P48 is a cytokine which induces monocyte differentia-tion and the induction of cytotoxic activity. In this study,the signal transduction events involved in the stimulation of monocytes with the membrane form of P48 (m...P48 is a cytokine which induces monocyte differentia-tion and the induction of cytotoxic activity. In this study,the signal transduction events involved in the stimulation of monocytes with the membrane form of P48 (mP48) were investigated. Monocyte stimulation with mP48 was found to involve the mobilization of intracellular calcium (Ca2+)and the activation and translocation of PKC from the cy-tosol to the membrane. Membane P48 induced a rapid rise of intracellular Ca2+ in a dose dependent maner. Simi-larly the stimulation of monocytes with P48 was found to involve the activation and translocation of PKC. The translocation of PKC was rapid (within 0-5 min) yet tran-sient with PKC activity returning to control levels by 8 min. The functional role of protein kineses in P48 induced TNF secretion was studied using various kinese inhibitors. The PKC inhibitors, H-7 and sphingosine, were found to inhibit P48 induced TNF secretion with 50% inhibition at 5μM HA1004, which inhibts cyclic nucleotide-dependent kinase (PKA, Ki 1.2μM), did not inhibit TNF secretion. H-8 (PKA inhibitor) was found to be an effective inhibitor of TNF secretion only at high concentrations(30μp. The Calmodulin-dependent kinase inhibitor, W7 (Ki 12μM)was found to be effective at concentration above 5μM.These findings suggest that P48-triggered TNF secretion involves transmembrane Ca2+ signaling and the subse-quent activation of at least two protein kineses, PKC and CaMK.展开更多
RNA-binding proteins(RBPs)are widely involved in the transcriptional and posttranscriptional regulation of multiple biological processes.The transcriptional regulatory ability of RBPs was indicated by the identificati...RNA-binding proteins(RBPs)are widely involved in the transcriptional and posttranscriptional regulation of multiple biological processes.The transcriptional regulatory ability of RBPs was indicated by the identification of chromatin-enriched RBPs(Che-RBPs).One of these proteins,KH-type splicing regulatory protein(KHSRP),is a multifunctional RBP that has been implicated in mRNA decay,alternative splicing,and miRNA biogenesis and plays an essential role in myeloid differentiation by facilitating the maturation of miR-129.In this study,we revealed that KHSRP regulates monocytic differentiation by regulating gene transcription and RNA splicing.KHSRP-occupied specific genomic sites in promoter and enhancer regions to regulate the expression of several hematopoietic genes through transcriptional activation and bound to pre-mRNA intronic regions to modulate alternative splicing during monocytic differentiation.Of note,KHSRP had co-regulatory effects at both the transcriptional and posttranscriptional levels on MOGOH and ADARB1.Taken together,our analyses revealed the dual DNA-and RNA-binding activities of KHSRP and have provided a paradigm to guide the analysis of other functional Che-RBPs in different biological systems.展开更多
Objective To investigate the expression of mRNA and proteins ofβ-catenin,TCF-4(ICAT)and Wnt signaling pathway-related genes in the monocytic differentiation of acute myeloid leukemia HL-60 cells induced by a new ster...Objective To investigate the expression of mRNA and proteins ofβ-catenin,TCF-4(ICAT)and Wnt signaling pathway-related genes in the monocytic differentiation of acute myeloid leukemia HL-60 cells induced by a new steroidal drug NSC67657.Methods Wright’s staining andα-NBE staining were used to observe the differentiation of HL-60 cells after 5 days of展开更多
Growth of the Pacific white shrimp Litopenaeus vannamei,the most important farmed crustacean,has consistently been a focal point for breeders.Over the past decades,some candidate genes for shrimp growth have been iden...Growth of the Pacific white shrimp Litopenaeus vannamei,the most important farmed crustacean,has consistently been a focal point for breeders.Over the past decades,some candidate genes for shrimp growth have been identified.However,fur-ther research is needed to elucidate the molecular regulatory mechanism of these genes.LvMmd2 was previously identified as a candidate gene that may inhibit the growth of L.vannamei.In this study,we analyzed the genotype and expression of the LvMmd2 gene in a breeding family and indicated its role as a growth-inhibiting gene.We found that LvMmd2 co-localized with its homolog LvPAQR3 at the Golgi apparatus.Using