CD147 deficiency in T cells prevents thymic involution by inhibiting the EMT process in TECs in the presence of TGFß.

Thymic involution during aging is a major cause of decreased T-cell production and reduced immunity. Here, we show that the loss of CD147 on T cells prevents thymic senescence, resulting in slowed shrinkage of the thymus with age and increased production of naive T cells. This phenotype is the result of slowing of the epithelial-mesenchymal transition (EMT) process in thymic epithelial cells (TECs), which eventually leads to reduced adipocyte accumulation. In an in vitro coculture system, we found that TGFß is an important factor in the EMT process in TECs and that it can reduce the expression of E-cadherin through p-Smad2/FoxC2 signaling. Moreover, CD147 on T cells can accelerate the decline in E-cadherin expression by interacting with Annexin A2 on TECs. In the presence of TGFß, Annexin A2 and E-cadherin colocalize on TECs. However, CD147 on T cells competitively binds to Annexin A2 on TECs, leading to the isolation of E-cadherin. Then, the isolated E-cadherin is easily phosphorylated by phosphorylated Src kinase, the phosphorylation of which was induced by TGFß, and finally, p-E-cadherin is degraded. Thus, in the thymus, the interaction between T cells and TECs contributes to thymic involution with age. In this study, we illuminate the mechanism underlying the triggering of the EMT process in TECs and show that inhibiting TGFß and/or CD147 may serve as a strategy to hinder age-related thymic involution.

First confirmatory study on PTPRQ as an autosomal dominant non-syndromic hearing loss gene.

BACKGROUND: Biallelic PTPRQ pathogenic variants have been previously reported as causative for autosomal recessive non-syndromic hearing loss. In 2018 the first heterozygous PTPRQ variant has been implicated in the development of autosomal dominant non-syndromic hearing loss (ADNSHL) in a German family. The study presented the only, so far known, PTPRQ pathogenic variant (c.6881G>A) in ADNSHL. It is located in the last PTPRQ coding exon and introduces a premature stop codon (p.Trp2294*). METHODS: A five-generation Polish family with ADNSHL was recruited for the study (n = 14). Thorough audiological, neurotological and imaging studies were carried out to precisely define the phenotype. Genomic DNA was isolated from peripheral blood samples or buccal swabs of available family members. Clinical exome sequencing was conducted for the proband. Family segregation analysis of the identified variants was performed using Sanger sequencing. Single nucleotide polymorphism array on DNA samples from the Polish and the original German family was used for genome-wide linkage analysis. RESULTS: Combining clinical exome sequencing and family segregation analysis, we have identified the same (NM_001145026.2:c.6881G>A, NP_001138498.1:p.Trp2294*) PTPRQ alteration in the Polish ADNSHL family. Using genome-wide linkage analysis, we found that the studied family and the original German family derive from a common ancestor. Deep phenotyping of the affected individuals showed that in contrast to the recessive form, the PTPRQ-related ADNSHL is not associated with vestibular dysfunction. In both families ADNSHL was progressive, affected mainly high frequencies and had a variable age of onset. CONCLUSION: Our data provide the first confirmation of PTPRQ involvement in ADNSHL. The finding strongly reinforces the inclusion of PTPRQ to the small set of genes leading to both autosomal recessive and dominant hearing loss.

Ptprj-as1 mediates inflammatory injury after intracerebral hemorrhage by activating NF-κB pathway.

