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Aging leads to skeletal muscle atrophy (i.e., sarcopenia), and muscle fiber loss is a critical component of this process. The mechanisms underlying these age‐related changes, however, remain unclear. We show here that mTORC1 signaling is activated in a subset of skeletal muscle fibers in aging mouse and human, colocalized with fiber damage. Activation of mTORC1 in TSC1 knockout mouse muscle fibers increases the content of morphologically abnormal mitochondria and causes progressive oxidative stress, fiber damage, and fiber loss over the lifespan. Transcriptomic profiling reveals that mTORC1's activation increases the expression of growth differentiation factors (GDF3, 5, and 15), and of genes involved in mitochondrial oxidative stress and catabolism. We show that increased GDF15 is sufficient to induce oxidative stress and catabolic changes, and that mTORC1 increases the expression of GDF15 via phosphorylation of STAT3. Inhibition of mTORC1 in aging mouse decreases the expression of GDFs and STAT3's phosphorylation in skeletal muscle, reducing oxidative stress and muscle fiber damage and loss. Thus, chronically increased mTORC1 activity contributes to age‐related muscle atrophy, and GDF signaling is a proposed mechanism. [ABSTRACT FROM AUTHOR]

Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 1 and CEACAM20, immunoglobulin superfamily members, are predominantly expressed in intestinal epithelial cells (IECs) and co-localized at the apical surface of these cells. We here showed that the expression of mouse CEACAM1 and CEACAM20 at both mRNA and protein levels was markedly reduced in IECs of the small intestine by the treatment of mice with antibiotics against Gram-positive bacteria. The expression of both proteins was also decreased in IECs of the small intestine from germ-free mice, compared with that from control specific-pathogen-free mice. Exposure of intestinal organoids to IFN-γ markedly increased the expression of either CEACAM1 or CEACAM20, whereas the exposure to TNF-α increased the expression of the former protein, but not that of the latter. In contrast, the expression of CEACAM20, but not of CEACAM1, in intestinal organoids was markedly increased by exposure to butyrate, a short-chain fatty acid produced by bacterial fermentation in the intestine. Collectively, our results suggest that Gram-positive bacteria promote the mRNA expression of CEACAM1 or CEACAM20 in the small intestine. Inflammatory cytokines or butyrate likely participates in such effects of commensal bacteria. [ABSTRACT FROM AUTHOR]

