Negative regulation of phagocytosis in macrophages by the CD47-SHPS-1 system

Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) is a transmembrane protein that is expressed predominantly in macrophages. Its extracellular region interacts with the transmembrane ligand CD47 expressed on the surface of adjacent cells, and its cytoplasmic region binds the protein tyrosine phosphatases SHP-1 and SHP-2. Phagocytosis of IgG- or complement-opsonized RBCs by peritoneal macrophages derived from mice that express a mutant SHPS-1 protein that lacks most of the cytoplasmic region was markedly enhanced compared with that apparent with wild-type macrophages. This effect was not observed either with CD47-deficient RBCs as the phagocytic target or in the presence of blocking Abs to SHPS-1. Depletion of SHPS-1 from wild-type macrophages by RNA interference also promoted FcgammaR-mediated phagocytosis of wild-type RBCs. Ligation of SHPS-1 on macrophages by CD47 on RBCs promoted tyrosine phosphorylation of SHPS-1 and its association with SHP-1, whereas tyrosine phosphorylation of SHPS-1 was markedly reduced in response to cross-linking of FcgammaRs. Treatment with inhibitors of PI3K or of Syk, but not with those of MEK or Src family kinases, abolished the enhancement of FcgammaR-mediated phagocytosis apparent in macrophages from SHPS-1 mutant mice. In contrast, FcgammaR-mediated tyrosine phosphorylation of Syk, Cbl, or the gamma subunit of FcR was similar in macrophages from wild-type and SHPS-1 mutant mice. These results suggest that ligation of SHPS-1 on macrophages by CD47 promotes the tyrosine phosphorylation of SHPS-1 and thereby prevents the FcgammaR-mediated disruption of the SHPS-1-SHP-1 complex, resulting in inhibition of phagocytosis. The inhibition of phagocytosis by the SHPS-1-SHP-1 complex may be mediated at the level of Syk or PI3K signaling.     

Enhanced phagocytosis of CD47-deficient red blood cells by splenic macrophages requires SHPS-1

The interaction of CD47 on red blood cells (RBCs) with SHPS-1 on macrophages is implicated to prevent the phagocytosis of the former cells by the latter cells. Indeed, the rate of clearance of transfused CD47-deficient (CD47(-/-)) RBCs from the bloodstream of wild-type mice was markedly increased compared with wild-type RBCs. Conversely, the rate of clearance of transfused wild-type RBCs was markedly increased in mice that expressed a mutant form of SHPS-1 lacking most of the cytoplasmic region of the protein. However, we here found that the clearance of CD47(-/-) RBCs in SHPS-1 mutant mice was minimal. In addition, the phagocytosis of CD47(-/-) RBCs by splenic macrophages from SHPS-1 mutant mice was markedly reduced compared with wild-type macrophages. These results thus suggest an additional role for CD47 on RBCs in the negative regulation of phagocytosis by macrophages and in determination of the life span of circulating RBCs.     

Tethering of apoptotic cells to phagocytes through binding of CD47 to Src homology 2 domain-bearing protein tyrosine phosphatase substrate-1

Apoptotic cells are swiftly phagocytosed by macrophages and immature dendritic cells. In this study, we found that one mouse macrophage cell line (BAM3) engulfed apoptotic thymocytes, but not a lymphoma cell line (WR19L). mAbs that inhibited the phagocytosis of apoptotic thymocytes by BAM3 were identified. Purification of the Ag revealed that it was Src homology 2 domain-bearing protein tyrosine phosphatase substrate-1 (SHPS-1). CD47, the ligand for SHPS-1, was expressed in mouse thymocytes, but not in WR19L. When WR19L was transformed with CD47, the transformants, after induction of apoptosis, could be phagocytosed by BAM3. The WR19L transformants expressing CD47 were more efficiently engulfed in vivo by splenic dendritic cells than the parental WR19L. Masking of the phosphatidylserine exposed on apoptotic thymocytes inhibited the engulfment, whereas the anti-SHPS-1 mAb inhibited not only the engulfment, but also the binding of apoptotic cells to phagocytes. These results indicate that macrophages require CD47 and phosphatidylserine on apoptotic cells for engulfment, and suggest that the interaction between CD47 and SHPS-1 works as a tethering step in the phagocytosis.     