OBJECTIVE: We aimed at investigating the expression of long non-coding RNA (lncRNA) Ptprj-as1 and the role of lncRNAPtprj-as1 in inflammatory cells after intracerebral hemorrhage and its potential mechanism. MATERIALS AND METHODS: The rat model of intracerebral hemorrhage was established. Expressions of Ptprj-as1 and inflammatory cytokines in inflammatory cells were detected by quantitative Real-time PCR (qRT-PCR). After BV2 cells were transfected with lentivirus, cell proliferation, migrative ability and apoptosis were detected by cell counting kit-8 (CCK-8) assay, transwell chamber and flow cytometry, respectively. Immunofluorescence was used to explore the ratio of M1 and M2 glial cells. The detection of tumor necrosis factor alpha (TNF-α) expression was performed using enzyme-linked immunosorbent assay (ELISA). Moreover, the expressions of key genes in NF-κB pathway were evaluated using Western blot. RESULTS: Ptprj-as1 was highly expressed in inflammatory tissues caused by intracerebral hemorrhage (ICH). Overexpressed Ptprj-as1 promoted the migration of BV2 cells and expression levels of inflammatory cytokines such as TNF-α, interleukin-1ß (IL-1ß), interleukin-6 (IL-6), iNOS and NO. Meanwhile, Ptprj-as1 enhanced the proportion of M1 glial cells, the mechanism of which might be related to the activation of NF-κB pathway. CONCLUSIONS: Ptprj-as1 activates NF-κB pathway in microglia and promotes the secretion of inflammatory cytokines, which is involved in inflammatory injury caused by intracerebral hemorrhage.

Identification of Protein Tyrosine Phosphatase Receptor Type O (PTPRO) as a Synaptic Adhesion Molecule that Promotes Synapse Formation.

The proper formation of synapses-specialized unitary structures formed between two neurons-is critical to mediating information flow in the brain. Synaptic cell adhesion molecules (CAMs) are thought to participate in the initiation of the synapse formation process. However, in vivo functional analysis demonstrates that most well known synaptic CAMs regulate synaptic maturation and plasticity rather than synapse formation, suggesting that either CAMs work synergistically in the process of forming synapses or more CAMs remain to be found. By screening for unknown CAMs using a co-culture system, we revealed that protein tyrosine phosphatase receptor type O (PTPRO) is a potent CAM that induces the formation of artificial synapse clusters in co-cultures of human embryonic kidney 293 cells and hippocampal neurons cultured from newborn mice regardless of gender. PTPRO was enriched in the mouse brain and localized to postsynaptic sites at excitatory synapses. The overexpression of PTPRO in cultured hippocampal neurons increased the number of synapses and the frequency of miniature EPSCs (mEPSCs). The knock-down (KD) of PTPRO expression in cultured neurons by short hairpin RNA (shRNA) reduced the number of synapses and the frequencies of the mEPSCs. The effects of shRNA KD were rescued by expressing either full-length PTPRO or a truncated PTPRO lacking the cytoplasmic domain. Consistent with these results, the N-terminal extracellular domain of PTPRO was required for its synaptogenic activity in the co-culture assay. Our data show that PTPRO is a synaptic CAM that serves as a potent initiator of the formation of excitatory synapses.SIGNIFICANCE STATEMENT The formation of synapses is critical for the brain to execute its function and synaptic cell adhesion molecules (CAMs) play essential roles in initiating the formation of synapses. By screening for unknown CAMs using a co-culture system, we revealed that protein tyrosine phosphatase receptor type O (PTPRO) is a potent CAM that induces the formation of artificial synapse clusters. Using loss-of-function and gain-of-function approaches, we show that PTPRO promotes the formation of excitatory synapses. The N-terminal extracellular domain of PTPRO was required for its synaptogenic activity in cultured hippocampal neurons and the co-culture assay. Together, our data show that PTPRO is a synaptic CAM that serves as a potent initiator of synapse formation.

Targeting Tie2 for Treatment of Diabetic Retinopathy and Diabetic Macular Edema.

