Regulation of Macrophage and Dendritic Cell Function by Chondroitin Sulfate in Innate to Antigen-Specific Adaptive Immunity.

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a linear acidic polysaccharide comprised of repeating disaccharides, modified with sulfate groups at various positions. Except for hyaluronan (HA), GAGs are covalently bound to core proteins, forming proteoglycans (PGs). With highly negative charges, GAGs interact with a variety of physiologically active molecules, including cytokines, chemokines, and growth factors, and control cell behavior during development and in the progression of diseases, including cancer, infections, and inflammation. Heparan sulfate (HS), another type of GAG, and HA are well reported as regulators for leukocyte migration at sites of inflammation. There have been many reports on the regulation of immune cell function by HS and HA; however, regulation of immune cells by CS has not yet been fully understood. This article focuses on the regulatory function of CS in antigen-presenting cells, including macrophages and dendritic cells, and refers to CSPGs, such as versican and biglycan, and the cell surface proteoglycan, syndecan.

Susceptibility to neurofibrillary tangles: role of the PTPRD locus and limited pleiotropy with other neuropathologies.

Tauopathies, including Alzheimer's disease (AD) and other neurodegenerative conditions, are defined by a pathological hallmark: neurofibrillary tangles (NFTs). NFT accumulation is thought to be closely linked to cognitive decline in AD. Here, we perform a genome-wide association study for NFT pathologic burden and report the association of the PTPRD locus (rs560380, P=3.8 × 10-8) in 909 prospective autopsies. The association is replicated in an independent data set of 369 autopsies. The association of PTPRD with NFT is not dependent on the accumulation of amyloid pathology. In contrast, we found that the ZCWPW1 AD susceptibility variant influences NFT accumulation and that this effect is mediated by an accumulation of amyloid ß plaques. We also performed complementary analyses to identify common pathways that influence multiple neuropathologies that coexist with NFT and found suggestive evidence that certain loci may influence multiple different neuropathological traits, including tau, amyloid ß plaques, vascular injury and Lewy bodies. Overall, these analyses offer an evaluation of genetic susceptibility to NFT, a common end point for multiple different pathologic processes.

Protein tyrosine phosphatase σ regulates autoimmune encephalomyelitis development.

Protein tyrosine phosphatases (PTPs) play essential roles in regulating signaling events in multiple cells by tyrosine dephosphorylation. One of them, PTPσ, appears important in regulating function of plasmacytoid dendritic cells (pDC). Here we report that PTPσ deletion in knockout mice and inhibition with a selective antagonist peptide exacerbated symptoms of experimental autoimmune encephalomyelitis (EAE) by enhancing axon and myelin damage in the spinal cord. PTPσ-/- mice displayed pro-inflammatory profiles in the spinal cord and lymphoid organs following MOG peptide immunization. PTPσ deletion promoted a pro-inflammatory phenotype in conventional DCs and directly regulated differentiation of CD4+ T cells. It also facilitated infiltration of T lymphocytes, activation of macrophages in the CNS and development of EAE. Therefore, PTPσ is a key negative regulator in EAE initiation and progression, which acts by regulating functions of DCs, T cells, and other immune cells. PTPσ may become an important molecular target for treating autoimmune disorders.

TGFß responsive tyrosine phosphatase promotes rheumatoid synovial fibroblast invasiveness.

OBJECTIVE: In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) ß-target gene, PTPRK, promotes RA FLS invasiveness. METHODS: Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The RPTPκ substrate was identified by pull-down assays. RESULTS: PTPRK expression was higher in FLS from patients with RA versus patients with osteoarthritis, resulting from increased TGFB1 expression in RA FLS. RPTPκ knockdown impaired RA FLS spreading, migration, invasiveness and responsiveness to platelet-derived growth factor, tumour necrosis factor and interleukin 1 stimulation. Furthermore, RPTPκ deficiency impaired the in vivo invasiveness of RA FLS. Molecular analysis revealed that RPTPκ promoted RA FLS migration by dephosphorylation of the inhibitory residue Y527 of SRC. CONCLUSIONS: By regulating phosphorylation of SRC, RPTPκ promotes the pathogenic action of RA FLS, mediating cross-activation of growth factor and inflammatory cytokine signalling by TGFß in RA FLS.

Receptor Protein Tyrosine Phosphatase Sigma (RPTP-σ) Increases pro-MMP Activity in a Trabecular Meshwork Cell Line Following Oxidative Stress Conditions.