Signal regulatory protein α ( SIRPα), an immunoglobulin superfamily protein that is expressed predominantly in myeloid lineage cells such as dendritic cells ( DCs) or macrophages, mediates cell-cell signaling. In the immune system, SIRPα is thought to be important for homeostasis of DCs, but it remains unclear whether SIRPα intrinsic to DCs is indeed indispensable for such functional role. Thus, we here generated the mice, in which SIRPα was specifically ablated in CD11c+ DCs ( SirpaΔ DC). SirpaΔ DC mice manifested a marked reduction of CD4+ CD8α- conventional DCs ( cDCs) in the secondary lymphoid organs, as well as of Langerhans cells in the epidermis. Such reduction of cDCs in SirpaΔ DC mice was comparable to that apparent with the mice, in which SIRPα was systemically ablated. Expression of SIRPα in DCs was well correlated with that of either endothelial cell-selective adhesion molecule ( ESAM) or Epstein-Barr virus-induced molecule 2 ( EBI2), both of which were also implicated in the regulation of DC homeostasis. Indeed, ESAM+ or EBI2+ cDCs were markedly reduced in the spleen of SirpaΔ DC mice. Thus, our results suggest that SIRPα intrinsic to CD11c+ DCs is essential for homeostasis of cDCs in the secondary lymphoid organs and skin. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphorylation is thought to be important for regulation of the proliferation, differentiation, and rapid turnover of intestinal epithelial cells (IECs). The role of protein tyrosine phosphatases in such homeostatic regulation of IECs has remained largely unknown, however. Src homology 2–containing protein tyrosine phosphatase (Shp2) is a ubiquitously expressed cytoplasmic protein tyrosine phosphatase that functions as a positive regulator of the Ras–mitogen-activated protein kinase (MAPK) signaling pathway operative downstream of the receptors for various growth factors and cytokines, and it is thereby thought to contribute to the regulation of cell proliferation and differentiation. We now show that mice lacking Shp2 specifically in IECs (Shp2 CKO mice) develop severe colitis and die as early as 3 to 4 weeks after birth. The number of goblet cells in both the small intestine and colon of Shp2 CKO mice was markedly reduced compared with that for control mice. Furthermore, Shp2 CKO mice showed marked impairment of both IEC migration along the crypt-villus axis in the small intestine and the development of intestinal organoids from isolated crypts. The colitis as well as the reduction in the number of goblet cells apparent in Shp2 CKO mice were normalized by expression of an activated form of K-Ras in IECs. Our results thus suggest that Shp2 regulates IEC homeostasis through activation of Ras and thereby protects against the development of colitis. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 121, 891-902. Abstract Signal regulatory protein α (SIRPα) is a neuronal membrane protein that undergoes tyrosine phosphorylation in the brain of mice in response to forced swim (FS) stress in cold water, and this response is implicated in regulation of depression-like behavior in the FS test. We now show that subjection of mice to the FS in warm (37°C) water does not induce the tyrosine phosphorylation of SIRPα in the brain. The rectal temperature ( Trec) of mice was reduced to 27° to 30°C by performance of the FS for 10 min in cold water, whereas it was not affected by the same treatment in warm water. The level of tyrosine phosphorylation of SIRPα in the brain was increased by administration of ethanol or picrotoxin, starvation, or cooling after anesthesia, all of which also induced hypothermia. Furthermore, the tyrosine phosphorylation of SIRPα in cultured hippocampal neurons was induced by lowering the temperature of the culture medium. CD47, a ligand of SIRPα, as well as Src family kinases or SH2 domain-containing protein phosphatase 2 (Shp2), might be important for the basal and the hypothermia-induced tyrosine phosphorylation of SIRPα. Hypothermia is therefore likely an important determinant of both the behavioral immobility and tyrosine phosphorylation of SIRPα observed in the FS test. [ABSTRACT FROM AUTHOR]

Mononuclear phagocytes such as dendritic cells (DCs) and macrophages in the lamina propria (LP) are thought to be important for both induction of inflammatory responses and maintenance of immunologic tolerance in the mammalian intestine. The molecular mechanisms by which these cells regulate intestinal immunity have remained poorly understood, however. Signal regulatory protein α (SIRPα) is a transmembrane protein that is specifically expressed in DCs, macrophages and neutrophils. Here, we show that SIRPα is abundant in CD11c CD11b LP cells of the mouse intestine. Whereas SIRPα did not appear to be important for the steady-state homeostasis of mucosal immunity in the intestine, the flagellin-stimulated production of IL-17 or interferon (IFN)-γ by LP cells of SIRPα mutant (MT) mice that lack the cytoplasmic region of the protein was markedly decreased compared with that observed with wild-type cells. Moreover, the flagellin-induced production of IL-6 by LP cells from SIRPα MT mice was also greatly reduced. SIRPα MT mice were also resistant to the development of colitis induced by IL-10 deficiency. Our data thus suggest that SIRPα expressed on CD11c LP cells is important for the production of IL-17 or IFN-γ in the LP as well as for the development of colitis induced by IL-10 deficiency. [ABSTRACT FROM AUTHOR]

Severe stress induces changes in neuronal function that are implicated in stress-related disorders such as depression. The molecular mechanisms underlying the response of the brain to stress remain primarily unknown, however. Signal regulatory protein α (SIRPα) is an Ig-superfamily protein that undergoes tyrosine phosphorylation and binds the protein tyrosine phosphatase Shp2. Here we show that mice expressing a form of SIRPα that lacks most of the cytoplasmic region manifest prolonged immobility (depression-like behavior) in the forced swim (FS) test. FS stress induced marked tyrosine phosphorylation of SIRPα in the brain of wild-type mice through activation of Src family kinases. The SIRPα ligand CD47 was important for such SIRPα phosphorylation, and CD47-deficient mice also manifested prolonged immobility in the FS test. Moreover, FS stress-induced tyrosine phosphorylation of both the NR2B subunit of the NMDA subtype of glutamate receptor and the K+-channel subunit Kvβ2 was regulated by SIRPα. Thus, tyrosine phosphorylation of SIRPα is important for regulation of depression-like behavior in the response of the brain to stress. [ABSTRACT FROM AUTHOR]

