Objective Leucine-rich repeats and immunoglobulin-like domains 1(LRIG1) is a newly identified human gene that inhibits the epidermal growth factor receptor(EGFR), which on combining with a ligand, can drive tumor grow...Objective Leucine-rich repeats and immunoglobulin-like domains 1(LRIG1) is a newly identified human gene that inhibits the epidermal growth factor receptor(EGFR), which on combining with a ligand, can drive tumor growth. This study investigated the interaction between human LRIG1 and EGFR and attempted to delineate the functions of as well as the mechanisms used by the extracellular(ECD) and cytoplasmic(CPD) domains of the human LRIG1 protein to downregulate human EGFR signaling activity.Methods Two constructed chimeric eukaryotic expression vectors, pIRES2-EGFP-3XFLAG-LRIG1-ET and p3FLAG-LRIG1-TC, encoding the extracellular and transmembrane regions(LRIG1-ET) and the transmembrane and cytoplasmic regions(LRIG1-TC), respectively, and the plasmid p3XFLAG-CMV-9-LRIG1 encoding full-length LRIG1(LRIG1-FL) were transfected into the human glioma cell line U251 or primary astrocytoma cells by using liposomes. The number and affinity of cell surface EGFR on transfected cells was determined by ^(125)I-EGF binding assay. Results The dissociation constant(KD) values for EGFR were higher, and the maximum increase was observed in the cells transfected into LRIG1-ET(1.36 folds). The number of maximal binding sites(Bmax) of the receptors was decreased in all transfected cells; the maximum decrease was noted in the cells transfected into LRIG1-FL(40.05%).Conclusion Both the ECD and CPD of LRIG1 are important to negate EGFR signaling. The ECD may interfere with the binding between EGFR and its ligand and facilitate the functions of CPD. The CPD may, when brought in proximity to EGFR, enhance receptor degradation. These two mechanisms can contribute to the downregulation of EGFR-mediated signaling by LRIG1.展开更多
AIM: To investigate the role of TR3 in induction of apoptosis in gastric cancer cells. METHODS: Human gastric cancer cell line, MGC80-3, was used. Expression of TR3 mRNA and its protein was detected by Northern blot a...AIM: To investigate the role of TR3 in induction of apoptosis in gastric cancer cells. METHODS: Human gastric cancer cell line, MGC80-3, was used. Expression of TR3 mRNA and its protein was detected by Northern blot and Western blot. Localization of TR3 protein was showed by immunofluorescence analysis under laser-scanning confocal microscope. Apoptotic morphology was observed by DAPI fluorescence staining, and apoptotic index was counted among 1000 cells randomly. Stable transfection assay was carried out by Lipofectamine. RESULTS: Treatment of MGC80-3 cells with TPA and VP-16 resulted in apoptosis, accompanied by the repression of Bcl-2 protein in a time-dependent manner. At the same time, TPA and VP-16 also up-regulated expression level of TR3 mRNA in MGC80-3 cells that expressed TR3 mRNA. When antisense-TR3 expression vector was transfected into the cells, expression of TR3 protein was repressed. In this case, TPA and VP-16 did not induce apoptosis. In addition, TPA and VP-16-induced apoptosis involved in translocation of TR3. In MGC80-3 cells, TR3 localized concentrative in nucleus, after treatment of cells with TPA and VP-16, TR3 translocated from nucleus to cytosol obviously. However, when this nuclear translocation was blocked by LMB, apoptosis was not occurred in MGC80-3 cells even in the presence of TPA and VP-16. CONCLUSION: Induction of apoptosis by TPA and VP-16 is through induction of TR3 expression and translocation of TR3 from nucleus to cytosol, which may be a novel signal pathway for TR3, and represent the new biological function of TR3 to exert its effect on apoptosis in gastric cancer cells.展开更多