Dual regulation of SIRPalpha phosphorylation by integrins and CD47

Signal regulatory protein alpha (SIRPalpha, SHPS-1) is a plasma membrane receptor for CD47 and a key regulator of phagocytosis, growth factor signaling, and migration. Phosphorylation of immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic tail is essential for the functional effects of SIRPalpha, at least in part, because the phosphorylated immunoreceptor tyrosine-based inhibition motifs recruit Src homology 2 domain-containing tyrosine phosphatases. Ligation by CD47 and integrin engagement both have been thought to regulate SIRPalpha phosphorylation. However, their distinct contributions have not been distinguished. Here, we show that the importance of CD47 varies with cell type, since ligation of CD47 is not necessary for SIRPalpha phosphorylation in myeloid cells, whereas it is required in endothelial cells. In contrast, integrin-mediated adhesion is required for SIRPalpha phosphorylation in both cell types. This shows that SIRPalpha phosphorylation is dually regulated and demonstrates a new mechanism for functional cooperation between integrins and the integrin-associated protein CD47.     

Differential involvement of Src family kinases in Fc gamma receptor-mediated phagocytosis

The tyrosine phosphorylation cascade originated from Fc gamma receptors (Fc gamma Rs) is essential for macrophage functions including phagocytosis. Although the initial step is ascribed to Src family tyrosine kinases, the role of individual kinases in phagocytosis signaling is still to be determined. In reconstitution experiments, we first showed that expression in the RAW 264.7 cell line of C-terminal Src kinase (Csk) inhibited and that of a membrane-anchored, gain-of-function Csk abolished the Fc gamma R-mediated signaling that leads to phagocytosis in a kinase-dependent manner. We next tested reconstruction of the signaling in the membrane-anchored, gain-of-function Csk-expressing cells by introducing Src family kinases the C-terminal negative regulatory sequence of which was replaced with a c-myc epitope. Those constructs derived from Lyn and Hck (a-Lyn and a-Hck) that associated with detergent-resistant membranes successfully reconstructed Fc gamma R-mediated Syk activation, filamentous actin rearrangement, and phagocytosis. In contrast, c-Src-derived construct (a-Src), that was excluded from detergent-resistant membranes, could not restore the series of phagocytosis signaling. Tyrosine phosphorylation of Vav and c-Cbl was restored in common by a-Lyn, a-Hck, and a-Src, but Fc gamma RIIB tyrosine phosphorylation, which is implicated in negative signaling, was reconstituted solely by a-Lyn and a-Hck. These findings suggest that Src family kinases are differentially involved in Fc gamma R-signaling and that selective kinases including Lyn and Hck are able to fully transduce phagocytotic signaling.     

Interaction between Src homology 2 domain bearing protein tyrosine phosphatase substrate-1 and CD47 mediates the adhesion of human B lymphocytes to nonactivated endothelial cells

CD47 modulates a variety of cell functions such as adhesion, spreading, and migration. Using a fusion protein consisting of the extracellular region of Src homology 2 domain bearing protein tyrosine phosphatase substrate-1 (SHPS-1) and the Fc portion of human Ig (SHPS-1-Ig) we investigated the effects of SHPS-1 as a ligand for CD47 on B lymphocytes. Although SHPS-1-Ig binding to human B cell lines was solely mediated via CD47, their binding capacity for soluble and immobilized SHPS-1-Ig varied among cell lines irrespective of the similar expression levels of CD47, suggesting that distinctive affinity/avidity states exist during B cell maturation. Nalm6 cell line and tonsilar B lymphocytes adhered to immobilized SHPS-1-Ig and showed polarization-like morphology. These effects of SHPS-1-Ig were blocked by anti-CD47 mAbs (B6H12 and SE5A5). Wortmannin, a phosphatidylinositol-3 kinase inhibitor, but not pertussis toxin significantly inhibited the polarization induced by the immobilized SHPS-1-Ig. Thus, SHPS-1 acts as an adhesive substrate via CD47 in human B lymphocyte. Immunohistochemical analyses indicated that SHPS-1 is expressed on high endothelial venule as well as macrophages in human tonsils. HUVECs also express SHPS-1 in the absence of any stimuli, and the adhesion of tonsilar B lymphocytes to nonactivated HUVECs was significantly inhibited by SE5A5, indicating that SHPS-1/CD47 interaction is involved in the adhesion. Our findings suggest that SHPS-1/CD47 interaction may contribute to the recruitment of B lymphocytes via endothelial cells under steady state conditions.     