Vascular endothelial-protein tyrosine phosphatase (VE-PTP) is a receptor-type protein tyrosine phosphatase with a single catalytic domain in its cytoplasmic region and multiple fibronectin type III-like domains in its extracellular region. VE-PTP is expressed specifically in endothelial cells and is implicated in regulation of angiogenesis. The molecular basis for such regulation by VE-PTP has remained largely unknown, however. We now show that forced expression of VE-PTP promoted cell spreading as well as formation of lamellipodia and filopodia in cultured fibroblasts plated on fibronectin. These effects of VE-PTP on cell morphology required its catalytic activity as well as activation of integrins and Ras. In addition, VE-PTP-induced cell spreading and lamellipodium formation were prevented by inhibition of Src family kinases or of Rac or Cdc42. Indeed, forced expression of VE-PTP increased the level of c-Src phosphorylation at tyrosine-416. Moreover, the VE-PTP-induced changes in cell morphology were suppressed by expression of dominant negative forms of FRG or Vav2, both of which are guanine nucleotide exchange factors for Rho family proteins and are activated by tyrosine phosphorylation. Forced expression of VE-PTP also enhanced fibronectin-dependent migration of cultured fibroblasts. Conversely, depletion of VE-PTP by RNA interference in human umbilical vein endothelial cells or mouse endothelioma cells inhibited cell spreading on fibronectin. These results suggest that VE-PTP, in cooperation with integrins, regulates the spreading and migration of endothelial cells during angiogenesis. J. Cell. Physiol. 224:195–204, 2010 © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Post-translational modification of protein tyrosine phosphatases (PTPs) is implicated in functional modulation of these enzymes. Stomach cancer–associated protein tyrosine phosphatase-1 (SAP-1), as well as protein tyrosine phosphatase receptor type O (PTPRO) and vascular endothelial-protein tyrosine phosphatase (VE-PTP) are receptor-type PTPs (RPTPs), which belong to the R3 subtype RPTP family. Here, we have shown that the carboxyl (COOH)-terminal region of SAP-1 undergoes tyrosine phosphorylation by the treatment with a PTP inhibitor. Src family kinases are important for the tyrosine phosphorylation of SAP-1. Either Grb2 or Fyn, through their Src homology-2 domains, bound to the tyrosine-phosphorylated SAP-1. Moreover, both PTPRO and VE-PTP underwent tyrosine phosphorylation in their COOH-terminal regions. Tyrosine phosphorylation of VE-PTP or PTPRO also promoted their complex formations with Grb2 or Fyn. Forced expression of SAP-1, PTPRO or VE-PTP promoted cell spreading and lamellipodium formation of fibroblasts that expressed an activated form of Ras. In contrast, such effects of non-tyrosine-phosphorylated forms of these RPTPs were markedly smaller than those of wild-type RPTPs. Our results thus suggest that tyrosine phosphorylation of R3 subtype RPTPs promotes their complex formations with Grb2 or Fyn and thus participates in the regulation of cell morphology. [ABSTRACT FROM AUTHOR]

