Takenori Kotani

Expression, localization, and biological function of the R3 subtype of receptor-type protein tyrosine phosphatases in mammals.

The R3 subtype of receptor-type protein tyrosine phosphatases (RPTPs) includes VE-PTP, DEP-1, PTPRO, and SAP-1. All of these enzymes share a similar structure, with a single catalytic domain and putative tyrosine phosphorylation sites in the cytoplasmic region and fibronectin type III-like domains in the extracellular region. The expression of each R3 RPTP is largely restricted to a single or limited number of cell types, with VE-PTP and DEP-1 being expressed in endothelial or hematopoietic cells, PTPRO in neurons and in podocytes of the renal glomerulus, and SAP-1 in gastrointestinal epithelial cells. In addition, these RPTPs are localized specifically at the apical surface of polarized cells. The structure, expression, and localization of the R3 RPTPs suggest that they perform tissue-specific functions and that they might act through a common mechanism that includes activation of Src family kinases. In this review, we describe recent insights into R3-subtype RPTPs, particularly those of mammals.

Dendritic Cell-Specific Ablation of the Protein Tyrosine Phosphatase Shp1 Promotes Th1 Cell Differentiation and Induces Autoimmunity

Dendritic cells (DCs) promote immune responses to foreign Ags and immune tolerance to self-Ags. Deregulation of DCs is implicated in autoimmunity, but the molecules that regulate DCs to protect against autoimmunity have remained unknown. In this study, we show that mice lacking the protein tyrosine phosphatase Shp1 specifically in DCs develop splenomegaly associated with more CD11c+ DCs. Splenic DCs from the mutant mice showed upregulation of CD86 and CCR7 expression and of LPS-induced production of proinflammatory cytokines. The mice manifested more splenic Th1 cells, consistent with the increased ability of their DCs to induce production of IFN-γ by Ag-specific T cells in vitro. The number of splenic CD5+CD19+ B-1a cells and the serum concentrations of Igs M and G2a were also increased in the mutant mice. Moreover, aged mutant mice developed glomerulonephritis and interstitial pneumonitis together with increased serum concentrations of autoantibodies. Shp1 is thus a key regulator of DC functions that protects against autoimmunity.

Signal Regulatory Protein α Regulates the Homeostasis of T Lymphocytes in the Spleen

The molecular basis for formation of lymphoid follicle and its homeostasis in the secondary lymphoid organs remains unclear. Signal regulatory protein α (SIRPα), an Ig superfamily protein that is predominantly expressed in dendritic cells or macrophages, mediates cell–cell signaling by interacting with CD47, another Ig superfamily protein. In this study, we show that the size of the T cell zone as well as the number of CD4+ T cells were markedly reduced in the spleen of mice bearing a mutant (MT) SIRPα that lacks the cytoplasmic region compared with those of wild-type mice. In addition, the expression of CCL19 and CCL21, as well as of IL-7, which are thought to be important for development or homeostasis of the T cell zone, was markedly decreased in the spleen of SIRPα MT mice. By the use of bone marrow chimera, we found that hematopoietic SIRPα is important for development of the T cell zone as well as the expression of CCL19 and CCL21 in the spleen. The expression of lymphotoxin and its receptor, lymphotoxin β receptor, as well as the in vivo response to lymphotoxin β receptor stimulation were also decreased in the spleen of SIRPα MT mice. CD47-deficient mice also manifested phenotypes similar to SIRPα MT mice. These data suggest that SIRPα as well as its ligand CD47 are thus essential for steady-state homeostasis of T cells in the spleen.

Promotion of cell spreading and migration by vascular endothelial-protein tyrosine phosphatase (VE-PTP) in cooperation with integrins

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

Tyrosine phosphorylation of R3 subtype receptor‐type protein tyrosine phosphatases and their complex formations with Grb2 or Fyn

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.

