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Cervical cancer (CC) is one of the most common malignant tumors. This study analyzed the impact of protein tyrosine phosphatase, receptor type B (PTPRB) on malignant behavior of CC and explored its possible molecular mechanism. RT-PCR, western blot and Immunohistochemistry were applied to examine the expression of PTPRB in CC specimens and cells. Aberrant PTPRB expression in CC and survival outcomes were constructed using The Cancer Genome Atlas (TCGA) database and tissue microarray cervical squamous cell carcinoma cohort. Cultured human CC cells were assayed for viability, apoptosis, migration, and invasion in vitro and in vivo. Kyoto Encyclopedia of Genes and Genomes (KEGG) assays and gene set enrichment analysis (GSEA) assays were used to delve into PTPRB-related pathways using TCGA datasets. The levels of proteins associated with the epithelial–mesenchymal transition (EMT) pathway and modulated by PTPRB were examined through Western blot. We found that the levels of PTPRB in CC tissues and cells were distinctly up-regulated. PTPRB was also an unfavorable prognostic factor for CC patients. Functionally, PTPRB knockdown exhibits tumor-suppressive function via reducing cell proliferation and metastasis and inducing cell apoptosis. KEGG assays and GSEA assays suggested PTPRB overexpression was associated with several tumor-related pathways. The results of Western blot assays suggested that N-cadherin was decreased in the PTPRB-knockdown CC cells, while E-cadherin was increased. Overall, PTPRB is highly expressed in CC and can effectively enhance the proliferation, metastasis and EMT process of tumor cells. PTPRB is expected to be a therapeutic target for CC.

Precise regulation of angiogenesis is required for organ development, wound repair, and tumor progression. Here, we identified a novel gene, nxhl (New XingHuo light), that is conserved in vertebrates and that plays a crucial role in vascular integrity and angiogenesis. Bioinformatic analysis uncovered its essential roles in development based on co-expression with several key developmental genes. Knockdown of nxhl in zebrafish causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels and decreased sprouting in the caudal vein plexus. The nxhl gene also affects human endothelial cell behavior in vitro. We found that nxhl functions in part by targeting VE-PTP through interaction with NCL (nucleolin). Loss of ptprb (a VE-PTP ortholo) in zebrafish resulted in defects similar to nxhl knockdown. Moreover, nxhl deficiency attenuates tumor invasion and proteins (including VE-PTP and NCL) associated with angiogenesis and EMT. These findings illustrate that nxhl can regulate angiogenesis via a novel nxhl–NCL–VE-PTP axis, providing a new therapeutic target for modulating vascular formation and function, especially for cancer treatment.

Vascular endothelial protein tyrosine phosphatase (VE-PTP, PTPRB) is a receptor type phosphatase that is crucial for the regulation of endothelial junctions and blood vessel development. We and others have shown recently that VE-PTP regulates vascular integrity by dephosphorylating substrates that are key players in endothelial junction stability, such as the angiopoietin receptor TIE2, the endothelial adherens junction protein VE-cadherin and the vascular endothelial growth factor receptor VEGFR2. Here, we have systematically searched for novel substrates of VE-PTP in endothelial cells by utilizing two approaches. First, we studied changes in the endothelial phosphoproteome on exposing cells to a highly VE-PTP-specific phosphatase inhibitor followed by affinity isolation and mass-spectrometric analysis of phosphorylated proteins by phosphotyrosine-specific antibodies. Second, we used a substrate trapping mutant of VE-PTP to pull down phosphorylated substrates in combination with SILAC-based quantitative mass spectrometry measurements. We identified a set of substrate candidates of VE-PTP, of which a remarkably large fraction (29%) is related to cell junctions. Several of those were found in both screens and displayed very high connectivity in predicted functional interaction networks. The receptor protein tyrosine kinase EPHB4 was the most prominently phosphorylated protein on VE-PTP inhibition among those VE-PTP targets that were identified by both proteomic approaches. Further analysis revealed that EPHB4 forms a ternary complex with VE-PTP and TIE2 in endothelial cells. VE-PTP controls the phosphorylation of each of these two tyrosine kinase receptors. Despite their simultaneous presence in a ternary complex, stimulating each of the receptors with their own specific ligand did not cross-activate the respective partner receptor. Our systematic approach has led to the identification of novel substrates of VE-PTP, of which many are relevant for the control of cellular junctions further promoting the importance of VE-PTP as a key player of junctional signaling.

