide and intracellular calcium release were employed to study the formulations influence on functional activity of mononuclear cell, human breast cancer (MCF-7) and co-culturing both. The optimized formulations remained stable in extreme conditions during 90 days, displaying biocompatible pH value, significant maintenance of its rheological profile, nanometric hydrodynamic diameter, negative zeta potential and no significant variations of IFN-γ dose (100 ng.mL-1). Additionaly, developed formulations did not alter the MN cell viability rates and increased the superoxide release, phagocytosis index and intracellular calcium release of mononuclear cells of human blood compared to the spontaneous group. Furthermore, the nanoemulsions were able to promote the death of MCF-7 human breast cancer or co-culturing. Our findings indicate that the produced formulations can improve the biological activity of IFN-γ
Zong Mei Bian - One of the best experts on this subject based on the ideXlab platform.
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dual involvement of caspase 4 in inflammatory and er stress induced apoptotic responses in human retinal pigment epithelial cells
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Zong Mei Bian, Susan G Elner, Victor M. ElnerAbstract:PURPOSE. To investigate the functional involvement of caspase-4 in human retinal pigment epithelial (hRPE) cells. METHODS. Expression and activation of caspase-4 in hRPE cells were measured after stimulation with proinflammatory Agents IL-I(3 (2 ng/mL), TNF-α (20 ng/mL), lipopolysaccharide (1000 ng/mL), interferon-γ (500 U/mL), or monocyte coculture in the absence or presence of Immunomodulating Agent cyclosporine (3 or 30 ng/mL), dexamethasone (10 μM), or IL-10 (100 U/mL) and endoplasmic reticulum (ER) stress inducer thapsigargin (25 nM) or tunicamycin (3 or 10 μM). The onset of ER stress was determined by expression of GRP78. The involvement of caspase-4 in inflammation and apoptosis was further examined by treating the cells with caspase-4 inhibitor Z-LEVD-fmk, caspase-1 and -4 inhibitor Z-YVAD-fmk, and pan-caspase inhibitor Z-VAD-fmk. RESULTS. Caspase-4 mRNA expression and protein activation were induced by all the proinflammatory Agents and ER stress inducers tested in this study. Caspase-4 activation was blocked or reduced by dexamethasone and IL-10. Elevated ER stress by proinflammatory Agents and ER stress inducers was shown by increased expression of the ER stress marker GRP78. The induced caspase-4 and caspase-3 activities by tunicamycin and the stimulated IL-8 protein expression by IL-1β were markedly reduced by caspase-4 inhibitor Z-LEVD-fmk. Although caspase-4 inhibitor Z-LEVD-fmk and caspase-1 and -4 inhibitor Z-YVAD-fmk reduced tunicamycin-induced hRPE apoptotic cell death by 59% and 86%, respectively, pan-caspase inhibitor Z-VAD-fmk completely abolished the induced apoptosis. CONCLUSIONS. Caspase-4 is dually involved in hRPE proinflammatory and proapoptotic responses. Various proinflammatory stimuli and ER stress induce hRPE caspase-4 mRNA synthesis and protein activation. ER stress-induced hRPE cell death is caspase and, in part, caspase-4 dependent.
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regulated expression of caspase 12 gene in human retinal pigment epithelial cells suggests its Immunomodulating role
Investigative Ophthalmology & Visual Science, 2008Co-Authors: Zong Mei Bian, Susan G Elner, Victor M. ElnerAbstract:Purpose To investigate the expression and regulation of the short form of caspase-12, caspase-12S, in human retinal pigment epithelial (hRPE) cells. Methods hRPE cells were stimulated by the proinflammatory Agents IL-1beta (2 ng/mL) and TNF-alpha (20 ng/mL); LPS (1000 ng/mL); coculture with monocytes; the Immunomodulating Agent cyclosporine (Cys; 30 ng/mL); the anti-inflammatory cytokine IL-10 (100 U/mL); and the endoplasmic reticulum (ER) stress inducers tunicamycin (3 or 10 muM) and thapsigargin (25 or 100 nM) for 6 hours or longer. The total RNAs were isolated and subjected to semiquantitative and quantitative real-time RT-PCR analysis. Effects of tunicamycin and thapsigargin on IL-1beta- and TNF-alpha-stimulated MCP-1 mRNA expression and protein production were further examined by RT-PCR and ELISA, respectively. Results RT-PCR results showed that caspase-12S is the predominant form of caspase-12 in the examined hRPE cells of this study, with expression at levels as high as those in many other human tissues such as pancreas, prostate, small intestine, lung, spleen, and kidney. Treatment with IL-1beta and TNF-alpha, as well as LPS and coculture with monocytes reduced hRPE caspase-12S mRNA expression within 6 hours. In contrast, hRPE exposure to cyclosporine (Cys) and the cytokine IL-10 for 6 hours increased caspase-12S mRNA expression. Compared to Cys and IL-10, the ER stress activators tunicamycin and thapsigargin were even more potent enhancers of hRPE caspase-12S gene expression. They also caused corresponding reductions in IL-1beta- and TNF-alpha-induced MCP-1 mRNA expression and protein production. Conclusions hRPE cells express a high level of caspase-12S. The regulated expression of caspase-12S suggests that this caspase recruitment domain (CARD)-only protein may be an endogenous dominant negative regulator that modulates inflammatory responses in hRPE cells.