Plants are exposed to numerous potential pathogenic microbes. To counter the threat, plants have evolved diverse patternrecognition receptors(PRRs), which are receptor kinases(RKs) and receptor proteins(RPs) specializ...Plants are exposed to numerous potential pathogenic microbes. To counter the threat, plants have evolved diverse patternrecognition receptors(PRRs), which are receptor kinases(RKs) and receptor proteins(RPs) specialized to detect conserved pathogen/microbe-associated molecular patterns(PAMPs/MAMPs). Although only a handful of RKs and RPs are known PRRs,they belong to the receptor-like kinase(RLK) and receptor-like protein(RLP) superfamilies that undergo lineage-specific expansion, suggesting that many of these RLKs and RLPs are potential PRRs. Analyses of existing PRRs have uncovered ligand-induced RLK-RK or RLK-RP oligomerization as a common mechanism for immune activation. PRRs can recruit additional components to form dynamic receptor complexes, which mediate specific cellular responses. Detailed analyses of these components are shedding light on molecular mechanisms underlying the regulation of PRR activity and downstream signaling.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
BACKGROUND： Most of the currently available information on purinergic receptors （P2Rs） involved in pain transmission is based on results obtained in dorsal root ganglion or the spinal cord. However, the mechanism o...BACKGROUND： Most of the currently available information on purinergic receptors （P2Rs） involved in pain transmission is based on results obtained in dorsal root ganglion or the spinal cord. However, the mechanism of P2Rs in trigeminal neuralgia remains unclear. OBJECTIVE： To investigate changes in the P2R-mediated calcium signaling pathway in nociceptive trigemJnal ganglion neurons. DESIGN, TIME AND SETTING： In vitro experiments were conducted at the Patch-Clamp Laboratory of Comprehensive Experiment Center of Anhui Medical University, China from September 2008 to June 2009. MATERIALS： Thapsigargin, caffeine, suramin, and adenosine 5＇-triphosphate were purchased from Sigma, USA. METHODS： Using Fura-2-based microfluorimetry, intracellular calcium concentration （[Ca^2＋]i） was measured in freshly isolated adult rat small trigeminal ganglion neurons before and after drug application. MAIN OUTCOME MEASURES： Fluorescent intensities were expressed as the ratio F340/F380 to observe [Ca^2＋]i changes. RESULTS： In normal extracellular solution and Ca^2＋-free solution, application of thapsigargin （1 μmol/L）, a sarcoplasmic reticulum Ca^2＋ pump adenosine 5＇-triphosphate inhibitor, as well as caffeine （20 mmol/L）, a ryanodine receptor agonist, triggered [Ca^2＋]i increase in small trigeminal ganglion neurons. A similar response was induced by application of adenosine 5＇-triphosphate （100 μmol/L）. In Ca^2＋-free conditions, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were inhibited in cells pre-treated with thapsigargin （P 〈 0.01）, but not by caffeine （P 〉 0.05）. In normal, extracellular solution, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were partly inhibited in cells pre-treated with thapsigargin （P 〈 0.05）. CONCLUSION： Inositol-1,4, 5-triphosphate （IP3）- and ryanodine-sensitive Ca^2＋ stores exist in rat nociceptive trigeminal ganglion neurons. Two pathways are involved in the purinoreceptor-mediated [Ca^2＋]i rise observed in nociceptive trigeminal ganglion neurons. One pathway involves the metabotropic P2Y receptors, which are associated with the IP3 sensitive Ca^2＋store, and the second pathway is coupled to ionotropic P2X receptors that induce the Ca^2＋ influx.展开更多
Caveolae, specialized vesicular invaginations of the plasma membrane ,have attracted increasing attentions to those who are studing siginal transduction .Many proteins involved in signal transduction have been found e...Caveolae, specialized vesicular invaginations of the plasma membrane ,have attracted increasing attentions to those who are studing siginal transduction .Many proteins involved in signal transduction have been found enriched in these microdomains.Caveolins,as marker proteins of caveolae,form a scaffold onto which many classes of signaling molecules can assemble at steady state or after ligand-induced activiton .In addition to concentrating these signal transducers within a distinct region of the plasma membrane ,caveolin binding may functionally regulate the activation state of caveolae-associated signaling molecules.Thus,an emerging notion is that caveolae are signaling processing centers which orchestrate signaling events at the cell surface that influence cell function .展开更多