Tie2 is a tyrosine kinase receptor located predominantly on vascular endothelial cells that plays a central role in vascular stability. Angiopoietin-1 (Angpt1), produced by perivascular cells, binds, clusters, and activates Tie2, leading to Tie2 autophosphorylation and downstream signaling. Activated Tie2 increases endothelial cell survival, adhesion, and cell junction integrity, thereby stabilizing the vasculature. Angiopoietin-2 (Angpt2) and vascular endothelial-protein tyrosine phosphatase (VE-PTP) are negative regulators increased by hypoxia; they inactivate Tie2, destabilizing the vasculature and increasing responsiveness to vascular endothelial growth factor (VEGF) and other inflammatory cytokines that stimulate vascular leakage and neovascularization. AKB-9778 is a small-molecule antagonist of VE-PTP which increases phosphorylation of Tie2 even in the presence of high Angpt2 levels. In preclinical studies, AKB-9778 reduced VEGF-induced leakage and ocular neovascularization (NV) and showed additive benefit when combined with VEGF suppression. In two clinical trials in diabetic macular edema (DME) patients, subcutaneous injections of AKB-9778 were safe and provided added benefit to VEGF suppression. Preliminary data suggest that AKB-9778 monotherapy improves diabetic retinopathy. These data suggest that Tie2 activation may be a valuable strategy to treat or prevent diabetic retinopathy.

Protein tyrosine phosphatase PTPRB regulates Src phosphorylation and tumour progression in NSCLC.

Protein tyrosine-phosphatases (PTPs) play important roles in various biological processes. Deregulation in PTP function has been implicated in carcinogenesis and tumour progression in many cancer types. However, the role of protein tyrosine phosphatase receptor type B (PTPRB) in non-small-cell lung cancer (NSCLC) tumorigenesis has not been investigated. Lentiviral vector expressing PTPRB cDNA or shRNA was infected into A549 and H1299 cell lines, followed by cell proliferation, colony formation, soft agar and invasion assays. A549 xenograft mouse model was used to evaluate in vivo function of PTPRB. Quantitative polymerase chain reaction (PCR) was used to measure PTPRB expression in NSCLC patient samples. Kaplan Meier analysis was performed to assess association between PTPRB expression and patient overall survival (OS). Multivariate analysis was performed to evaluate prognostic significance of PTPRB. Overexpression of PTPRB reduced cell proliferation rate, colony formation efficiency, soft agar growth and cell invasion in A549 and H1299 cells, as well as tumour growth rate in A549 xenograft. Knockdown of PTPRB increased Src phosphorylation and cell invasion, which was reversed by Src inhibitor PP2. Additionally, PTPRB was down-regulated in NSCLC patient and was associated with patient OS. PTPRB regulates Src phosphorylation and tumorigenesis in NSCLC. PTPRB may serve as an independent prognostic biomarker for NSCLC patients.

PTPRO-mediated autophagy prevents hepatosteatosis and tumorigenesis.

Autophagy plays a critical role in the progression of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). Protein tyrosine phosphatase receptor type O (PTPRO) was recently identified as a tumor suppressor, but little is known about its role in NASH. Here, we investigated the role of PTPRO-dependent autophagy in insulin resistance, lipid metabolism, and hepatocarcinogenesis. Wild-type (WT) and ptpro-/- mice were fed a high-fat diet (HFD) for another 16 weeks after diethylnitrosamine (DEN) injection to induce NASH. Ptpro-/- mice exhibited severe liver injury, insulin resistance, hepatosteatosis and autophagy deficiency compared with WT littermates. PTPRO deletion also promoted the induction of lipogenic target genes and decreases in ß-oxidation-related genes. Increased activation of AKT and accumulation of cytoplasmic p53 was detected in ptpro-/- mice, which in combination repressed autophagy. Intriguingly, hyperinsulinemia involving AKT activation was also exacerbated in HFD-fed mice due to PTPRO deletion. Activation of AKT induced stabilization of the MDMX/MDM2 heterocomplex, thus promoting p53 accumulation in the cytoplasm. Inhibition of AKT restored autophagy and p53 accumulation in hepatocytes, indicating that AKT acts upstream of p53. Due to hyperinsulinemia and autophagy deficiency, a HFD could aggravate steatohepatitis in ptpro-/- mice. Importantly, the expression of PTPRO was much decreased in human steatohepatitis, which was associated with increased p62 accumulation. Together, these data indicate that PTPRO regulates insulin and lipid metabolism via the PI3K/Akt/MDM4/MDM2/P53 axis by affecting autophagy.

Protein tyrosine phosphatase receptor type O expression in the tumor niche correlates with reduced tumor growth, angiogenesis, circulating tumor cells and metastasis of breast cancer.