PURPOSE: To elucidate the role of phosphatases in the eye drainage system by overexpressing the receptor tyrosine phosphatase sigma (RPTP-σ) in a human normal trabecular meshwork (NTM) cell line. METHODS: The efficacy, expression, and location of RPTP-σ were evaluated following its transfection in NTM cells (NTM(T)) and in NTM control cells. The cells were also analyzed for viability, matrix metalloproteinase (MMP) activity, and phosphatase activity following oxidative stress conditions. Assays were conducted in the presence or absence of a specific RPTP-σ inhibitor. RESULTS: Transfection efficacy measurements revealed that RPTP-σ expression measured via GFP fluorescence was significantly higher (×3.8) in NTM(T) cells than in control cells. Western blot analyses showed that RPTP-σ expression was significantly higher (×2.25) in NTM(T) cells than in control cells. No significant differences were observed in cell viability between NTM(T) and control cells after oxidative stress. We found that pro-MMP-2 and pro-MMP-9 showed a significantly higher activity (×2.18 and ×1.9; respectively) in NTM(T) cells than in control cells. Serine/threonine phosphatase activity in NTM(T) cells was significantly increased following oxidative stress. The specific phosphatase inhibitor PTP-IV inhibited 15% of the RPTP-σ expression in NTM cells and 31% in NTMT cells. The activity of pro-MMP-9, pro-MMP-2, and MMP-9 was significantly inhibited (48%, 35%, and 78% respectively). CONCLUSIONS: The findings indicate that RPTP-σ is expressed constituently in NTM cells and that oxidative stress changes the general phosphatase balance in NTM cells. In addition, the results show that expression levels of RPTP-σ affect the activity of various forms of MMP.

Protein tyrosine phosphatase receptor S acts as a metastatic suppressor in hepatocellular carcinoma by control of epithermal growth factor receptor-induced epithelial-mesenchymal transition.

UNLABELLED: Hepatocellular carcinoma (HCC) is the third-most lethal cancer worldwide. Understanding the molecular pathogenesis of HCC recurrence and metastasis is the key to improve patients' prognosis. In this study, we report that protein tyrosine phosphatase receptor S (PTPRS) is significantly down-regulated in nearly 80% of HCCs, and its expression negatively correlates with aggressive pathological features, such as larger tumor size and advanced stage. In addition, PTPRS deficiency is independently associated with shorter survival and increased recurrence in patients, although 16.7% of HCCs show intratumor heterogeneous expression of PTPRS. Restoration of wild-type, but not mutant, PTPRS expression significantly inhibits HCC cell migration and invasion in vitro as well as lung metastasis in vivo, whereas knockdown of its expression significantly promotes invasion and metastasis. Notably, PTPRS-regulated HCC invasiveness is accompanied by typical changes of epithelial-mesenchymal transition (EMT). Moreover, PTPRS forms a complex with epithermal growth factor receptor (EGFR) and regulates its tyrosine residues' phosphorylation. Ectopic expression of EGFR reverses the metastasis-inhibiting effects of PTPRS, whereas silencing of EGFR or inhibiting phosphorylation of key molecules in EGFR downstream pathways reinhibits EMT and metastasis caused by PTPRS down-regulation. Meanwhile, promoter hypermethylation of PTPRS is frequently detected in HCC samples and cell lines. Treatment with a demethylation agent, 5-aza-2'-deoxycytidine, recovers PTPRS expression in a dose-dependent manner. CONCLUSIONS: Epigenetic inactivation of PTPRS may increase phosphorylation and activity of EGFR signaling to promote EMT and metastasis in HCC.

Protein tyrosine phosphatase PTPRT as a regulator of synaptic formation and neuronal development.

PTPRT/RPTPρ is the most recently isolated member of the type IIB receptor-type protein tyrosine phosphatase family and its expression is restricted to the nervous system. PTPRT plays a critical role in regulation of synaptic formation and neuronal development. When PTPRT was overexpressed in hippocampal neurons, synaptic formation and dendritic arborization were induced. On the other hand, knockdown of PTPRT decreased neuronal transmission and attenuated neuronal development. PTPRT strengthened neuronal synapses by forming homophilic trans dimers with each other and heterophilic cis complexes with neuronal adhesion molecules. Fyn tyrosine kinase regulated PTPRT activity through phosphorylation of tyrosine 912 within the membrane-proximal catalytic domain of PTPRT. Phosphorylation induced homophilic cis dimerization of PTPRT and resulted in the inhibition of phosphatase activity. BCR-Rac1 GAP and Syntaxin-binding protein were found as new endogenous substrates of PTPRT in rat brain. PTPRT induced polymerization of actin cytoskeleton that determined the morphologies of dendrites and spines by inhibiting BCR-Rac1 GAP activity. Additionally, PTPRT appeared to regulate neurotransmitter release through reinforcement of interactions between Syntaxin-binding protein and Syntaxin, a SNARE protein. In conclusion, PTPRT regulates synaptic function and neuronal development through interactions with neuronal adhesion molecules and the dephosphorylation of synaptic molecules.

