SHIP-1 is an SH2 domain containing inositol-5-phosphatase that appears to be a negative regulator of hematopoiesis. To the potential effects of SHIP-1 on MMP2 secretion and migration of cancer cells, three murine SHIP...SHIP-1 is an SH2 domain containing inositol-5-phosphatase that appears to be a negative regulator of hematopoiesis. To the potential effects of SHIP-1 on MMP2 secretion and migration of cancer cells, three murine SHIP-1 mutants were made： △SH2-SHIP-1, △Ptase-SHIP-1, △Cter-SHIP-1. These mutant forms were subcloned as well as the wild type （WT） of murine SHIP-1 cDNA were subcloned into pcDNA3 expression vector, then transfected into and overexpressed SHIP-1 and its mutants in a Src-transformed 3Y1 cellline （SR3Y1）. The results showed that overexpression of wild type of SHIP-1 does not affect the MMP2 secretion in both SR3Y1 and 3Y1 cells, but can induce MMP9 secretion, while either WT SHIP-1, the SH2 domain, phosphatase domain, or C terminus deletion mutants could significantly block the MMP2 and MMP9 secretion in SR3Y1 cells and suppress cell invasion ability. The results confirmed SHIP-1 as a negative regulator for cell migration and invasion in transformed cells, and implied that it may function through each of its three domains.展开更多
AIM To explore the mechanism by which microRNA-155 (miR-155) regulates the pathogenesis of experimental colitis. METHODS A luciferase assay was performed to confirm the binding of miR-155 to the SHIP-1 3'-UTR. MiR...AIM To explore the mechanism by which microRNA-155 (miR-155) regulates the pathogenesis of experimental colitis. METHODS A luciferase assay was performed to confirm the binding of miR-155 to the SHIP-1 3'-UTR. MiR-155 mimics, negative controls and SHIP-1 expression/knockdown vectors were established and then utilized in gain-and loss-of-function studies performed in raw264.7 cells and primary bone marrow-derived macrophages (BMDMs). Thereafter, dextran sulfate sodium (DSS)-induced colitis mouse model with or without antagomiR-155 treatment was established, and the levels of miR-155 and SHIP-1, as well as the pro-inflammatory capabilities, were measured by western blot, quantitative polymerase chain reaction, and immunohistochemistry. RESULTS MiR-155 directly bound to the 3'-UTR of SHIP-1 mRNA and induced a significant decrease in SHIP-1 expression in both raw264.7 cells and primary BMDMs. MiR-155 markedly promoted cell proliferation and proinflammatory secretions including IL-6, TNF-alpha, IL-1 beta, and IFN-gamma, whereas these effects could be reversed by the restoration of SHIP-1 expression. In vivo studies showed that antagomiR-155 administration could alleviate DSS-induced intestinal inflammation in Balb/c mice. Moreover, significantly increased SHIP-1 expression, as well as decreased Akt activation and in-flammatory response, were observed in the antagomiR-155-treated mice. CONCLUSION MiR-155 promotes experimental colitis by repressing SHIP-1 expression. Thus, the inhibition of miR-155 might be a promising strategy for therapy.展开更多
Toll-like receptor （TLR）-mediated inflammatory response could negatively affect bone metabolism. In this study, we determined how osteogenesis is regulated during inflammatory responses that are downstream of TLR si...Toll-like receptor （TLR）-mediated inflammatory response could negatively affect bone metabolism. In this study, we determined how osteogenesis is regulated during inflammatory responses that are downstream of TLR signaling. Human primary osteoblasts were cultured in collagen gels. Pam3CSK4 （P3C） and Escherichia coli lipopolysaccharide （EcLPS） were used as TLR2 and TLR4 ligand respectively. Porphyromonas gingivalis LPS having TLR2 activity with either TLR4 agonism （Pg1690） or TLR4 antagonism （Pg1449） and mutant E. coli LPS （LPxE/LPxF/WSK） were used. IL-lp, SH2-containing inositol phosphatase-1 （SHIP1） that has regulatory roles in osteogenesis, alkaline phosphatase and mineralization were analyzed. 3α-Aminocholestane （3AC） was used to inhibit SHIP1. Our results suggest that osteoblasts stimulated by P3C, poorly induced IL-1β but strongly upregulated SHIP1 and enhanced osteogenic mediators. On the contrary, EcLPS significantly induced IL-1β and osteogenic mediators were not induced. While Pg1690 downmodulated osteogenic mediators, Pg1449 enhanced osteogenic responses, suggesting that TLR4 signaling annuls osteogenesis even with TLR2 activity. Interestingly, mutant E. coli LPS that induces weak inflammation upregulated osteogenesis, but SHIP1 was not induced. Moreover, inhibiting SHIP1 significantly upregulated TLR2-mediated inflammatory response and downmodulated osteogenesis. In conclusion, these results suggest that induction of weak inflammatory response through TLR2 （with SHIP1 activity） and mutant TLR4 ligands could enhance osteogenesis.展开更多
基金the Sasagawa Medical Fellowship from Japan-Sino Medical Association with funds from the Nippon Foundation.