Protein tyrosine phosphatase receptor type O (PTPRO) has been recognized as a tumor suppressor in various types of cancer cells. However, little attention has been given to the role of PTPRO expression in the tumor microenvironment. We aimed to reveal the role of PTPRO in the breast cancer niche. Py8119 mouse breast cancer cells were implanted orthotopically into female wild-type or ptpro-/- C57Bl/6 mice. We observed that the loss of PTPRO in the tumor niche was correlated with larger tumor volume, more metastases, increased number of circulating tumor cells (CTCs), less apoptosis and reduced necrosis rates in the orthotopic mouse model of breast cancer. The tumor microenvironment in the ptpro-/- mice also showed increased microvessel density. Moreover, an intracardiac injection mouse model was used to determine the role of PTPRO in the pre-metastatic niche. Notably, more metastases were observed in the mice of the ptpro-/- group. Taken together, PTPRO expression in the tumor niche prevents tumor growth and the formation of metastases of breast cancer, in part by attenuating tumor-associated angiogenesis and inducing the apoptosis and necrosis of tumor cells.

Blockage of PTPRJ promotes cell growth and resistance to 5-FU through activation of JAK1/STAT3 in the cervical carcinoma cell line C33A.

Gene therapy is a promising therapeutic approach for chemoresistant cervical cancers. Therapeutic interventions targeting the key factors contributing to the initiation and progression of cervical cancer may be a more effective treatment strategy. In the present study, we firstly determined the expression of protein tyrosine phosphatase receptor J (PTPRJ) in 8-paired human cervical tumor and non-tumor tissues. We observed a striking downregulation of PTPRJ in the human cervical tumor tissues. Next, we investigated the roles and the function mechanism of PTPRJ in the human cervical carcinoma cell line C33A by loss- and gain-of-function experiments. Our study indicated that C33A cells with loss of PTPRJ expression showed a significantly increased cell viability, rising growth and migration rate, as well as a G1-S transition. We obtained the opposite results when we overexpressed PTPRJ in C33A cells. Our further study indicated that PTPRJ levels were highly correlated with cell survival when the C33A cells were treated with 5-fluorouracil (5-FU), an important chemotherapeutic agent for cervical cancer. In addition, the signaling pathway screening assay showed an obvious alteration of the Janus kinase 1/signal transducer and activator of transcription 3 (JAK1/STAT3) pathway. PTPRJ negatively regulated the activation of the JAK1/STAT3 pathway by decreasing the phosphorylation levels of JAK1 and STAT3. In addition, PTPRJ also regulated the expression of the downstream factors of STAT3, such as cyclin D, Bax, VEGF and MMP2. Our results suggest that PTPRJ may be a promising gene therapy target and its therapeutic potential can be fulfilled when used alone, or in combination with other anticancer agents.

PTPROt-mediated regulation of p53/Foxm1 suppresses leukemic phenotype in a CLL mouse model.

The gene encoding PTPROt (truncated isoform of protein tyrosine phosphatase receptor-type O) is methylated and suppressed in chronic lymphocytic leukemia (CLL). PTPROt exhibits in vitro tumor-suppressor characteristics through the regulation of B-cell receptor (BCR) signaling. Here we generated transgenic (Tg) mice with B-cell-specific expression of PTPROt. Although lymphocyte development is normal in these mice, crossing them with TCL1 Tg mouse model of CLL results in a survival advantage compared with the TCL1 Tg mice. Gene expression profiling of splenic B-lymphocytes before detectable signs of CLL followed by Ingenuity Pathway Analysis revealed that the most prominently regulated functions in TCL1 Tg vs non-transgenic (NTg) and TCL1 Tg vs PTPROt/TCL1 double Tg are the same and also biologically relevant to this study. Further, enhanced expression of the chemokine Ccl3, the oncogenic transcription factor Foxm1 and its targets in TCL1 Tg mice were significantly suppressed in the double Tg mice, suggesting a protective function of PTPROt against leukemogenesis. This study also showed that PTPROt-mediated regulation of Foxm1 involves activation of p53, a transcriptional repressor of Foxm1, which is facilitated through suppression of BCR signaling. These results establish the in vivo tumor-suppressive function of PTPROt and identify p53/Foxm1 axis as a key downstream effect of PTPROt-mediated suppression of BCR signaling.