Protein tyrosine phosphatase-σ regulates hematopoietic stem cell-repopulating capacity.

Hematopoietic stem cell (HSC) function is regulated by activation of receptor tyrosine kinases (RTKs). Receptor protein tyrosine phosphatases (PTPs) counterbalance RTK signaling; however, the functions of receptor PTPs in HSCs remain incompletely understood. We found that a receptor PTP, PTPσ, was substantially overexpressed in mouse and human HSCs compared with more mature hematopoietic cells. Competitive transplantation of bone marrow cells from PTPσ-deficient mice revealed that the loss of PTPσ substantially increased long-term HSC-repopulating capacity compared with BM cells from control mice. While HSCs from PTPσ-deficient mice had no apparent alterations in cell-cycle status, apoptosis, or homing capacity, these HSCs exhibited increased levels of activated RAC1, a RhoGTPase that regulates HSC engraftment capacity. shRNA-mediated silencing of PTPσ also increased activated RAC1 levels in wild-type HSCs. Functionally, PTPσ-deficient BM cells displayed increased cobblestone area-forming cell (CAFC) capacity and augmented transendothelial migration capacity, which was abrogated by RAC inhibition. Specific selection of human cord blood CD34⁺CD38⁻CD45RA⁻lin⁻ PTPσ⁻ cells substantially increased the repopulating capacity of human HSCs compared with CD34⁺CD38⁻CD45RA⁻lin⁻ cells and CD34⁺CD38⁻CD45RA⁻lin⁻PTPσ⁺ cells. Our results demonstrate that PTPσ regulates HSC functional capacity via RAC1 inhibition and suggest that selecting for PTPσ-negative human HSCs may be an effective strategy for enriching human HSCs for transplantation.

Structural basis for extracellular cis and trans RPTPσ signal competition in synaptogenesis.

Receptor protein tyrosine phosphatase sigma (RPTPσ) regulates neuronal extension and acts as a presynaptic nexus for multiple protein and proteoglycan interactions during synaptogenesis. Unknown mechanisms govern the shift in RPTPσ function, from outgrowth promotion to synaptic organization. Here, we report crystallographic, electron microscopic and small-angle X-ray scattering analyses, which reveal sufficient inter-domain flexibility in the RPTPσ extracellular region for interaction with both cis (same cell) and trans (opposite cell) ligands. Crystal structures of RPTPσ bound to its postsynaptic ligand TrkC detail an interaction surface partially overlapping the glycosaminoglycan-binding site. Accordingly, heparan sulphate and heparin oligomers compete with TrkC for RPTPσ binding in vitro and disrupt TrkC-dependent synaptic differentiation in neuronal co-culture assays. We propose that transient RPTPσ ectodomain emergence from the presynaptic proteoglycan layer allows capture by TrkC to form a trans-synaptic complex, the consequent reduction in RPTPσ flexibility potentiating interactions with additional ligands to orchestrate excitatory synapse formation.

Structural basis for LAR-RPTP/Slitrk complex-mediated synaptic adhesion.

Synaptic adhesion molecules orchestrate synaptogenesis. The presynaptic leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) regulate synapse development by interacting with postsynaptic Slit- and Trk-like family proteins (Slitrks), which harbour two extracellular leucine-rich repeats (LRR1 and LRR2). Here we identify the minimal regions of the LAR-RPTPs and Slitrks, LAR-RPTPs Ig1-3 and Slitrks LRR1, for their interaction and synaptogenic function. Subsequent crystallographic and structure-guided functional analyses reveal that the splicing inserts in LAR-RPTPs are key molecular determinants for Slitrk binding and synapse formation. Moreover, structural comparison of the two Slitrk1 LRRs reveal that unique properties on the concave surface of Slitrk1 LRR1 render its specific binding to LAR-RPTPs. Finally, we demonstrate that lateral interactions between adjacent trans-synaptic LAR-RPTPs/Slitrks complexes observed in crystal lattices are critical for Slitrk1-induced lateral assembly and synaptogenic activity. Thus, we propose a model in which Slitrks mediate synaptogenic functions through direct binding to LAR-RPTPs and the subsequent lateral assembly of LAR-RPTPs/Slitrks complexes.