Ichiro Azuma - One of the best experts on this subject based on the ideXlab platform.
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mdp lys l18 a synthetic muramyl dipeptide derivative enhances antitumor activity of an inactivated tumor vaccine
Journal of Microbiology and Biotechnology, 2000Co-Authors: Yung Choon Yoo, Seungyong Park, Kyungbok Lee, Ichiro AzumaAbstract:The adjuvant effect of a muramyl dipeptide (MDP) derivative, MDP-Lys (L18), on enhancing of antitumor immunity induced by X-irradiated tumor cells against highly metastatic B16-BL6 melanoma cells was examined in mice. Mice immunized intradermally (i.d.) with a mixture of X-irradiated B16-BL6 cells and MDP-Lys (L18) [Vac+MDP-Lys (L18)] followed by an intravenous (i.v.) inoculation of 10 4 viable tumor cells 7 days after immunization, showed a significant inhibition of experimental lung metastasis of B 16-BL6 melanoma cells. The most effective immunization for the prophylactic inhibition of tumor metastasis was obtained from the mixture of 100 μg of MDP-Lys (L18) and 10 4 X-irradiatied tumor vaccine. Furthermore, immunization of mice with Vac+MDP-Lys (L18), 3 days after tumor challenge, resulted in a significant inhibition of lung metastasis of B16-BL6 melanoma cells in an experimental lung metastasis model. Similarly, the administration of Vac+MDP-Lys (L18), 1 or 7 days after tumor removal, markedly inhibited tumor metastasis of B16-BL6 in a spontaneous lung metastasis model. When Vac+MDP-Lys (L18) was i.d. administered 3 days after subcutaneous (s.c.) inoculation of tumor cells (5 × 10 5 /site) on the back, mice treated with Vac+MDP-Lys (L18) showed inhibition of significantly tumor growth on day 20. These results suggest that MDP-Lys (L18) is able to enhance antitumor activity induced by X-irradiated tumor vaccine to reduce lung metastasis of tumor cells, and is a potent Immunomodulating Agent which may be applied prophylactically as well as therapeutically to treatment of cancer metastasis.
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mdp lys l18 a lipophilic derivative of muramyl dipeptide inhibits the metastasis of haematogenous and non haematogenous tumours in mice
Vaccine, 1994Co-Authors: Yoo Yung Choon, Ikuo Saiki, Katsuaki Sato, Ichiro AzumaAbstract:Abstract The antimetastatic effects of MDP-Lys(L18), a lipophilic derivative of muramyl dipeptide (MDP), against three different types of highly metastatic murine tumour cells, B16-BL6 melanoma, colon 26-M3.1 carcinoma and L5178Y-ML25 T lymphoma, were examined in C57BL/6, Balb/c and CDF1 mice, respectively. The administration of 100 μg of MDP-Lys(L18) 2 or 4 days before tumour inoculation led to a significant decrease in lung metastasis of B16-BL6 melanoma or colon 26-M3.1 carcinoma cells. MDP-Lys(L18) was also effective in the inhibition of liver metastasis of L5178Y-ML25 lymphoma cells by administration 2 or 4 days before tumour inoculation. The prophylactic effect of 100 μg of MDP-Lys(L18) on tumour metastasis was evident for the different administration routes, i.e. subcutaneous, intravenous or intranasal injection, or oral administration. It is of prime interest that oral administration of 1 mg of MDP-Lys(L18) induced a significant decrease in lung metastasis of B16-BL6 melanoma cells. Administration ofMDP-Lys(L18) 4 days before assay led to induction of tumoricidal activity by peritoneal macrophages and growth inhibition by the sera against B16-BL6 or L929 cells. When MDP-Lys(L18) was subcutaneously administered five times after tumour inoculation to test therapeutic effect in an experimental and spontaneous metastasis model using B16-BL6 melanoma, the consecutive administrations of MDP-Lys(L18) significantly inhibited lung metastasis in tumour-bearing mice. These results suggest that MDP-Lys(L18) is able to enhance host resistance to reduce tumour metastasis and is a potent Immunomodulating Agent which may be applied prophylactically or therapeutically for the treatment of cancer metastasis.