基金Supported by the grants of the National Natural Science Foundation of China(No.30973073 and 81172402)
文摘Objective Leucine-rich repeats and immunoglobulin-like domains 1(LRIG1) is a newly identified human gene that inhibits the epidermal growth factor receptor(EGFR), which on combining with a ligand, can drive tumor growth. This study investigated the interaction between human LRIG1 and EGFR and attempted to delineate the functions of as well as the mechanisms used by the extracellular(ECD) and cytoplasmic(CPD) domains of the human LRIG1 protein to downregulate human EGFR signaling activity.Methods Two constructed chimeric eukaryotic expression vectors, pIRES2-EGFP-3XFLAG-LRIG1-ET and p3FLAG-LRIG1-TC, encoding the extracellular and transmembrane regions(LRIG1-ET) and the transmembrane and cytoplasmic regions(LRIG1-TC), respectively, and the plasmid p3XFLAG-CMV-9-LRIG1 encoding full-length LRIG1(LRIG1-FL) were transfected into the human glioma cell line U251 or primary astrocytoma cells by using liposomes. The number and affinity of cell surface EGFR on transfected cells was determined by ^(125)I-EGF binding assay. Results The dissociation constant(KD) values for EGFR were higher, and the maximum increase was observed in the cells transfected into LRIG1-ET(1.36 folds). The number of maximal binding sites(Bmax) of the receptors was decreased in all transfected cells; the maximum decrease was noted in the cells transfected into LRIG1-FL(40.05%).Conclusion Both the ECD and CPD of LRIG1 are important to negate EGFR signaling. The ECD may interfere with the binding between EGFR and its ligand and facilitate the functions of CPD. The CPD may, when brought in proximity to EGFR, enhance receptor degradation. These two mechanisms can contribute to the downregulation of EGFR-mediated signaling by LRIG1.
基金the National Outstanding Youth Science foundation of China (B type,39825502)the National Natural Science Foundation of China (39880015,30170477)the Natural Science Foundation of Fujian Province (C0110004).
文摘AIM: To investigate the role of TR3 in induction of apoptosis in gastric cancer cells. METHODS: Human gastric cancer cell line, MGC80-3, was used. Expression of TR3 mRNA and its protein was detected by Northern blot and Western blot. Localization of TR3 protein was showed by immunofluorescence analysis under laser-scanning confocal microscope. Apoptotic morphology was observed by DAPI fluorescence staining, and apoptotic index was counted among 1000 cells randomly. Stable transfection assay was carried out by Lipofectamine. RESULTS: Treatment of MGC80-3 cells with TPA and VP-16 resulted in apoptosis, accompanied by the repression of Bcl-2 protein in a time-dependent manner. At the same time, TPA and VP-16 also up-regulated expression level of TR3 mRNA in MGC80-3 cells that expressed TR3 mRNA. When antisense-TR3 expression vector was transfected into the cells, expression of TR3 protein was repressed. In this case, TPA and VP-16 did not induce apoptosis. In addition, TPA and VP-16-induced apoptosis involved in translocation of TR3. In MGC80-3 cells, TR3 localized concentrative in nucleus, after treatment of cells with TPA and VP-16, TR3 translocated from nucleus to cytosol obviously. However, when this nuclear translocation was blocked by LMB, apoptosis was not occurred in MGC80-3 cells even in the presence of TPA and VP-16. CONCLUSION: Induction of apoptosis by TPA and VP-16 is through induction of TR3 expression and translocation of TR3 from nucleus to cytosol, which may be a novel signal pathway for TR3, and represent the new biological function of TR3 to exert its effect on apoptosis in gastric cancer cells.