Protein tyrosine phosphatase receptor type O (Ptpro) regulates cerebellar formation during zebrafish development through modulating Fgf signaling.

Protein activities controlled by receptor protein tyrosine phosphatases (RPTPs) play comparably important roles in transducing cell surface signals into the cytoplasm by protein tyrosine kinases. Previous studies showed that several RPTPs are involved in neuronal generation, migration, and axon guidance in Drosophila, and the vertebrate hippocampus, retina, and developing limbs. However, whether the protein tyrosine phosphatase type O (ptpro), one kind of RPTP, participates in regulating vertebrate brain development is largely unknown. We isolated the zebrafish ptpro gene and found that its transcripts are primarily expressed in the embryonic and adult central nervous system. Depletion of zebrafish embryonic Ptpro by antisense morpholino oligonucleotide knockdown resulted in prominent defects in the forebrain and cerebellum, and the injected larvae died on the 4th day post-fertilization (dpf). We further investigated the function of ptpro in cerebellar development and found that the expression of ephrin-A5b (efnA5b), a Fgf signaling induced cerebellum patterning factor, was decreased while the expression of dusp6, a negative-feedback gene of Fgf signaling in the midbrain-hindbrain boundary region, was notably induced in ptpro morphants. Further analyses demonstrated that cerebellar defects of ptpro morphants were partially rescued by inhibiting Fgf signaling. Moreover, Ptpro physically interacted with the Fgf receptor 1a (Fgfr1a) and dephosphorylated Fgfr1a in a dose-dependant manner. Therefore, our findings demonstrate that Ptpro activity is required for patterning the zebrafish embryonic brain. Specifically, Ptpro regulates cerebellar formation during zebrafish development through modulating Fgf signaling.

Estrogen-sensitive PTPRO expression represses hepatocellular carcinoma progression by control of STAT3.

UNLABELLED: Protein tyrosine phosphatase receptor type O (PTPRO), one of the receptor types of phosphotyrosine phosphatases (PTP), was recently described as a tumor suppressor in various kinds of cancers. We aimed to clarify the role of PTPRO in hepatocellular carcinoma (HCC). It was demonstrated in 180 pairs (120 male and 60 female) of clinical HCC specimens that the PTPRO level was significantly reduced, as compared with adjacent tissue, and the PTPRO level in male adjacent tissue was lower than in female. We further found that estrogen receptor alpha (ERα) could up-regulate PTPRO expression as a transcription factor. Moreover, an in vitro study showed that cell proliferation was inhibited and apoptosis was promoted in PTPRO-transduced HCC cell lines, whereas an in vivo study represented that tumor number and size was increased in ptpro(-/-) mice. As a result of its tumor-suppressive position, PTPRO was proved to down-regulate signal transducers and activators of transcription (STAT3) activity dependent on Janus kinase 2 (JAK2) and phosphoinositide 3-kinase (PI3K) dephosphorylation. CONCLUSIONS: PTPRO expression results in pathological deficiency and gender bias in HCC, which could be attributed to ERα regulation. The suppressive role of PTPRO in HCC could be ascribed to STAT3 inactivation.

Dominant role of the protein-tyrosine phosphatase CD148 in regulating platelet activation relative to protein-tyrosine phosphatase-1B.