Susan G Elner - One of the best experts on this subject based on the ideXlab platform.
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dual involvement of caspase 4 in inflammatory and er stress induced apoptotic responses in human retinal pigment epithelial cells
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Zong Mei Bian, Susan G Elner, Victor M. ElnerAbstract:PURPOSE. To investigate the functional involvement of caspase-4 in human retinal pigment epithelial (hRPE) cells. METHODS. Expression and activation of caspase-4 in hRPE cells were measured after stimulation with proinflammatory Agents IL-I(3 (2 ng/mL), TNF-α (20 ng/mL), lipopolysaccharide (1000 ng/mL), interferon-γ (500 U/mL), or monocyte coculture in the absence or presence of Immunomodulating Agent cyclosporine (3 or 30 ng/mL), dexamethasone (10 μM), or IL-10 (100 U/mL) and endoplasmic reticulum (ER) stress inducer thapsigargin (25 nM) or tunicamycin (3 or 10 μM). The onset of ER stress was determined by expression of GRP78. The involvement of caspase-4 in inflammation and apoptosis was further examined by treating the cells with caspase-4 inhibitor Z-LEVD-fmk, caspase-1 and -4 inhibitor Z-YVAD-fmk, and pan-caspase inhibitor Z-VAD-fmk. RESULTS. Caspase-4 mRNA expression and protein activation were induced by all the proinflammatory Agents and ER stress inducers tested in this study. Caspase-4 activation was blocked or reduced by dexamethasone and IL-10. Elevated ER stress by proinflammatory Agents and ER stress inducers was shown by increased expression of the ER stress marker GRP78. The induced caspase-4 and caspase-3 activities by tunicamycin and the stimulated IL-8 protein expression by IL-1β were markedly reduced by caspase-4 inhibitor Z-LEVD-fmk. Although caspase-4 inhibitor Z-LEVD-fmk and caspase-1 and -4 inhibitor Z-YVAD-fmk reduced tunicamycin-induced hRPE apoptotic cell death by 59% and 86%, respectively, pan-caspase inhibitor Z-VAD-fmk completely abolished the induced apoptosis. CONCLUSIONS. Caspase-4 is dually involved in hRPE proinflammatory and proapoptotic responses. Various proinflammatory stimuli and ER stress induce hRPE caspase-4 mRNA synthesis and protein activation. ER stress-induced hRPE cell death is caspase and, in part, caspase-4 dependent.
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regulated expression of caspase 12 gene in human retinal pigment epithelial cells suggests its Immunomodulating role
Investigative Ophthalmology & Visual Science, 2008Co-Authors: Zong Mei Bian, Susan G Elner, Victor M. ElnerAbstract:Purpose To investigate the expression and regulation of the short form of caspase-12, caspase-12S, in human retinal pigment epithelial (hRPE) cells. Methods hRPE cells were stimulated by the proinflammatory Agents IL-1beta (2 ng/mL) and TNF-alpha (20 ng/mL); LPS (1000 ng/mL); coculture with monocytes; the Immunomodulating Agent cyclosporine (Cys; 30 ng/mL); the anti-inflammatory cytokine IL-10 (100 U/mL); and the endoplasmic reticulum (ER) stress inducers tunicamycin (3 or 10 muM) and thapsigargin (25 or 100 nM) for 6 hours or longer. The total RNAs were isolated and subjected to semiquantitative and quantitative real-time RT-PCR analysis. Effects of tunicamycin and thapsigargin on IL-1beta- and TNF-alpha-stimulated MCP-1 mRNA expression and protein production were further examined by RT-PCR and ELISA, respectively. Results RT-PCR results showed that caspase-12S is the predominant form of caspase-12 in the examined hRPE cells of this study, with expression at levels as high as those in many other human tissues such as pancreas, prostate, small intestine, lung, spleen, and kidney. Treatment with IL-1beta and TNF-alpha, as well as LPS and coculture with monocytes reduced hRPE caspase-12S mRNA expression within 6 hours. In contrast, hRPE exposure to cyclosporine (Cys) and the cytokine IL-10 for 6 hours increased caspase-12S mRNA expression. Compared to Cys and IL-10, the ER stress activators tunicamycin and thapsigargin were even more potent enhancers of hRPE caspase-12S gene expression. They also caused corresponding reductions in IL-1beta- and TNF-alpha-induced MCP-1 mRNA expression and protein production. Conclusions hRPE cells express a high level of caspase-12S. The regulated expression of caspase-12S suggests that this caspase recruitment domain (CARD)-only protein may be an endogenous dominant negative regulator that modulates inflammatory responses in hRPE cells.