Role of the CD47-SHPS-1 system in regulation of cell migration

SHPS-1 is a transmembrane protein whose extracellular region interacts with CD47 and whose cytoplasmic region undergoes tyrosine phosphorylation and there by binds the protein tyrosine phosphatase SHP-2. Formation of this complex is implicated in regulation of cell migration by an unknown mechanism. A CD47-Fc fusion protein or antibodies to SHPS-1 inhibited migration of human melanoma cells or of CHO cells overexpressing SHPS-1. Overexpression of wild-type SHPS-1 promoted CHO cell migration, whereas expression of the SHPS-1-4F mutant, which lacks the phosphorylation sites required for SHP-2 binding, had no effect. Antibodies to SHPS-1 failed to inhibit migration of CHO cells expressing SHPS-1-4F. SHPS-1 ligands induced the dephosphorylation of SHPS-1 and dissociation of SHP-2. Antibodies to SHPS-1 also enhanced Rho activity and induced both formation of stress fibers and adoption of a less polarized morphology in melanoma cells. Our results suggest that engagement of SHPS-1 by CD47 prevents the positive regulation of cell migration by this protein. The CD47- SHPS-1 system and SHP-2 might thus contribute to the inhibition of cell migration by cell-cell contact.     

Mycobacterium avium inhibition of IFN-gamma signaling in mouse macrophages: Toll-like receptor 2 stimulation increases expression of dominant-negative STAT1 beta by mRNA stabilization

Mycobacterial infections of macrophages have been shown to inhibit the ability of the macrophage to respond to IFN-gamma. We previously reported that Mycobacterium avium infection of mouse macrophages decreases IFN-gamma-induced STAT1 tyrosine phosphorylation and STAT1 DNA binding. Because macrophages respond to M. avium through Toll-like receptor 2 (TLR2), we determined whether TLR2 stimulation inhibits the response to IFN-gamma. Treatment of mouse RAW264.7 macrophages with TLR2 agonists inhibited the induction of IFN-gamma-inducible genes by IFN-gamma. In contrast to M. avium infection, TLR2 agonists did not inhibit the IFN-gamma induction of DNA-binding activity of STAT1 and the tyrosine phosphorylation of STAT1alpha. Instead, IFN-gamma induction of RAW264.7 cells treated with TLR2 agonists resulted in an increase in the tyrosine phosphorylation of the dominant-negative STAT1beta. TLR2 stimulation of RAW264.7 cells increased both STAT1beta protein and mRNA expression, suggesting that the increased STAT1beta phosphorylation results from increased STAT1beta expression. Because STAT1alpha and STAT1beta mRNA have different 3' untranslated regions, and 3' untranslated regions can regulate mRNA stability, we examined the effects of TLR2 stimulation on mRNA stability. TLR2 stimulation of RAW264.7 cells increased the stability of STAT1beta mRNA, while not affecting the stability of STAT1alpha mRNA. The ability of STAT1beta to function as a dominant negative was confirmed by overexpression of STAT1beta in RAW264.7 macrophages by transient transfection, which inhibited IFN-gamma-induced gene expression. These findings suggest that M. avium infection of mouse macrophages inhibits IFN-gamma signaling through a TLR2-dependent increase in STAT1beta expression by mRNA stablization and a TLR2-independent inhibition of STAT1 tyrosine phosphorylation.     

Resistance of B16 melanoma cells to CD47-induced negative regulation of motility as a result of aberrant N-glycosylation of SHPS-1

The adhesion receptor SHPS-1 activates the protein-tyrosine-phosphatase SHP-2 and thereby promotes integrin-mediated reorganization of the cytoskeleton. SHPS-1 also contributes to cell-cell communication through association with CD47. Although functional alteration of SHPS-1 is implicated in cellular transformation, the role of the CD47-SHPS-1 interaction in carcinogenesis has been unclear. A soluble SHPS-1 ligand (CD47-Fc) has now been shown to bind to Melan-a non-tumorigenic melanocytes but not to syngeneic B16F10 melanoma cells. Treatment of B16F10 cells with 1-deoxymannojirimycin, which prevents N-glycan processing, restored the ability of SHPS-1 derived from these cells to bind CD47-Fc in vitro, indicating that aberrant N-glycosylation of SHPS-1 impairs CD47 binding in B16F10 cells. CD47-Fc inhibited the migration of Melan-a cells but not that of B16F10 cells. However, a monoclonal antibody that reacts with SHPS-1 on both Melan-a and B16F10 cells inhibited the migration of both cell types similarly. CD47 binding induced proteasome-mediated degradation of SHPS-1 in a tyrosine phosphorylation-independent manner. Furthermore, overexpression of SHPS-1 reduced the level of tyrosine phosphorylation of focal adhesion kinase, and this effect was reversed by CD47 binding. These results suggest that CD47 binds to and thereby down-regulates SHPS-1 on adjacent cells, resulting in inhibition of cell motility. Resistance to this inhibitory mechanism may contribute to the highly metastatic potential of B16 melanoma.     