OBJECTIVE: The receptor-like protein-tyrosine phosphatase (PTP) CD148 and the nontransmembrane PTP1-B have been shown to be net positive regulators of Src family kinases in platelets. In the present study, we compared the relative contributions of these PTPs in platelet activation by the major glycoprotein, glycoprotein VI, α(IIb)ß(3), and C-type lectin-like receptor 2 (CLEC-2). METHODS AND RESULTS: PTP-1B-deficient mouse platelets responded normally to the glycoprotein VI-specific agonist collagen-related peptide and antibody-mediated CLEC-2 activation. However, they exhibited a marginal reduction in α(IIb)ß(3)-mediated Src family kinase activation and tyrosine phosphorylation. In contrast, CD148-deficient platelets exhibited a dramatic reduction in activation by glycoprotein VI and α(IIb)ß(3) and a marginal reduction in response to activation by CLEC-2, which was further enhanced in the absence of PTP-1B. These defects were associated with reduced activation of Src family kinase and spleen tyrosine kinase, suggesting a causal relationship. Under arteriolar flow conditions, there was defective aggregate formation in the absence of PTP-1B and, to a greater extent, CD148 and a severe abrogation of both adhesion and aggregation in the absence of both PTPs. CONCLUSIONS: Findings from this study demonstrate that CD148 plays a dominant role in activating Src family kinases in platelets relative to PTP-1B. Both PTPs are required for optimal platelet activation and aggregate formation under high arterial shear rates.

Polymorphisms of protein tyrosine phosphatase CD148 influence FcγRIIA-dependent platelet activation and the risk of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is due primarily to IgG antibodies specific to platelet factor 4/heparin complexes (PF4/Hs) that activate platelets via FcγRIIA. CD148 is a protein tyrosine phosphatase that regulates Src kinases and collagen-induced platelet activation. Three polymorphisms affecting CD148 (Q276P, R326Q, and D872E) were studied in HIT patients and 2 control groups, with or without antibodies to PF4/Hs. Heterozygote status for CD148 276P or 326Q alleles was less frequent in HIT patients, suggesting a protective effect of these polymorphisms. Aggregation tests performed with collagen, HIT plasma, and monoclonal antibodies cross-linking FcγRIIA showed consistent hyporesponsiveness of platelets expressing the 276P/326Q alleles. In addition, platelets expressing the 276P/326Q alleles exhibited a greater sensitivity to the Src family kinases inhibitor dasatinib in response to collagen or ALB6 cross-linking FcγRIIA receptors. Moreover, the activatory phosphorylation of Src family kinases was considerably delayed as well as the phosphorylation of Linker for activation of T cells and phospholipase Cγ2, 2 major signaling proteins downstream from FcγRIIA. In conclusion, this study shows that CD148 polymorphisms affect platelet activation and probably exert a protective effect on the risk of HIT in patients with antibodies to PF4/Hs.

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.

CD148 enhances platelet responsiveness to collagen by maintaining a pool of active Src family kinases.

SUMMARY BACKGROUND: We have previously shown that the receptor-like protein tyrosine phosphatase (PTP) CD148 is essential for initiating glycoprotein VI (GPVI) signaling in platelets. We proposed that CD148 does so by dephosphorylating the C-terminal inhibitory tyrosine of Src family kinases (SFKs). However, this mechanism is complicated by CD148-deficient mouse platelets having a concomitant reduction in GPVI expression. OBJECTIVES: To investigate the effect of CD148 on GPVI signaling independent of the decrease in GPVI expression and to further establish the molecular basis of the activatory effect of CD148 and downregulation of GPVI. METHODS: CD148-deficient mouse platelets were investigated for functional and biochemical defects. The DT40/NFAT-lucifierase reporter assay was used to analyze the effect of CD148 on GPVI signaling. CD148-SFK interactions and dephosphorylation were quantified using biochemical assays. RESULTS: CD148-deficient mouse platelets exhibited reduced collagen-mediated aggregation, secretion and spreading in association with reduced expression of GPVI and FcR gamma-chain and reduced tyrosine phosphorylation. The phosphorylation status of SFKs suggested a global reduction in SFK activity in resting CD148-deficient platelets. Studies in a cell model confirmed that CD148 inhibits GPVI signaling independent of a change in receptor expression and through a mechanism dependent on tyrosine dephosphorylation. Recombinant CD148 dephosphorylated the inhibitory tyrosines of Fyn, Lyn and Src in vitro, although paradoxically it also dephosphorylated the activation loop of SFKs. CONCLUSIONS: CD148 plays a critical role in regulating GPVI/FcR gamma-chain expression and maintains a pool of active SFKs in platelets by directly dephosphorylating the C-terminal inhibitory tyrosines of SFKs that is essential for platelet activation.