SHP-2 is a cytoplasmic protein tyrosine phosphatase (PTP) that contains two Src homology 2 (SH2) domains. Although PTPs are generally considered to be negative regulators on the basis of their ability to oppose the effects of protein tyrosine kinases, SHP-2 is unusual in that it promotes the activation of the Ras-MAPK signaling pathway by receptors for various growth factors and cytokines. The molecular basis for the activation of SHP-2 is also unique: In the basal state, the NH2-terminal SH2 domain of SHP-2 interacts with the PTP domain, resulting in autoinhibition of PTP activity; the binding of SHP-2 via its SH2 domains to tyrosine-phosphorylated growth factor receptors or docking proteins, however, results in disruption of this intramolecular interaction, leading to exposure of the PTP domain and catalytic activation. Indeed, SHP-2 proteins with artificial mutations in the NH2-terminal SH2 domain have been shown to act as dominant active mutants in vitro. Such activating mutations of PTPN11 (human SHP-2 gene) were subsequently identified in individuals with Noonan syndrome, a human developmental disorder that is sometimes associated with juvenile myelomonocytic leukemia. Furthermore, somatic mutations of PTPN11 were found to be associated with pediatric leukemia. SHP-2 is also thought to participate in the development of other malignant disorders, but in a manner independent of such activating mutations. Biochemical and functional studies of SHP-2 and genetic analysis of PTPN11 in human disorders have thus converged to provide new insight into the pathogenesis of cancer as well as potential new targets for cancer treatment. ( Cancer Sci 2009; 100: 1786–1793) [ABSTRACT FROM AUTHOR]

SAP-1 (PTPRH) is a receptor-type protein tyrosine phosphatase (RPTP) with a single catalytic domain in its cytoplasmic region and fibronectin type III-like domains in its extracellular region. The cellular localization and biological functions of this RPTP have remained unknown, however. We now show that mouse SAP-1 mRNA is largely restricted to the gastrointestinal tract and that SAP-1 protein localizes to the microvilli of the brush border in gastrointestinal epithelial cells. The expression of SAP-1 in mouse intestine is minimal during embryonic development but increases markedly after birth. SAP-1-deficient mice manifested no marked changes in morphology of the intestinal epithelium. In contrast, SAP-1 ablation inhibited tumorigenesis in mice with a heterozygous mutation of the adenomatous polyposis coli gene. These results thus suggest that SAP-1 is a microvillus-specific RPTP that regulates intestinal tumorigenesis. [ABSTRACT FROM AUTHOR]

SHPS-1 is a transmembrane protein predominantly expressed in macrophages. The possible role of SHPS-1 in regulation of Toll-like receptor (TLR)-dependent production of proinflammatory cytokines by macrophages has remained unknown, however. We now show that expression either of a mutant version of mouse SHPS-1 (SHPS-1–4F) in which the four tyrosine phosphorylation sites in the cytoplasmic region are replaced by phenylalanine or of a chimeric protein comprising the extracellular and transmembrane regions of human CD8 fused to the cytoplasmic region of SHPS-1–4F (CD8–4F) markedly promoted the production of tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid [poly(I : C)] in RAW264.7 macrophages. In contrast, expression of a mutant form of SHPS-1 that lacks most of the cytoplasmic region did not promote such responses. Expression of SHPS-1–4F promoted the LPS- or poly(I : C)-induced activation of NF-κB. LPS and poly(I : C) each induced the tyrosine phosphorylation of SHPS-1 through a Src family kinase and the association of SHPS-1 with SHP-1 and SHP-2. These results suggest that LPS or poly(I : C) induces tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with SHP-1 and SHP-2 in a manner dependent on a Src family kinase. SHPS-1 then negatively regulates TLR4- or TLR3-dependent cytokine production through inhibition of NF-κB-dependent signaling. [ABSTRACT FROM AUTHOR]

The development of axons and dendrites is controlled by small GTP-binding proteins of the Rho family, but the upstream signaling mechanisms responsible for such regulation remain unclear. We have now investigated the role of the transmembrane protein cluster of differentiation 47 (CD47) in this process with hippocampal neurons. CD47-deficient neurons manifested markedly impaired development of dendrites and axons, whereas overexpression of CD47 promoted such development. Interaction of SH2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) with CD47 also induced the formation of dendritic filopodia and spines. These effects of CD47 were prevented by inhibition of either cell division cycle 42 (Cdc42) or Rac. In CD47-deficient neurons, autophosphorylation of Src was markedly reduced. In addition, overexpression of CD47 promoted the autophosphorylation of Src. Inhibition of Src family kinases indeed prevented CD47-promoted dendritic development. Inhibition of either FGD1-related Cdc42-guanine nucleotide exchange factor (GEF) (FRG) or Vav2, which is a GEF for Cdc42 and Rac and is activated by Src, also prevented the effects of CD47 on dendritic development. These results indicate that CD47 promotes development of dendrites and axons in hippocampal neurons in a manner dependent, at least in part, on activation of Cdc42 and Rac mediated by Src as well as by FRG and Vav2. [ABSTRACT FROM AUTHOR]