co-immunoprecipitation(Co-IP)and DUAL membrane system yeast two-hybrid(MbY2H),we indicated the interactions between LvMmd2 and LvPAQR3,LvPAQR3 and LvRaf1,as well as LvMmd2 and LvRho.These results suggest that LvMmd2 directly and indirectly regulates the Ras signaling pathway.Furthermore,we show that the LvMmd2 gene may indirectly affect the PI3K/AKT,insulin,and Hippo signaling pathways to regulate cell proliferation and differentiation via LvPAQR3 and LvRaf1.Through transcriptome and MbY2H analyses,we have also revealed the interaction between LvMmd2 and proteins involved in growth,immunity,protein transport,synthesis,and modification.These findings demonstrate the various molecular pathways through which LvMmd2 regulates L.vannamei growth.This study provides insights into the mechanism of shrimp growth regulated by Mmd2,enhances our understanding of LvMmd2 function,and highlights its potential application in shrimp breeding.展开更多
During liver injury,intrahepatic macrophage compartment is augmented by circulating monocytes that infiltrate the liver driven by C-C motif chemokine ligand/C-C motif chemokine receptor(CCL/CCR)axis including CCL1‒CCR...During liver injury,intrahepatic macrophage compartment is augmented by circulating monocytes that infiltrate the liver driven by C-C motif chemokine ligand/C-C motif chemokine receptor(CCL/CCR)axis including CCL1‒CCR8 axis,thereby contributing to liver inflammation.Numerous small molecular receptor antagonists,including R243,have been developed for targeting CCR8;however,these agents face challenges in clinical translation,potentially attributed to their poor pharmacokinetic profiles,lack of target specificity,and potential adverse effects.In this study,we designed four CCR8 antagonizing peptides(AP8i-AP8iv)and performed molecular characterization in silico and therapeutic investigation in vitro and in vivo.Based on in silico docking,molecular dynamic simulation using homology build model and in-vitro(competitive)binding studies,AP8ii(YEWRFYHG)evidenced highly favorable and selective interactions at the CCR8-active site.AP8ii inhibited CCL1-driven chemotaxis and LPS/IFNg-induced pro-inflammatory activation of monocytes-macrophages in vitro.In a CCl4-induced acute liver injury mouse model,AP8ii treatment decreased intrahepatic infiltration of circulating monocytes.Moreover,AP8ii reduced liver inflammation,as indicated by decreased F4/80,IL6 and iNOS expression,diminished ALT levels,and attenuated fibrosis,as indicated by reduced collagen-I expression.In conclusion,we report a novel CCR8-antagonizing peptide that inhibited CCL1-driven intrahepatic monocytes infiltration and differentiation into pro-inflammatory phenotype,consequently ameliorating liver inflammation and fibrogenesis in an acute liver injury mouse model.展开更多
文摘P48 is a cytokine which induces monocyte differentia-tion and the induction of cytotoxic activity. In this study,the signal transduction events involved in the stimulation of monocytes with the membrane form of P48 (mP48) were investigated. Monocyte stimulation with mP48 was found to involve the mobilization of intracellular calcium (Ca2+)and the activation and translocation of PKC from the cy-tosol to the membrane. Membane P48 induced a rapid rise of intracellular Ca2+ in a dose dependent maner. Simi-larly the stimulation of monocytes with P48 was found to involve the activation and translocation of PKC. The translocation of PKC was rapid (within 0-5 min) yet tran-sient with PKC activity returning to control levels by 8 min. The functional role of protein kineses in P48 induced TNF secretion was studied using various kinese inhibitors. The PKC inhibitors, H-7 and sphingosine, were found to inhibit P48 induced TNF secretion with 50% inhibition at 5μM HA1004, which inhibts cyclic nucleotide-dependent kinase (PKA, Ki 1.2μM), did not inhibit TNF secretion. H-8 (PKA inhibitor) was found to be an effective inhibitor of TNF secretion only at high concentrations(30μp. The Calmodulin-dependent kinase inhibitor, W7 (Ki 12μM)was found to be effective at concentration above 5μM.These findings suggest that P48-triggered TNF secretion involves transmembrane Ca2+ signaling and the subse-quent activation of at least two protein kineses, PKC and CaMK.
基金This work was supported by the National Key Research and Development Program of China(2019YFA0801800,2021YFA1102400,2019YFA0802600 and 2021YFA0805703)the National Natural Science Foundation of China(81530007,31900072,31725013,82022001,82122005,81970103 and 81970101)CAMS Innovation Fund for Medical Sciences[2021-I2M-1-019 and 2021-I2M-1-040].