基金supported by Chinese Ministry of Science and Technology(2015CB910201)Chinese Academy of Sciences Strategic Priority Research Program(XDB11020200)
文摘Plants are exposed to numerous potential pathogenic microbes. To counter the threat, plants have evolved diverse patternrecognition receptors(PRRs), which are receptor kinases(RKs) and receptor proteins(RPs) specialized to detect conserved pathogen/microbe-associated molecular patterns(PAMPs/MAMPs). Although only a handful of RKs and RPs are known PRRs,they belong to the receptor-like kinase(RLK) and receptor-like protein(RLP) superfamilies that undergo lineage-specific expansion, suggesting that many of these RLKs and RLPs are potential PRRs. Analyses of existing PRRs have uncovered ligand-induced RLK-RK or RLK-RP oligomerization as a common mechanism for immune activation. PRRs can recruit additional components to form dynamic receptor complexes, which mediate specific cellular responses. Detailed analyses of these components are shedding light on molecular mechanisms underlying the regulation of PRR activity and downstream signaling.
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the National Natural Science Foundation of China, No.30670694 the Natural Science Foundation of Anhui Province Department of Education in China, No.2006KJ361B+2 种基金 the National Science Fund for Distinguished Young Scholars of Anhui Medical University, No.GJJQ-0801 the Scientific Research Foundation for Doctor of Anhui Medical University, No. XJ2005006the Special Foundation for Young Scientists in Higher Education Institutions of Anhui Province, No.2010SQRL078
文摘BACKGROUND： Most of the currently available information on purinergic receptors （P2Rs） involved in pain transmission is based on results obtained in dorsal root ganglion or the spinal cord. However, the mechanism of P2Rs in trigeminal neuralgia remains unclear. OBJECTIVE： To investigate changes in the P2R-mediated calcium signaling pathway in nociceptive trigemJnal ganglion neurons. DESIGN, TIME AND SETTING： In vitro experiments were conducted at the Patch-Clamp Laboratory of Comprehensive Experiment Center of Anhui Medical University, China from September 2008 to June 2009. MATERIALS： Thapsigargin, caffeine, suramin, and adenosine 5＇-triphosphate were purchased from Sigma, USA. METHODS： Using Fura-2-based microfluorimetry, intracellular calcium concentration （[Ca^2＋]i） was measured in freshly isolated adult rat small trigeminal ganglion neurons before and after drug application. MAIN OUTCOME MEASURES： Fluorescent intensities were expressed as the ratio F340/F380 to observe [Ca^2＋]i changes. RESULTS： In normal extracellular solution and Ca^2＋-free solution, application of thapsigargin （1 μmol/L）, a sarcoplasmic reticulum Ca^2＋ pump adenosine 5＇-triphosphate inhibitor, as well as caffeine （20 mmol/L）, a ryanodine receptor agonist, triggered [Ca^2＋]i increase in small trigeminal ganglion neurons. A similar response was induced by application of adenosine 5＇-triphosphate （100 μmol/L）. In Ca^2＋-free conditions, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were inhibited in cells pre-treated with thapsigargin （P 〈 0.01）, but not by caffeine （P 〉 0.05）. In normal, extracellular solution, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were partly inhibited in cells pre-treated with thapsigargin （P 〈 0.05）. CONCLUSION： Inositol-1,4, 5-triphosphate （IP3）- and ryanodine-sensitive Ca^2＋ stores exist in rat nociceptive trigeminal ganglion neurons. Two pathways are involved in the purinoreceptor-mediated [Ca^2＋]i rise observed in nociceptive trigeminal ganglion neurons. One pathway involves the metabotropic P2Y receptors, which are associated with the IP3 sensitive Ca^2＋store, and the second pathway is coupled to ionotropic P2X receptors that induce the Ca^2＋ influx.
文摘Caveolae, specialized vesicular invaginations of the plasma membrane ,have attracted increasing attentions to those who are studing siginal transduction .Many proteins involved in signal transduction have been found enriched in these microdomains.Caveolins,as marker proteins of caveolae,form a scaffold onto which many classes of signaling molecules can assemble at steady state or after ligand-induced activiton .In addition to concentrating these signal transducers within a distinct region of the plasma membrane ,caveolin binding may functionally regulate the activation state of caveolae-associated signaling molecules.Thus,an emerging notion is that caveolae are signaling processing centers which orchestrate signaling events at the cell surface that influence cell function .
