Objective： To investigate the role of peroxisome proliferator-activated receptors δ （PPARδ） in inflammatory reaction and its possible mechanism in adipocyte. Methods：Lentivirus-mediated RNA interference （RNAi）...Objective： To investigate the role of peroxisome proliferator-activated receptors δ （PPARδ） in inflammatory reaction and its possible mechanism in adipocyte. Methods：Lentivirus-mediated RNA interference （RNAi） was used to block the expression of PPARδ in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α（TNFα, 20 ng/ml） for 4 h. The expression of PPARδ, nuclear factor κB （NFκB） and C reactive protein （CRP） were determined by Western blot analysis. Results：The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα but had no effect on normal cells. Conclusion： PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.展开更多
Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type ...Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type calcium channels.T-type calcium channel blockers,such as pimozide and mibefradil,have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury.However,the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear.Here,in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons.The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons.We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury.Cav3.2 knockout markedly reduced infarct volume and brain water content,and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury.Additionally,Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress,inflammatory response,and neuronal apoptosis.In the hippocampus of Cav3.2-knockout mice,calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury.These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling.Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.展开更多
Background：Mitofusin-2 （MFN2）,a well-known mitochondrial fusion protein,has been shown to participate in innate immunity,but its role in mediating adaptive immunity remains poorly characterized.In this study,we exp...Background：Mitofusin-2 （MFN2）,a well-known mitochondrial fusion protein,has been shown to participate in innate immunity,but its role in mediating adaptive immunity remains poorly characterized.In this study,we explored the potential role of MFN2 in mediating the immune function of T lymphocytes.Methods：We manipulated MFN2 gone expression in Jurkat cells via lentiviral transduction of MFN2 small interfering RNA （siRNA） or full-length MFN2.After transduction,the immune response and its underlying mechanism were determined in Jurkat cells.One-way analysis of variance and Student＇s t-test were performed to determine the statistical significance between the groups.Results：Overexpression of MFN2 enhanced the immune response of T lymphocytes by upregulating Ca2＋ （359.280 ± 10.130 vs.266.940 ± 10.170,P =0.000）,calcineurin （0.513 ± 0.014 vs.0.403 ± 0.020 nmol/L,P =0.024）,and nuclear factor of activated T cells （NFATs） activation （1.040 ± 0.086 vs.0.700 ± 0.115,P =0.005）,whereas depletion of MFN2 impaired the immune function ofT lymphocytes by downregulating Ca2＋ （141.140 ± 14.670 vs.267.060 ± 9.230,P =0.000）,calcineurin （0.054 ± 0.030 nmol/L vs.0.404 ± 0.063 nmol/L,P =0.000）,and NFAT activation （0.500 ± 0.025 vs.0.720 ± 0.061,P =0.012）.Furthermore,upregulated calcineurin partially reversed the negative effects ofMFN2 siRNA on T cell-mediated immunity evidenced by elevations in T cell proliferation （1.120 ± 0.048 vs.0.580 ± 0.078,P =0.040）,interleukin-2 （IL-2） production （473.300 ± 24.100 vs.175.330 ± 12.900 pg/ml,P =0.000）,and the interferon-γ/IL-4 ratio （3.080 ± 0.156 vs.0.953 ± 0.093,P =0.000）.Meanwhile,calcineurin activity inhibitor depleted the positive effects of overexpressed MFN2 on T cells function.Conclusions：Our findings suggest that MFN2 may regulate T cell immune functions primarily through the Ca2＋-calcineurin-NFAT pathway.MFN2 may represent a potential therapeutic target for T cell immune dysfunction-related diseases.展开更多
Osteoclast differentiation depends on receptor activator of nuclear factor-κB(RANK) signaling,which can be divided into triggering,amplifying and targeting phases based on how active the master regulator nuclear fact...Osteoclast differentiation depends on receptor activator of nuclear factor-κB(RANK) signaling,which can be divided into triggering,amplifying and targeting phases based on how active the master regulator nuclear factor of activated T-cells cytoplasmic 1(NFATc1) is. The triggering phase is characterized by immediateearly RANK signaling induced by RANK ligand(RANKL) stimulation mediated by three adaptor proteins,tumor necrosis factor receptor-associated factor 6,Grb-2-associated binder-2 and phospholipase C(PLC)γ2,leading to activation of IκB kinase,mitogen-activated protein kinases and the transcription factors nuclear factor(NF)-κB and activator protein-1(AP-1). Mice lacking NF-κB p50/p52 or the AP-1 subunit c-Fos(encoded by Fos) exhibit severe osteopetrosis due to a differentiation block in the osteoclast lineage. The amplification phase occurs about 24 h later in a RANKLinduced osteoclastogenic culture when Ca2+ oscillation starts and the transcription factor NFATc1 is abundantly produced. In addition to Ca2+ oscillation-dependent nuclear translocation and transcriptional auto-induction of NFATc1,a Ca2+ oscillation-independent,osteoblastdependent mechanism stabilizes NFATc1 protein in dif-ferentiating osteoclasts. Osteoclast precursors lacking PLCγ2,inositol-1,4,5-trisphosphate receptors,regulator of G-protein signaling 10,or NFATc1 show an impaired transition from the triggering to amplifying phases. The final targeting phase is mediated by activation of numerous NFATc1 target genes responsible for cell-cell fusion and regulation of bone-resorptive function. This review focuses on molecular mechanisms for each of the three phases of RANK signaling during osteoclast differentiation.展开更多
Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathw...Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular freecalcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulindependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways.展开更多
文摘Objective： To investigate the role of peroxisome proliferator-activated receptors δ （PPARδ） in inflammatory reaction and its possible mechanism in adipocyte. Methods：Lentivirus-mediated RNA interference （RNAi） was used to block the expression of PPARδ in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α（TNFα, 20 ng/ml） for 4 h. The expression of PPARδ, nuclear factor κB （NFκB） and C reactive protein （CRP） were determined by Western blot analysis. Results：The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα but had no effect on normal cells. Conclusion： PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.