文摘RNA-binding proteins(RBPs)are widely involved in the transcriptional and posttranscriptional regulation of multiple biological processes.The transcriptional regulatory ability of RBPs was indicated by the identification of chromatin-enriched RBPs(Che-RBPs).One of these proteins,KH-type splicing regulatory protein(KHSRP),is a multifunctional RBP that has been implicated in mRNA decay,alternative splicing,and miRNA biogenesis and plays an essential role in myeloid differentiation by facilitating the maturation of miR-129.In this study,we revealed that KHSRP regulates monocytic differentiation by regulating gene transcription and RNA splicing.KHSRP-occupied specific genomic sites in promoter and enhancer regions to regulate the expression of several hematopoietic genes through transcriptional activation and bound to pre-mRNA intronic regions to modulate alternative splicing during monocytic differentiation.Of note,KHSRP had co-regulatory effects at both the transcriptional and posttranscriptional levels on MOGOH and ADARB1.Taken together,our analyses revealed the dual DNA-and RNA-binding activities of KHSRP and have provided a paradigm to guide the analysis of other functional Che-RBPs in different biological systems.
文摘Objective To investigate the expression of mRNA and proteins ofβ-catenin,TCF-4(ICAT)and Wnt signaling pathway-related genes in the monocytic differentiation of acute myeloid leukemia HL-60 cells induced by a new steroidal drug NSC67657.Methods Wright’s staining andα-NBE staining were used to observe the differentiation of HL-60 cells after 5 days of
基金funded by the National Key R&D Program of China(2022YFF1000304)the National Natural Sciences Foundation of China(32273102 and 31972782)+1 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences(Grant No.XDA24030105)the Taishan Scholars Program.
文摘Growth of the Pacific white shrimp Litopenaeus vannamei,the most important farmed crustacean,has consistently been a focal point for breeders.Over the past decades,some candidate genes for shrimp growth have been identified.However,fur-ther research is needed to elucidate the molecular regulatory mechanism of these genes.LvMmd2 was previously identified as a candidate gene that may inhibit the growth of L.vannamei.In this study,we analyzed the genotype and expression of the LvMmd2 gene in a breeding family and indicated its role as a growth-inhibiting gene.We found that LvMmd2 co-localized with its homolog LvPAQR3 at the Golgi apparatus.Using co-immunoprecipitation(Co-IP)and DUAL membrane system yeast two-hybrid(MbY2H),we indicated the interactions between LvMmd2 and LvPAQR3,LvPAQR3 and LvRaf1,as well as LvMmd2 and LvRho.These results suggest that LvMmd2 directly and indirectly regulates the Ras signaling pathway.Furthermore,we show that the LvMmd2 gene may indirectly affect the PI3K/AKT,insulin,and Hippo signaling pathways to regulate cell proliferation and differentiation via LvPAQR3 and LvRaf1.Through transcriptome and MbY2H analyses,we have also revealed the interaction between LvMmd2 and proteins involved in growth,immunity,protein transport,synthesis,and modification.These findings demonstrate the various molecular pathways through which LvMmd2 regulates L.vannamei growth.This study provides insights into the mechanism of shrimp growth regulated by Mmd2,enhances our understanding of LvMmd2 function,and highlights its potential application in shrimp breeding.
文摘During liver injury,intrahepatic macrophage compartment is augmented by circulating monocytes that infiltrate the liver driven by C-C motif chemokine ligand/C-C motif chemokine receptor(CCL/CCR)axis including CCL1‒CCR8 axis,thereby contributing to liver inflammation.Numerous small molecular receptor antagonists,including R243,have been developed for targeting CCR8;however,these agents face challenges in clinical translation,potentially attributed to their poor pharmacokinetic profiles,lack of target specificity,and potential adverse effects.In this study,we designed four CCR8 antagonizing peptides(AP8i-AP8iv)and performed molecular characterization in silico and therapeutic investigation in vitro and in vivo.Based on in silico docking,molecular dynamic simulation using homology build model and in-vitro(competitive)binding studies,AP8ii(YEWRFYHG)evidenced highly favorable and selective interactions at the CCR8-active site.AP8ii inhibited CCL1-driven chemotaxis and LPS/IFNg-induced pro-inflammatory activation of monocytes-macrophages in vitro.In a CCl4-induced acute liver injury mouse model,AP8ii treatment decreased intrahepatic infiltration of circulating monocytes.Moreover,AP8ii reduced liver inflammation,as indicated by decreased F4/80,IL6 and iNOS expression,diminished ALT levels,and attenuated fibrosis,as indicated by reduced collagen-I expression.In conclusion,we report a novel CCR8-antagonizing peptide that inhibited CCL1-driven intrahepatic monocytes infiltration and differentiation into pro-inflammatory phenotype,consequently ameliorating liver inflammation and fibrogenesis in an acute liver injury mouse model.