Slitrks control excitatory and inhibitory synapse formation with LAR receptor protein tyrosine phosphatases.

The balance between excitatory and inhibitory synaptic inputs, which is governed by multiple synapse organizers, controls neural circuit functions and behaviors. Slit- and Trk-like proteins (Slitrks) are a family of synapse organizers, whose emerging synaptic roles are incompletely understood. Here, we report that Slitrks are enriched in postsynaptic densities in rat brains. Overexpression of Slitrks promoted synapse formation, whereas RNAi-mediated knockdown of Slitrks decreased synapse density. Intriguingly, Slitrks were required for both excitatory and inhibitory synapse formation in an isoform-dependent manner. Moreover, Slitrks required distinct members of the leukocyte antigen-related receptor protein tyrosine phosphatase (LAR-RPTP) family to trigger synapse formation. Protein tyrosine phosphatase σ (PTPσ), in particular, was specifically required for excitatory synaptic differentiation by Slitrks, whereas PTPδ was necessary for inhibitory synapse differentiation. Taken together, these data suggest that combinatorial interactions of Slitrks with LAR-RPTP family members maintain synapse formation to coordinate excitatory-inhibitory balance.

Functional analysis of the putative tumor suppressor PTPRD in neuroblastoma cells.

The gene encoding PTPδ is mutated or downregulated in human cancers including neuroblastoma. Here, we functionally tested the tumor-suppressive potential of PTPδ in neuroblastoma cell lines by reconstitution of both short and long PTPδ isoforms. We did not observe any significant difference in colony forming ability between cells expressing wild-type or catalytically inactive PTPδ. Although endogenous PTPδ expression was very low in neuroblastoma cells, it was also low in mouse embryo adrenal glands, suggesting that PTPδ may have little developmental function in early adrenal neuroblasts. This study, therefore, questions the significance of PTPδ as a tumor suppressor protein in neuroblastoma.

LAR receptor tyrosine phosphatases and HSPGs guide peripheral sensory axons to the skin.

BACKGROUND: Peripheral axons of somatosensory neurons innervate the skin early in development to detect touch stimuli. Embryological experiments had suggested that the skin produces guidance cues that attract sensory axons, but neither the attractants nor their neuronal receptors had previously been identified. RESULTS: To investigate peripheral axon navigation to the skin, we combined live imaging of developing zebrafish Rohon-Beard (RB) neurons with molecular loss-of-function manipulations. Simultaneously knocking down two members of the leukocyte antigen-related (LAR) family of receptor tyrosine phosphatases expressed in RB neurons, or inhibiting their function with dominant-negative proteins, misrouted peripheral axons to internal tissues. Time-lapse imaging indicated that peripheral axon guidance, rather than outgrowth or maintenance, was defective in LAR-deficient neurons. Peripheral axons displayed a similar misrouting phenotype in mutants defective in heparan sulfate proteoglycan (HSPG) production and avoided regions in which HSPGs were locally degraded. CONCLUSIONS: HSPGs and LAR family receptors are required for sensory axon guidance to the skin. Together, our results support a model in which peripheral HSPGs are attractive ligands for LAR receptors on RB neurons.

Leukocyte common antigen-related phosphatase is a functional receptor for chondroitin sulfate proteoglycan axon growth inhibitors.

Chondroitin sulfate proteoglycans (CSPGs) are a family of extracellular matrix molecules with various functions in regulating tissue morphogenesis, cell division, and axon guidance. A number of CSPGs are highly upregulated by reactive glial scar tissues after injuries and form a strong barrier for axonal regeneration in the adult vertebrate CNS. Although CSPGs may negatively regulate axonal growth via binding and altering activity of other growth-regulating factors, the molecular mechanisms by which CSPGs restrict axonal elongation are not well understood. Here, we identified a novel receptor mechanism whereby CSPGs inhibit axonal growth via interactions with neuronal transmembrane leukocyte common antigen-related phosphatase (LAR). CSPGs bind LAR with high affinity in transfected COS-7 cells and coimmunoprecipitate with LAR expressed in various tissues including the brain and spinal cord. CSPG stimulation enhances activity of LAR phosphatase in vitro. Deletion of LAR in knock-out mice or blockade of LAR with sequence-selective peptides significantly overcomes neurite growth restrictions of CSPGs in neuronal cultures. Intracellularly, CSPG-LAR interaction mediates axonal growth inhibition of neurons partially via inactivating Akt and activating RhoA signals. Systemic treatments with LAR-targeting peptides in mice with thoracic spinal cord transection injuries induce significant axon growth of descending serotonergic fibers in the vicinity of the lesion and beyond in the caudal spinal cord and promote locomotor functional recovery. Identification of LAR as a novel CSPG functional receptor provides a therapeutic basis for enhancing axonal regeneration and functional recovery after CNS injuries in adult mammals.