基金supported by the Natural Science Foundation of Anhui Province of China,No.2208085Y32Scientific Research Plan Project of Anhui Province of China,No.2022AH020076the Chen Xiao-Ping Foundation for the Development of Science and Technology of Hubei Province,No.CXPJJH12000005-07-115(all to CT).
文摘Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type calcium channels.T-type calcium channel blockers,such as pimozide and mibefradil,have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury.However,the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear.Here,in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons.The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons.We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury.Cav3.2 knockout markedly reduced infarct volume and brain water content,and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury.Additionally,Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress,inflammatory response,and neuronal apoptosis.In the hippocampus of Cav3.2-knockout mice,calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury.These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling.Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.
基金This study was supported by grants from the Natural Science Foundation of Zhejiang Province （No.LY13HI50006 and No. LY13H150004）, the National Natural Science Foundation （No. 81571937 and No. 81772112）, and the Key Construction Academic Subject （Medical Innovation） of Zhejiang Province （No. 11-CX26）.
文摘Background：Mitofusin-2 （MFN2）,a well-known mitochondrial fusion protein,has been shown to participate in innate immunity,but its role in mediating adaptive immunity remains poorly characterized.In this study,we explored the potential role of MFN2 in mediating the immune function of T lymphocytes.Methods：We manipulated MFN2 gone expression in Jurkat cells via lentiviral transduction of MFN2 small interfering RNA （siRNA） or full-length MFN2.After transduction,the immune response and its underlying mechanism were determined in Jurkat cells.One-way analysis of variance and Student＇s t-test were performed to determine the statistical significance between the groups.Results：Overexpression of MFN2 enhanced the immune response of T lymphocytes by upregulating Ca2＋ （359.280 ± 10.130 vs.266.940 ± 10.170,P =0.000）,calcineurin （0.513 ± 0.014 vs.0.403 ± 0.020 nmol/L,P =0.024）,and nuclear factor of activated T cells （NFATs） activation （1.040 ± 0.086 vs.0.700 ± 0.115,P =0.005）,whereas depletion of MFN2 impaired the immune function ofT lymphocytes by downregulating Ca2＋ （141.140 ± 14.670 vs.267.060 ± 9.230,P =0.000）,calcineurin （0.054 ± 0.030 nmol/L vs.0.404 ± 0.063 nmol/L,P =0.000）,and NFAT activation （0.500 ± 0.025 vs.0.720 ± 0.061,P =0.012）.Furthermore,upregulated calcineurin partially reversed the negative effects ofMFN2 siRNA on T cell-mediated immunity evidenced by elevations in T cell proliferation （1.120 ± 0.048 vs.0.580 ± 0.078,P =0.040）,interleukin-2 （IL-2） production （473.300 ± 24.100 vs.175.330 ± 12.900 pg/ml,P =0.000）,and the interferon-γ/IL-4 ratio （3.080 ± 0.156 vs.0.953 ± 0.093,P =0.000）.Meanwhile,calcineurin activity inhibitor depleted the positive effects of overexpressed MFN2 on T cells function.Conclusions：Our findings suggest that MFN2 may regulate T cell immune functions primarily through the Ca2＋-calcineurin-NFAT pathway.MFN2 may represent a potential therapeutic target for T cell immune dysfunction-related diseases.
基金Supported by Grants from MEXT Japan,No.23790265(to Kuroda Y)and No.21390425(to Matsuo K)
文摘Osteoclast differentiation depends on receptor activator of nuclear factor-κB(RANK) signaling,which can be divided into triggering,amplifying and targeting phases based on how active the master regulator nuclear factor of activated T-cells cytoplasmic 1(NFATc1) is. The triggering phase is characterized by immediateearly RANK signaling induced by RANK ligand(RANKL) stimulation mediated by three adaptor proteins,tumor necrosis factor receptor-associated factor 6,Grb-2-associated binder-2 and phospholipase C(PLC)γ2,leading to activation of IκB kinase,mitogen-activated protein kinases and the transcription factors nuclear factor(NF)-κB and activator protein-1(AP-1). Mice lacking NF-κB p50/p52 or the AP-1 subunit c-Fos(encoded by Fos) exhibit severe osteopetrosis due to a differentiation block in the osteoclast lineage. The amplification phase occurs about 24 h later in a RANKLinduced osteoclastogenic culture when Ca2+ oscillation starts and the transcription factor NFATc1 is abundantly produced. In addition to Ca2+ oscillation-dependent nuclear translocation and transcriptional auto-induction of NFATc1,a Ca2+ oscillation-independent,osteoblastdependent mechanism stabilizes NFATc1 protein in dif-ferentiating osteoclasts. Osteoclast precursors lacking PLCγ2,inositol-1,4,5-trisphosphate receptors,regulator of G-protein signaling 10,or NFATc1 show an impaired transition from the triggering to amplifying phases. The final targeting phase is mediated by activation of numerous NFATc1 target genes responsible for cell-cell fusion and regulation of bone-resorptive function. This review focuses on molecular mechanisms for each of the three phases of RANK signaling during osteoclast differentiation.
基金Supported by The Breast Cancer Campaign and the Research Institute in Healthcare Sciences (Armesilla AL)The Wellcome Trust (Emerson M)
文摘Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular freecalcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulindependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways.