SHPS-1 induces aggregation of Ba/F3 pro-B cells via an interaction with CD47

SHPS-1 (SH2-domain bearing protein tyrosine phosphatase (SHP) substrate-1), a member of the inhibitory-receptor superfamily that is abundantly expressed in macrophages and neural tissue, appears to regulate intracellular signaling events downstream of receptor protein-tyrosine kinases and integrin-extracellular matrix molecule interactions. To investigate the function of SHPS-1 in a hematopoietic cell line, SHPS-1 was expressed in Ba/F3 cells, an IL-3-dependent pro-B-cell line that lacks endogenous SHPS-1 protein. Interestingly, expression of either SHPS-1, or a mutant lacking the intracellular domain of SHPS-1 (DeltaCT SHPS-1), resulted in the rapid formation of macroscopic Ba/F3 cell aggregates. As the integrin-associated protein/CD47 was shown to be a SHPS-1 ligand in neural cells, we investigated whether CD47 played a role in the aggregation of SHPS-1-expressing Ba/F3 cells. In support of this idea, aggregate formation was inhibited by an anti-CD47 Ab. Furthermore, erythrocytes from control, but not from CD47-deficient mice, were able to form rosettes on SHPS-1-expressing Ba/F3 cells. Because erythrocytes do not express integrins, this result suggested that SHPS-1-CD47 interactions can take place in the absence of a CD47-integrin association. We also present evidence that the amino-terminal Ig domain of SHPS-1 mediates the interaction with CD47. Although SHPS-1-CD47 binding likely triggers bidirectional intracellular signaling processes, these results demonstrate that this interaction can also mediate cell-cell adhesion.     

Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 regulates the migration of Langerhans cells from the epidermis to draining lymph nodes

Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1) is a member of the signal regulatory protein family in which the extracellular region interacts with its ligand, CD47. Recent studies have demonstrated that SHPS-1 plays an important role in cell migration and cell adhesion. We demonstrate in this study, using immunohistochemical and flow cytometric analyses, that murine Langerhans cells (LCs) express SHPS-1. Treatment of mice ears with 2,4-dinitro-1-fluorobenzene significantly reduced the number of epidermal LCs, and that reduction could be reversed by pretreatment with mAb to SHPS-1 or the CD47-Fc fusion protein. Treatment with the SHPS-1 mAb in vivo reduced the number of FITC-bearing cells in the lesional lymph nodes after the application of FITC to the skin. The SHPS-1 mAb inhibited the in vivo TNF-alpha-induced migration of LCs. The emigration of dendritic cells expressing I-A(b+) from skin explants to the medium was also reduced by the SHPS-1 mAb. We further demonstrate that the chemotaxis of a murine dendritic cell line, XS52, by macrophage inflammatory protein-3beta was significantly inhibited by treatment with the SHPS-1 mAb or CD47-Fc recombinant protein. Finally, we show that migration of LCs was attenuated in mutant mice that lack the intracellular domain of SHPS-1. These observations show that the ligation of SHPS-1 with the SHPS-1 mAb or with CD47-Fc abrogates the migration of LCs in vivo and in vitro, which suggests that the SHPS-1-CD47 interaction may negatively regulate LC migration.     

SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages.