Brown adipocytes have an important role in the regulation of energy balance through uncoupling protein-1 (UCP-1)-mediated nonshivering thermogenesis. Although brown adipocytes have been highlighted as a new therapeutic target for the treatment of metabolic diseases, such as obesity and type II diabetes in adult humans, the molecular mechanism underlying brown adipogenesis is not fully understood. We recently found that protein tyrosine phosphatase receptor type B (PTPRB) expression dramatically decreased during brown adipogenic differentiation. In this study, we investigated the functional roles of PTPRB and its regulatory mechanism during brown adipocyte differentiation. Ectopic expression of PTPRB led to a reduced brown adipocyte differentiation by suppressing the tyrosine phosphorylation of VEGFR2, whereas a catalytic inactive PTPRB mutant showed no effects on differentiation and phosphorylation. Consistently, the expression of brown adipocyte-related genes, such as UCP-1, PGC-1α, PRDM16, PPAR-γ, and CIDEA, were significantly inhibited by PTPRB overexpression. Overall, these results suggest that PTPRB functions as a negative regulator of brown adipocyte differentiation through its phosphatase activity-dependent mechanism and may be used as a target protein for the regulation of obesity and type II diabetes.

Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the Membrane Yeast Two-Hybrid (MYTH) and the Mammalian Membrane Two-Hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.

Elevated intraocular pressure (IOP) due to insufficient aqueous humor outflow through the trabecular meshwork and Schlemm’s canal (SC) is the most important risk factor for glaucoma, a leading cause of blindness worldwide. We previously reported loss of function mutations in the receptor tyrosine kinase TEK or its ligand ANGPT1 cause primary congenital glaucoma in humans and mice due to failure of SC development. Here, we describe a novel approach to enhance canal formation in these animals by deleting a single allele of the gene encoding the phosphatase PTPRB during development. Compared to Tek haploinsufficient mice, which exhibit elevated IOP and loss of retinal ganglion cells, Tek+/-;Ptprb+/- mice have elevated TEK phosphorylation, which allows normal SC development and prevents ocular hypertension and RGC loss. These studies provide evidence that PTPRB is an important regulator of TEK signaling in the aqueous humor outflow pathway and identify a new therapeutic target for treatment of glaucoma.

Background Accumulating evidence indicates that aberrant microRNA (miRNA) expression contributes to osteosarcoma progression. This study aimed to elucidate the association between miR-624-5p expression and osteosarcoma (OS) development and to investigate its underlying mechanism. Methods We analyzed GSE65071 from the GEO database and found miR-624-5p was the most upregulated miRNA. The expression of miR-624-5p and its specific target gene were determined in human OS specimens and cell lines by RT-PCR and western blot. The effects of miR-624-5p depletion or ectopic expression on OS proliferation, migration and invasion were evaluated in vitro using CCK-8 proliferation assay, colony formation assay, transwell assay, would-healing assay and 3D spheroid BME cell invasion assay respectively. We investigated in vivo effects of miR-624-5p using a mouse tumorigenicity model. Besides, luciferase reporter assays were employed to identify interactions between miR-624-5p and its specific target gene. Results miR-624-5p expression was upregulated in OS cells and tissues, and overexpressing miR-624-5p led to a higher malignant level of OS, including cell proliferation, migration and invasion in vitro and in vivo. Protein tyrosine phosphatase receptor type B (PTPRB) was negatively correlated with miR-624-5p expression in OS tissues. Using the luciferase reporter assay and Western blotting, PTPRB was confirmed as a downstream target of miR-624-5p. PTPRB restored the effects of miR-624-5p on OS migration and invasion. The Hippo signaling pathway was identified as being involved in the miR-624-5p/PTPRB axis. Conclusions In conclusion, our results suggest that miR-624-5p is a negative regulator of PTPRB and a risk factor for tumor metastasis in OS progression.