Protein tyrosine phosphatase σ regulates the synapse number of zebrafish olfactory sensory neurons.

The formation and refinement of synaptic connections are key steps of neural development to establish elaborate brain networks. To investigate the functional role of protein tyrosine phosphatase (PTP) σ, we employed an olfactory sensory neuron (OSN)-specific gene manipulation system in combination with in vivo imaging of transparent zebrafish embryos. Knockdown of PTPσ enhanced the accumulation of synaptic vesicles in the axon terminals of OSNs. The exaggerated accumulation of synaptic vesicles was restored to the normal level by the OSN-specific expression of PTPσ, indicating that presynaptic PTPσ is responsible for the regulation of synaptic vesicle accumulation. Consistently, transient expression of a dominant-negative form of PTPσ in OSNs enhanced the accumulation of synaptic vesicles. The exaggerated accumulation of synaptic vesicles was reproduced in transgenic zebrafish lines carrying an OSN-specific expression vector of the dominant-negative PTPσ. By electron microscopic analysis of the transgenic line, we found the significant increase of the number of OSN-mitral cell synapses in the central zone of the olfactory bulb. The density of docked vesicles at the active zone was also increased significantly. Our results suggest that presynaptic PTPσ controls the number of OSN-mitral cell synapses by suppressing their excessive increase.

Tumour suppressor function of protein tyrosine phosphatase receptor-T.

It has long been thought that PTPs (protein tyrosine phosphatases) normally function as tumour suppressors. Recent high-throughput mutational analysis identified loss-of-function mutations in six PTPs in human colon cancers, providing critical cancer genetics evidence that PTPs can act as tumour suppressor genes. PTPRT (protein tyrosine phosphatase receptor-T), a member of the family of type IIB receptor-like PTPs, is the most frequently mutated PTP among them. Consistent with the notion that PTPRT is a tumour suppressor, PTPRT knockout mice are hypersensitive to AOM (azoxymethane)-induced colon cancer. The present review focuses on the physiological and pathological functions of PTPRT as well as the cellular pathways regulated by this phosphatase.

Regulatory role of leukocyte-common-antigen-related molecule (LAR) in thymocyte differentiation.

The strength of interaction between the antigenic peptide-loaded MHC (MHC/p) and the TCR determines T-cell fate in the thymus. A high avidity interaction between the TCR and the MHC/p induces apoptosis of self-reactive T cells (negative selection), whereas a moderate avidity interaction rescues thymocytes from apoptosis and permits further differentiation to mature T cells (positive selection). Leukocyte common antigen-related molecule (LAR), a receptor-like protein tyrosine phosphatase, is expressed on immature thymocytes, but its role in thymocyte differentiation has not yet been fully elucidated. We analyzed LAR-deficient mice and demonstrated that LAR deficiency affected the differentiation and expansion of immature thymocytes as well as positive and negative selection. Furthermore, LAR deficiency resulted in a lower Ca2+ response. The results indicate that LAR is an important modulator of TCR signaling that controls thymocyte differentiation.

Synapse formation regulated by protein tyrosine phosphatase receptor T through interaction with cell adhesion molecules and Fyn.

The receptor-type protein tyrosine phosphatases (RPTPs) have been linked to signal transduction, cell adhesion, and neurite extension. PTPRT/RPTPrho is exclusively expressed in the central nervous system and regulates synapse formation by interacting with cell adhesion molecules and Fyn protein tyrosine kinase. Overexpression of PTPRT in cultured neurons increased the number of excitatory and inhibitory synapses by recruiting neuroligins that interact with PTPRT through their ecto-domains. In contrast, knockdown of PTPRT inhibited synapse formation and withered dendrites. Incubation of cultured neurons with recombinant proteins containing the extracellular region of PTPRT reduced the number of synapses by inhibiting the interaction between ecto-domains. Synapse formation by PTPRT was inhibited by phosphorylation of tyrosine 912 within the membrane-proximal catalytic domain of PTPRT by Fyn. This tyrosine phosphorylation reduced phosphatase activity of PTPRT and reinforced homophilic interactions of PTPRT, thereby preventing the heterophilic interaction between PTPRT and neuroligins. These results suggest that brain-specific PTPRT regulates synapse formation through interaction with cell adhesion molecules, and this function and the phosphatase activity are attenuated through tyrosine phosphorylation by the synaptic tyrosine kinase Fyn.