Inhibitory immunoreceptors downregulate signaling by recruiting Src homology 2 (SH2) domain-containing tyrosine and/or lipid phosphatases to activating receptor complexes [1]. There are indications that some inhibitory receptors might also perform other functions [2] [3]. In adherent macrophages, two inhibitory receptors, SHPS-1 and PIR-B, are the major proteins binding to the tyrosine phosphatase SHP-1. SHPS-1 also associates with two tyrosine-phosphorylated proteins (pp55 and pp130) and a protein tyrosine kinase [4]. Here, we have identified pp55 and pp130 as the adaptor molecules SKAP55hom/R (Src-kinase-associated protein of 55 kDa homologue) and FYB/SLAP-130 (Fyn-binding protein/SLP-76-associated protein of 130 kDa), respectively, and the tyrosine kinase activity as PYK2. Two distinct SHPS-1 complexes were formed, one containing SKAP55hom/R and FYB/SLAP-130, and the other containing PYK2. Recruitment of FYB/SLAP-130 to SHPS-1 required SKAP55hom/R, whereas PYK2 associated with SHPS-1 independently. Formation of both complexes was independent of SHP-1 and tyrosine phosphorylation of SHPS-1. Finally, tyrosine phosphorylation of members of the SHPS-1 complexes was regulated by integrin-mediated adhesion. Thus, SHPS-1 provides a scaffold for the assembly of multi-protein complexes that might both transmit adhesion-regulated signals and help terminate such signals through SHP-1-directed dephosphorylation. Other inhibitory immunoreceptors might have similar scaffold-like functions.     

Regulation of multiple functions of SHPS-1, a transmembrane glycoprotein,by its cytoplasmic region

SHPS-1 is a receptor-type transmembrane glycoprotein, which contains four tyrosine residues in its cytoplasmic region, and the phosphorylation of these tyrosine residues serves the binding sites for SHP-2 protein-tyrosine phosphatase. Its extracellular region interacts with another membrane protein, CD47, thereby constituting a cell-cell communication system. We analyzed this ligand-receptor interaction using Chinese hamster ovary (CHO) cells expressing wild-type (WT) or mutant SHPS-1. The binding affinity of an SHPS-1 mutant such as deltaCyto, that lacked most of cytoplasmic region, or 4F, in which all four tyrosine residues in cytoplasmic region were substituted with phenylalanine, for a recombinant CD47-Fc was greater than that of WT. In addition, oligomerization of deltaCyto or 4F mutant by binding of CD47-Fc was greater than WT. Chemical cross-linking of SHPS-1 indicated that SHPS-1 formed a cis-dimer. Furthermore, WT cells exhibited a less polarized cell shape with decreased formation of actin stress fibers, compared with parental CHO cells and mutant SHPS-1 expressing cells. Prominent lamellipodium formation and membrane ruffling were also observed at leading edges of migrating WT cells but not at those of other mutant SHPS-1 expressing cells. These results suggest that the binding affinity of SHPS-1 to CD47, clustering ability of SHPS-1, and cytoskeletal reorganization are regulated by the cytoplasmic region of SHPS-1.     

SHPS-1 deficiency induces robust neuroprotection against experimental stroke by attenuating oxidative stress

Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), also known as Signal-regulatory protein alpha (SIRPα) or SIRPA is a transmembrane protein that is predominantly expressed in neurons, dendritic cells, and macrophages. This study was conducted to investigate the role of SHPS-1 in the oxidative stress and brain damage induced by acute focal cerebral ischemia. Wild-type (WT) and SHPS-1 mutant (MT) mice were subjected to middle cerebral artery occlusion (60 min) followed by reperfusion. SHPS-1 MT mice had significantly reduced infarct volumes and improved neurological function after brain ischemia. In addition, neural injury and oxidative stress were inhibited in SHPS-1 MT mice. The mRNA and protein levels of the antioxidant genes nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase 1 were up-regulated in SHPS-1 MT mice. The SHPS-1 mutation suppressed the phosphorylation of SHP-1 and SHP-2 and increased the phosphorylation of Akt and GSK3β. These results provide the first demonstration that SHPS-1 plays an important role in the oxidative stress and brain injury induced by acute cerebral ischemia. The activation of Akt signaling and the up-regulation of Nrf2 and heme oxygenase 1 likely account for the protective effects that were observed in the SHPS-1 MT mice.     