Dysregulation of protein tyrosine phosphatase, receptor type B (PTPRB) correlates with the development of a variety of tumors. Here we show that PTPRB promotes metastasis of colorectal cancer (CRC) cells via inducing epithelial-mesenchymal transition (EMT). We find that PTPRB is expressed at significantly higher levels in CRC tissues compared to adjacent nontumor tissues and in CRC cell lines with high invasion. PTPRB knockdown decreased the number of invasive CRC cells in an in vitro wound healing model, and also reduced tumor metastasis in vivo. Conversely, PTPRB overexpression promoted CRC cell invasion in vitro and metastasis in vivo. PTPRB overexpression decreased vimentin expression and promoted E-cadherin expression, consistent with promotion of EMT, while PTPRB knockdown had the opposite effect. Hypoxic conditions induced EMT and promoted invasion in CRC cells, but these effects were eliminated by PTPRB knockdown. EMT blockade via TWIST1 knockdown inhibited the migration and invasiveness of CRC cells, and even increased PTPRB expression could not reverse this effect. Altogether, these data support the conclusion that PTPRB promotes invasion and metastasis of CRC cells via inducing EMT, and that PTPRB would be a novel therapeutic target for the treatment of CRC.

Background: Central serous chorioretinopathy (CSC) is a chorioretinal disease characterized by fluid accumulation between the neuroretina and retinal pigment epithelium with unknown etiology. Family studies have suggested a heritable component for CSC with an autosomal dominant inheritance pattern. Therefore, exome sequencing was performed on familial cCSC to indentify the genetic components contributing to familial cCSC. Methods: Exome sequencing was performed on 72 individuals of 18 families with CSC. In these families, we determined whether rare genetic variants (minor allele frequency T; p.T1382I (rs61758735) in the PTPRB gene, was also associated with CSC in a large case–control cohort sequenced previously (p = 0.009). Additionally, in 28 genes two or more different heterozygous variants segregated in two or more families, but no gene showed consistent associations in both the family gene-burden results and gene-burden analysis in the case–control cohort. Conclusion: We identified potential candidate genes for familial CSC and managed to exclude Mendelian inheritance of variants in one or a limited number of genes. Instead, familial CSC may be a heterogeneous Mendelian disease caused by variants in many different genes, or alternatively CSC may represent a complex disease to which both environmental factors and genetics contribute.

Growing evidence suggests that aberrant microRNA (miRNA) expression contributes to hepatocellular carcinoma (HCC) development and progression. However, the potential role and mechanism of miR-665 in the progression of liver cancer remains largely unknown. Our current study showed that miR-665 expression was upregulated in HCC cells and tissues. High expression of miR-665 exhibited more severe tumor size, vascular invasion and Edmondson grading in HCC patients. Gain- or loss-of-function assays demonstrated that miR-665 promoted cell proliferation, migration, invasion, and the epithelial–mesenchymal transition (EMT) of HCC cells in vitro and in vivo. Tyrosine phosphatase receptor type B (PTPRB) was downregulated in HCC tissues, and was negatively correlated with miR-665 expression. Through western blotting and luciferase reporter assay, PTPRB was identified as a direct downstream target of miR-665. Restoration of PTPRB reverses the effects of miR-665 on HCC migration, invasion, and cell proliferation. A mechanistic study showed that PTPTRB mediated the functional role of miR-665 through regulation of the Hippo signaling pathway. In conclusion, our results suggested that miR-665 was a negative regulator of the PTPRB and could promote tumor proliferation and metastasis in HCC through decreasing Hippo signaling pathway activity, which can be a potential target for HCC treatment.