Essential roles of SIRPα in homeostatic regulation of skin dendritic cells

Signal regulatory protein α (SIRPα) is an immunoglobulin superfamily protein that is predominantly expressed in dendritic cells (DCs). Its cytoplasmic region binds SHP-1 or SHP-2 protein tyrosine phosphatases, while its extracellular region interacts with CD47, another immunoglobulin superfamily protein, constituting cell-cell signaling. SIRPα was previously shown to be important for development of contact hypersensitivity, likely as a result of its positive regulation of the priming by DCs of CD4(+) T cells. However, the mechanism by which SIRPα regulates DC functions remains unknown. Here we found that the number of I-A(+) cells, which represent migratory DCs such as Langerhans cells (LCs) or dermal DCs from the skin, in the peripheral lymph nodes (LNs) was markedly decreased in mice expressing a mutant form of SIRPα that lacks the cytoplasmic region compared with that of wild-type (WT) mice. In addition, an increase of fluorescein isothiocyanate (FITC)-bearing I-A(+) cells in the draining lymph nodes (LNs) after skin-painting with FITC was markedly blunted in SIRPα mutant mice. However, migratory ability, as well as expression of CCR7, of bone marrow-derived DCs prepared from SIRPα mutant mice were not impaired. By contrast, the number of I-A(+) LCs in the epidermis of SIRPα mutant mice was markedly decreased compared with that of WT mice. In addition, the mRNA expression of transforming growth factor-β receptor II in LCs of SIRPα mutant mice was markedly decreased compared with that of WT mice. These results suggest that SIRPα is important for homeostasis of LCs in the skin, as well as of migratory DCs in the LNs, but unlikely for migration of these cells from the skin to draining LNs.

Expression of PTPRO in the interneurons of adult mouse olfactory bulb

PTPRO is a receptor‐type protein tyrosine phosphatase (PTP) with a single catalytic domain in its cytoplasmic region and multiple fibronectin type III‐like domains in its extracellular region. In the chick, PTPRO mRNA has been shown to be particularly abundant in embryonic brain, and PTPRO is implicated in axon growth and guidance during embryonic development. However, the temporal and spatial expression of PTPRO protein in the mammalian CNS, particularly in the juvenile and adult mammalian brain, has not been evaluated in any detail. By immunohistofluorescence analysis with a monoclonal antibody to PTPRO, we show that PTPRO is widely expressed throughout the mouse brain from embryonic day 16 to postnatal day 1, while expression is largely confined to the olfactory bulb (OB) and olfactory tubercle in the adult brain. In the OB, PTPRO protein is expressed predominantly in the external plexiform layer, the granule cell layer, and the glomerular layer (GL). In these regions, expression of PTPRO is predominant in interneurons such as γ‐aminobutyric acid (GABA)‐ergic or calretinin (CR)‐positive granule cells. In addition, PTPRO is expressed in GABAergic, CR‐positive, tyrosine hydroxylase‐positive, or neurocalcin‐positive periglomerular cells in the GL. Costaining of PTPRO with other neuronal markers suggests that PTPRO is likely to be localized to the dendrites or dendritic spines of these olfactory interneurons. Thus, PTPRO might participate in regulation of dendritic morphology or synapse formation of interneurons in the adult mouse OB. J. Comp. Neurol. 518:119–136, 2010.

Role of SIRPα in regulation of mucosal immunity in the intestine.

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.

Hypothermia-induced tyrosine phosphorylation of SIRPα in the brain

J. Neurochem. (2012) 121, 891–902.

Requirement of SIRPα for protective immunity against Leishmania major.

Signal regulatory protein α (SIRPα) is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is abundantly expressed on dendritic cells and macrophages. Wild-type (WT) C57BL/6 mice are known to be resistant to Leishmania major infection. We here found that C57BL/6 mice that express a mutant version of SIRPα lacking most of the cytoplasmic region manifested increased susceptibility to L. major infection, characterized by the marked infiltration of inflammatory cells in the infected lesions. The numbers of the parasites in footpads, draining lymph nodes and spleens were also markedly increased in the infected SIRPα mutant mice, compared with those for the infected WT mice. In addition, soluble leishmanial antigen-induced production of IFN-γ by splenocytes of the infected SIRPα mutant mice was markedly reduced. By contrast, the ability of macrophages of SIRPα mutant mice to produce nitric oxide in response to IFN-γ was almost equivalent to that of macrophages from WT mice. These results suggest that SIRPα is indispensable for protective immunity against L. major by the induction of Th1 response.