Regulation by Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 of alpha-galactosylceramide-induced antimetastatic activity and Th1 and Th2 responses of NKT cells

Interaction of alpha-galactosylceramide (alpha-GalCer) presented by CD1d on dendritic cells (DCs) with the invariant TCR of NKT cells activates NKT cells. We have now investigated the role of Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), a transmembrane protein abundantly expressed on DCs, in regulation of NKT cells with the use of mice that express a mutant form of SHPS-1. The suppression by alpha-GalCer of experimental lung metastasis was markedly attenuated in SHPS-1 mutant mice compared with that apparent in wild-type (WT) mice. The antimetastatic effect induced by adoptive transfer of alpha-GalCer-pulsed DCs from SHPS-1 mutant mice was also reduced compared with that apparent with WT DCs. Both the production of IFN-gamma and IL-4 as well as cell proliferation in response to alpha-GalCer in vitro were greatly attenuated in splenocytes or hepatic mononuclear cells from SHPS-1 mutant mice compared with the responses of WT cells. Moreover, CD4+ mononuclear cells incubated with alpha-GalCer and CD11c+ DCs from SHPS-1 mutant mice produced markedly smaller amounts of IFN-gamma and IL-4 than did those incubated with alpha-GalCer and CD11c+ DCs from WT mice. SHPS-1 on DCs thus appears to be essential for alpha-GalCer-induced antimetastatic activity and Th1 and Th2 responses of NKT cells. Moreover, our recent findings suggest that SHPS-1 on DCs is also essential for the priming of CD4+ T cells by DCs.     

Kinesin 5B is necessary for delivery of membrane and receptors during FcγR-mediated phagocytosis

FcγR-mediated phagocytosis is a cellular event that is evolutionary conserved to digest IgG-opsonized pathogens. Pseudopod formation during phagocytosis is a limiting step in managing the uptake of particles, and in this paper, we show that the conventional kinesin is involved in both receptor and membrane delivery to the phagocytic cup. Expression of a mutant kinesin isoform (GFP dominant negative mutant of kinesin H chain [EGFP-Kif5B-DN]) in RAW264.7 cells significantly reduced binding of IgG-sheep RBCs when macrophages were faced with multiple encounters with opsonized particles. Scanning electron microscopy analysis of EGFP-Kif5B-DN-expressing cells challenged with two rounds of IgG-sheep RBCs showed sparse, extremely thin pseudopods. We saw disrupted Rab11 trafficking to the phagocytic cup in EGFP-Kif5B-DN-transfected cells. Our particle overload assays also implicated phagosome membrane recycling in pseudopod formation. We observed reduced phagosome fission and trafficking in mutant kinesin-expressing cells, as well as reduced cell surface expression of FcγRs and Mac-1 receptors. In conclusion, anterograde trafficking via kinesin is essential for both receptor recycling from the phagosome and delivery of Rab11-containing membrane stores to effect broad and functional pseudopods during FcγR-mediated phagocytosis.     

Receptor activator of NF-kappa B ligand stimulates recruitment of SHP-1 to the complex containing TNFR-associated factor 6 that regulates osteoclastogenesis

Receptor activator of NF-kappaB ligand (RANKL) is essential for differentiation and function of osteoclasts. The negative signaling pathways downstream of RANKL are not well characterized. By retroviral transduction of RAW264.7 cells with a dominant negative Src homology 2 domain-containing phosphatase-1 (SHP-1)(C453S), we studied the role of tyrosine phosphatase SHP-1 in RANKL-induced osteoclastogenesis. Over-expression of SHP-1(C453S) significantly enhanced the number of tartrate-resistant acid phosphatase-positive multinuclear osteoclast-like cells in response to RANKL in a dose-dependent manner. RANKL induced the recruitment of SHP-1 to a complex containing TNFR-associated factor (TRAF)6. GST pull down experiments indicated that the association of SHP-1 with TRAF6 is mediated by SHP-1 lacking the two Src homology 2 domains. RANKL-stimulated IkappaB-alpha phosphorylation, IkappaB-alpha degradation and DNA binding ability of NF-kappaB were increased after over-expression of SHP-1(C453S). However, RANKL-induced phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, was unchanged. In addition, SHP-1 regulated RANKL-stimulated tyrosine phosphorylation of p85 subunit of phosphatidylinositol 3 kinase and the phosphorylation of Akt. Increased numbers of osteoclasts contribute to severe osteopenia in Me(v)/Me(v) mice due to mutation of SHP-1. Like RAW264.7 cells expressing SHP-1(C453S), the bone marrow macrophages of Me(v)/Me(v) mice generated much more osteoclast-like cells than that of littermate controls in response to RANKL. Furthermore compared with controls, RANKL induces enhanced association of TRAF6 and RANK in both RAW264.7 cells expressing SHP-1(C453S) and bone marrow macrophages from Me(v)/Me(v) mice. Therefore, SHP-1 plays a role in signals downstream of RANKL by recruitment to the complex containing TRAF6 and these observations may help to understand the mechanism of osteoporosis in Me(v)/Me(v) mice.     