Diabetic nephropathy is a leading cause of kidney failure. VE-PTP phosphatase expression is increased in the endothelium of rodents with diabetes and hypertension. Genetic deletion of VE-PTP reduces kidney injury in diabetic mice, suggesting it may be a therapeutic target., Diabetic nephropathy is a leading cause of end-stage kidney failure. Reduced angiopoietin-TIE2 receptor tyrosine kinase signaling in the vasculature leads to increased vascular permeability, inflammation, and endothelial cell loss and is associated with the development of diabetic complications. Here, we identified a mechanism to explain how TIE2 signaling is attenuated in diabetic animals. Expression of vascular endothelial protein tyrosine phosphatase VE-PTP (also known as PTPRB), which dephosphorylates TIE2, is robustly up-regulated in the renal microvasculature of diabetic rodents, thereby reducing TIE2 activity. Increased VE-PTP expression was dependent on hypoxia-inducible factor transcriptional activity in vivo. Genetic deletion of VE-PTP restored TIE2 activity independent of ligand availability and protected kidney structure and function in a mouse model of severe diabetic nephropathy. Mechanistically, inhibition of VE-PTP activated endothelial nitric oxide synthase and led to nuclear exclusion of the FOXO1 transcription factor, reducing expression of pro-inflammatory and pro-fibrotic gene targets. In sum, we identify inhibition of VE-PTP as a promising therapeutic target to protect the kidney from diabetic injury., Graphical Abstract

PTPRB is a transmembrane protein tyrosine phosphatase known to regulate blood vessel remodelling and angiogenesis. Here, we demonstrate that PTPRB negatively regulates branching morphogenesis in the mouse mammary epithelium. We show that Ptprb is highly expressed in adult mammary stem cells and also, although at lower levels, in oestrogen receptor-positive luminal cells. During mammary development, Ptprb expression is downregulated during puberty, a period of extensive ductal outgrowth and branching. In vivo shRNA knockdown of Ptprb in the cleared mammary fat pad transplant assay resulted in smaller epithelial outgrowths with an increased branching density and also increased branching in an in vitro organoid assay. Organoid branching was dependent on stimulation by FGF2, and Ptprb knockdown in mammary epithelial cells resulted in a higher level of fibroblast growth factor receptor (FGFR) activation and ERK1/2 phosphorylation, both at baseline and following FGF2 stimulation. Therefore, PTPRB regulates branching morphogenesis in the mammary epithelium by modulating the response of the FGFR signalling pathway to FGF stimulation. Considering the importance of branching morphogenesis in multiple taxa, our findings have general importance outside mammary developmental biology.

Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signaling genes, as a key driver of angiosarcoma. Here we employed whole-genome, whole-exome and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored predominantly truncating mutations in 10 of 39 tumors (26%). PLCG1, a signal transducer of tyrosine kinases, encoded a recurrent, likely activating p.Arg707Gln missense variant in 3 of 34 cases (9%). Overall, 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signaling in angiosarcoma.

Background: Cerebrovascular white matter lesions (WMLs) are associated with cognitive impairment in patients with subcortical vascular dementia. We performed a comprehensive gene expression analysis to elucidate genes associated with WML development in a chronic cerebral hypoperfusion rat model. Methods: Brains of rats with bilateral carotid ligation (2VO, n = 10) and sham-operated rats (n = 5-10/group) were removed on days 1, 7, or 28 after surgery. Total RNA isolated from the corpus callosum was evaluated by microarray analysis and quantitative reverse transcription-polymerase chain reaction. Results: On days 7 and 28, WMLs exhibited histologic changes. On day 7, 16 genes were differentially expressed between groups. mRNA levels of Ptprb, Kcnj8, Crispld2, Bcl6b, and Gja5 were differentially expressed in 2VO rats on day 7, but then returned to normal, whereas mRNA levels of Vwf and Trappc6a were upregulated after day 7. Immunohistochemistry showed that GJA5 and vWF were detected in endothelial cells, KCNJ8 in endothelial cells and astrocytes, CRISPLD2 in neurons and astrocytes, and TRAPPC6A in neurons. Conclusion: Our findings indicate novel genes that may be associated with WML development in the chronic cerebral hypoperfusion rat model, and suggest an important role of neurovascular dysfunction in the pathophysiology.