CD47 is an integrin-associated penta-transmembrane protein that possesses an immunoglobulin-like domain in its extracellular region. We have now investigated the role of CD47 in the regulation of epithelial cell spreading and migration. CD47 is colocalized with E-cadherin at cell–cell adhesion sites of epithelial cells. A Ca2+ switch experiment showed that CD47 was endocytosed and then relocalized to cell–cell adhesion sites in a similar manner to E-cadherin. Such polarized localization of CD47 required the multiple spanning region of this protein. Forced expression of CD47 induced cell spreading with marked lamellipodium formation and resulted in both partial disruption of cell–cell adhesion and enhancement of the hepatocyte growth factor-stimulated scattering of Madin–Darby canine kidney cells. The CD47-induced cell spreading was blocked by inhibition of Src and mitogen-activated protein kinase kinase. Thus, these results suggest that CD47 participates in the regulation of cell–cell adhesion and cell migration through reorganization of the actin cytoskeleton in epithelial cells. This function of CD47 is mediated by the activation of Src and mitogen-activated protein kinase kinase. ( Cancer Sci 2006; 97: 889–895) [ABSTRACT FROM AUTHOR]

Polarized localization of membrane proteins to axons or dendrites is important for a variety of neuronal functions, including neurite outgrowth and synaptogenesis during neural development. Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) and its ligand cluster of differentiation 47 (CD47), both of which are members of the Ig superfamily of proteins, are thought to constitute an intercellular communication system in the CNS, although the physiological functions of this CD47-SHPS-1 system remain unknown. To provide insight into these functions, we have now examined the localization of SHPS-1 and CD47 in cultured hippocampal neurons. Endogenous SHPS-1 was detected at the surface of both axons and dendrites, whereas endogenous CD47 was localized predominantly to the surface of dendrites. Forced expression of these two proteins confirmed their distinct localizations, The extracellular regions of SHPS-1 and CD47 were responsible, at least in part, for their axonal and dendritic localizations, respectively; however, the axonal localization of SHPS-1 was not mediated by any one of the three Ig domains in its extracellular region. Overexpression of SHPS-1 and CD47 in distinct neurons resulted in marked accumulation of these proteins at sites of contact between SHPS-1-expressing axons and CD47-expressing dendrites. Such contact sites exhibited an enlarged structure but did not contain the synaptic marker protein vesicle-associated membrane protein-2. These results suggest that differential localization of SHPS-1 and CD47 at axons and dendrites generates a directional intercellular communication system that potentially contributes to regulation of synaptogenesis and the formation of neural networks. [ABSTRACT FROM AUTHOR]

CD47 and SHPS-1 are transmembrane proteins that interact with each other through their extracellular regions and constitute a bidirectional cell-cell communication system (the CD47-SHPS-1 system). We have now shown that the trans-interaction of CD47 and SHPS-1 that occurred on contact of CD47-expressing CHO cells and SHPS-1-expressing CHO cells resulted in endocytosis of the ligand-receptor complex into either cell type. Such trans-endocytosis of CD47 by SHPS-1-expressing cells was found to be mediated by clathrin and dynamin. A juxtamembrane region of SHPS-1 was indispensable for efficient trans-endocytosis of CD47, which was also regulated by Rac and Cdc42, probably through reorganization of the actin cytoskeleton. Inhibition of trans-endocytosis of CD47 promoted the aggregation of CD47-expressing cells with the cells expressing SHPS-1. Moreover, CD47 expressed on the surface of cultured mouse hippocampal neurons was shown to undergo trans-endocytosis by neighboring astrocytes expressing endogenous SHPS-1. These results suggest that trans-endocytosis of CD47 is responsible for removal of the CD47-SHPS-1 complex from the cell surface and hence regulates the function of the CD47-SHPS-1 system, at least in neurons and glial cells. [ABSTRACT FROM AUTHOR]