Embryonic stem cell tumor model reveals role of vascular endothelial receptor tyrosine phosphatase in regulating Tie2 pathway in tumor angiogenesis.

Inhibiting angiogenesis has become an effective approach for treating cancer and other diseases. However, our understanding of signaling pathways in tumor angiogenesis has been limited by the embryonic lethality of many gene knockouts. To overcome this limitation, we used the plasticity of embryonic stem (ES) cells to develop a unique approach to study tumor angiogenesis. Murine ES cells can be readily manipulated genetically; in addition, ES cells implanted subcutaneously in mice develop into tumors that contain a variety of cell types (teratomas). We show that ES cells differentiate into bona fide endothelial cells within the teratoma, and that these ES-derived endothelial cells form part of the functional tumor vasculature. Using this powerful and flexible system, the Angiopoietin/Tie2 system is shown to have a key role in the regulation of tumor vessel size. Endothelial differentiation in the ES teratoma model allows gene-targeting methods to be used in the study of tumor angiogenesis.

An unbiased screen identifies DEP-1 tumor suppressor as a phosphatase controlling EGFR endocytosis.

BACKGROUND: The epidermal growth factor (EGF) stimulates rapid tyrosine phosphorylation of the EGF receptor (EGFR). This event precedes signaling from both the plasma membrane and from endosomes, and it is essential for recruitment of a ubiquitin ligase, CBL, that sorts activated receptors to endosomes and degradation. Because hyperphosphorylation of EGFR is involved in oncogenic pathways, we performed an unbiased screen of small interfering RNA (siRNA) oligonucleotides targeting all human tyrosine phosphatases. RESULTS: We report the identification of PTPRK and PTPRJ (density-enhanced phosphatase-1 [DEP-1]) as EGFR-targeting phosphatases. DEP-1 is a tumor suppressor that dephosphorylates and thereby stabilizes EGFR by hampering its ability to associate with the CBL-GRB2 ubiquitin ligase complex. DEP-1 silencing enhanced tyrosine phosphorylation of endosomal EGFRs and, accordingly, increased cell proliferation. In line with functional interactions, EGFR and DEP-1 form physical associations, and EGFR phosphorylates a substrate-trapping mutant of DEP-1. Interestingly, the interactions of DEP-1 and EGFR are followed by physical segregation: whereas EGFR undergoes endocytosis, DEP-1 remains confined to the cell surface. CONCLUSIONS: EGFR and DEP-1 physically interact at the cell surface and maintain bidirectional enzyme-substrate interactions, which are relevant to their respective oncogenic and tumor-suppressive functions. These observations highlight the emerging roles of vesicular trafficking in malignant processes.

Dimerization of tyrosine phosphatase PTPRO decreases its activity and ability to inactivate TrkC.

Receptor-protein tyrosine phosphatases (RPTPs), like receptor tyrosine kinases, regulate neuronal differentiation. While receptor tyrosine kinases are dimerized and activated by extracellular ligands, the extent to which RPTPs dimerize, and the effects of dimerization on phosphatase activity, are poorly understood. We have examined a neuronal type III RPTP, PTPRO; we find that PTPRO can form dimers in living cells, and that disulfide linkages in PTPROs intracellular domain likely regulate dimerization. Dimerization of PTPROs transmembrane and intracellular domains, achieved by ligand binding to a chimeric fusion protein, decreases activity toward artificial peptides and toward a putative substrate, tropomyosin-related kinase C (TrkC). Dephosphorylation of TrkC by PTPRO may be physiologically relevant, as it is efficient, and TrkC and PTPRO can be co-precipitated from transfected cells. Inhibition of PTPROs phosphatase activity by dimerization is interesting, as dimerization of a related RPTP, CD148/PTPRJ, increases activity. Thus, our results suggest a complex relationship between dimerization and activity in type III RPTPs.