Promotion of neurite and filopodium formation by CD47: roles of integrins, Rac, and Cdc42

Axon extension during development is guided by many factors, but the signaling mechanisms responsible for its regulation remain largely unknown. We have now investigated the role of the transmembrane protein CD47 in this process in N1E-115 neuroblastoma cells. Forced expression of CD47 induced the formation of neurites and filopodia. Furthermore, an Fc fusion protein containing the extracellular region of the CD47 ligand SHPS-1 induced filopodium formation, and this effect was enhanced by CD47 overexpression. SHPS-1-Fc also promoted neurite and filopodium formation triggered by serum deprivation. Inhibition of Rac or Cdc42 preferentially blocked CD47-induced formation of neurites and filopodia, respectively. Overexpression of CD47 resulted in the activation of both Rac and Cdc42. The extracellular region of CD47 was sufficient for the induction of neurite formation by forced expression, but the entire structure of CD47 was required for enhancement of filopodium formation by SHPS-1-Fc. Neurite formation induced by CD47 was also inhibited by a mAb to the integrin beta3 subunit. These results indicate that the interaction of SHPS-1 with CD47 promotes neurite and filopodium formation through the activation of Rac and Cdc42, and that integrins containing the beta3 subunit participate in the effect of CD47 on neurite formation.     

人参根提取物增强巨噬细胞RAW264.7的自噬水平并提高其增殖和吞噬能力

研究人参根提取物对巨噬细胞RAW264.7的增殖能力、吞噬能力和自噬水平的影响以及其相关性。用细胞计数试剂(CCK-8)检测不同浓度的人参根以及加入对巨噬细胞RAW264.7增殖的影响;采用中性红吞噬实验检测人参根提取物对巨噬细胞吞噬活性的影响;采用吖啶橙染色法(AO染色法)检测自噬体的形成;采用免疫印迹法(Western blot)检测自噬相关蛋白LC3B、ATG7的表达变化以及自噬通路相关蛋白AMPK、AKT、mTOR及ULK1磷酸化的变化。引入自噬诱导剂rapamycin和自噬抑制剂CQ探讨人参根提取物影响细胞自噬与细胞增殖、吞噬能力的相关性。结果显示,与空白对照组比较,各组人参根提取物可促进巨噬细胞RAW264.7的增殖及吞噬作用;参根提取物能够增加巨噬细胞RAW264.7酸性自噬体的数量,提高LC3B、ATG7的表达、增加AMPK和ULK1的蛋白磷酸化水平,时降低AKT,mTOR的磷酸化水平。Rapamycin进一步增强巨噬细胞的增殖以及吞噬能力,而CQ则减弱了人参根提取物所引起的巨噬细胞的增殖以及吞噬能力提高。以上结果表明人参根提取物可能通过增强巨噬细胞RAW264.7的自噬水平,提高其增殖和吞噬能力,具有激活巨噬细胞增强机体免疫力的潜在作用。     

Ectodomain shedding of SHPS-1 and its role in regulation of cell migration

SHPS-1 is a transmembrane protein whose cytoplasmic region undergoes tyrosine phosphorylation and then binds the protein-tyrosine phosphatase SHP-2. Formation of the SHPS-1-SHP-2 complex is implicated in regulation of cell migration. In addition, SHPS-1 and its ligand CD47 constitute an intercellular recognition system that contributes to inhibition of cell migration by cell-cell contact. The ectodomain of SHPS-1 has now been shown to be shed from cells in a reaction likely mediated by a metalloproteinase. This process was promoted by activation of protein kinase C or of Ras, and the released ectodomain exhibited minimal CD47-binding activity. Metalloproteinases catalyzed the cleavage of a recombinant SHPS-1-Fc fusion protein in vitro, and the primary cleavage site was localized to the juxtamembrane region of SHPS-1. Forced expression of an SHPS-1 mutant resistant to ectodomain shedding impaired cell migration, cell spreading, and reorganization of the actin cytoskeleton. It also increased the tyrosine phosphorylation of paxillin and FAK triggered by cell adhesion. These results suggest that shedding of the ectodomain of SHPS-1 plays an important role in regulation of cell migration and spreading by this protein.