文摘SHIP-1 is an SH2 domain containing inositol-5-phosphatase that appears to be a negative regulator of hematopoiesis. To the potential effects of SHIP-1 on MMP2 secretion and migration of cancer cells, three murine SHIP-1 mutants were made： △SH2-SHIP-1, △Ptase-SHIP-1, △Cter-SHIP-1. These mutant forms were subcloned as well as the wild type （WT） of murine SHIP-1 cDNA were subcloned into pcDNA3 expression vector, then transfected into and overexpressed SHIP-1 and its mutants in a Src-transformed 3Y1 cellline （SR3Y1）. The results showed that overexpression of wild type of SHIP-1 does not affect the MMP2 secretion in both SR3Y1 and 3Y1 cells, but can induce MMP9 secretion, while either WT SHIP-1, the SH2 domain, phosphatase domain, or C terminus deletion mutants could significantly block the MMP2 and MMP9 secretion in SR3Y1 cells and suppress cell invasion ability. The results confirmed SHIP-1 as a negative regulator for cell migration and invasion in transformed cells, and implied that it may function through each of its three domains.
文摘AIM To explore the mechanism by which microRNA-155 (miR-155) regulates the pathogenesis of experimental colitis. METHODS A luciferase assay was performed to confirm the binding of miR-155 to the SHIP-1 3'-UTR. MiR-155 mimics, negative controls and SHIP-1 expression/knockdown vectors were established and then utilized in gain-and loss-of-function studies performed in raw264.7 cells and primary bone marrow-derived macrophages (BMDMs). Thereafter, dextran sulfate sodium (DSS)-induced colitis mouse model with or without antagomiR-155 treatment was established, and the levels of miR-155 and SHIP-1, as well as the pro-inflammatory capabilities, were measured by western blot, quantitative polymerase chain reaction, and immunohistochemistry. RESULTS MiR-155 directly bound to the 3'-UTR of SHIP-1 mRNA and induced a significant decrease in SHIP-1 expression in both raw264.7 cells and primary BMDMs. MiR-155 markedly promoted cell proliferation and proinflammatory secretions including IL-6, TNF-alpha, IL-1 beta, and IFN-gamma, whereas these effects could be reversed by the restoration of SHIP-1 expression. In vivo studies showed that antagomiR-155 administration could alleviate DSS-induced intestinal inflammation in Balb/c mice. Moreover, significantly increased SHIP-1 expression, as well as decreased Akt activation and in-flammatory response, were observed in the antagomiR-155-treated mice. CONCLUSION MiR-155 promotes experimental colitis by repressing SHIP-1 expression. Thus, the inhibition of miR-155 might be a promising strategy for therapy.
基金supported by Elam M. and Georgina E.Hack Memorial Research Funds,Department of Periodontics,University of Washington,Seattle,WA,USAsupported by WVCTSI funds,West Virginia University,Morgantown,WV,USA
文摘Toll-like receptor （TLR）-mediated inflammatory response could negatively affect bone metabolism. In this study, we determined how osteogenesis is regulated during inflammatory responses that are downstream of TLR signaling. Human primary osteoblasts were cultured in collagen gels. Pam3CSK4 （P3C） and Escherichia coli lipopolysaccharide （EcLPS） were used as TLR2 and TLR4 ligand respectively. Porphyromonas gingivalis LPS having TLR2 activity with either TLR4 agonism （Pg1690） or TLR4 antagonism （Pg1449） and mutant E. coli LPS （LPxE/LPxF/WSK） were used. IL-lp, SH2-containing inositol phosphatase-1 （SHIP1） that has regulatory roles in osteogenesis, alkaline phosphatase and mineralization were analyzed. 3α-Aminocholestane （3AC） was used to inhibit SHIP1. Our results suggest that osteoblasts stimulated by P3C, poorly induced IL-1β but strongly upregulated SHIP1 and enhanced osteogenic mediators. On the contrary, EcLPS significantly induced IL-1β and osteogenic mediators were not induced. While Pg1690 downmodulated osteogenic mediators, Pg1449 enhanced osteogenic responses, suggesting that TLR4 signaling annuls osteogenesis even with TLR2 activity. Interestingly, mutant E. coli LPS that induces weak inflammation upregulated osteogenesis, but SHIP1 was not induced. Moreover, inhibiting SHIP1 significantly upregulated TLR2-mediated inflammatory response and downmodulated osteogenesis. In conclusion, these results suggest that induction of weak inflammatory response through TLR2 （with SHIP1 activity） and mutant TLR4 ligands could enhance osteogenesis.