Eph receptors play pivotal roles in the axon guidance of retinal ganglion cells (RGCs) at the optic chiasm and the establishment of the topographic retinocollicular map. We previously demonstrated that protein tyrosine phosphatase receptor type O (PTPRO) is specifically involved in the control of retinotectal projections in chicks through the dephosphorylation of EphA and EphB receptors. We subsequently revealed that all the mouse R3 subfamily members (PTPRB, PTPRH, PTPRJ, and PTPRO) of the receptor protein tyrosine phosphatase (RPTP) family inhibited Eph receptors as their substrates in cultured mammalian cells. We herein investigated the functional roles of R3 RPTPs in the projection of mouse retinal axon of both sexes.PtproandPtprjwere expressed in mouse RGCs; however,Ptprjexpression levels were markedly higher than those ofPtpro. Consistent with their expression levels, Eph receptor activity was significantly enhanced inPtprj-knock-out (Ptprj-KO) retinas. InPtprj-KO andPtprj/Ptpro-double-KO (DKO) mice, the number of retinal axons that projected ipsilaterally or to the contralateral eye was significantly increased. Furthermore, retinal axons inPtprj-KO and DKO mice formed anteriorly shifted ectopic terminal zones in the superior colliculus (SC). We found that c-Abl (Abelson tyrosine kinase) was downstream of ephrin–Eph signaling for the repulsion of retinal axons at the optic chiasm and in the SC. c-Abl was identified as a novel substrate for PTPRJ and PTPRO, and the phosphorylation of c-Abl was upregulated inPtprj-KO and DKO retinas. Thus, PTPRJ regulates retinocollicular projections in mice by controlling the activity of Eph and c-Abl kinases.SIGNIFICANCE STATEMENTCorrect retinocollicular projection is a prerequisite for proper vision. Eph receptors have been implicated in retinal axon guidance at the optic chiasm and the establishment of the topographic retinocollicular map. We herein demonstrated that protein tyrosine phosphatase receptor type J (PTPRJ) regulated retinal axonal projections by controlling Eph activities. The retinas ofPtprj-knock-out (KO) andPtpro/Ptprjdouble-KO mice exhibited significantly enhanced Eph activities over those in wild-type mice, and their axons showed defects in pathfinding at the chiasm and retinocollicular topographic map formation. We also revealed that c-Abl (Abelson tyrosine kinase) downstream of Eph receptors was regulated by PTPRJ. These results indicate that the regulation of the ephrin–Eph–c-Abl axis by PTPRJ plays pivotal roles in the proper central projection of retinal axons during development.

Subtype R3 phosphotyrosine phosphatase receptors (R3 RPTPs) are single-spanning membrane proteins characterized by a unique modular composition of extracellular fibronectin repeats and a single cytoplasmatic protein tyrosine phosphatase (PTP) domain. Vertebrate R3 RPTPs consist of five members: PTPRB, PTPRJ, PTPRH and PTPRO, which dephosphorylate tyrosine residues, and PTPRQ, which dephosphorylates phophoinositides. R3 RPTPs are considered novel therapeutic targets in several pathologies such as ear diseases, nephrotic syndromes and cancer. R3 RPTP vertebrate receptors, as well as their known invertebrate counterparts from animal models: PTP52F, PTP10D and PTP4e from the fruitfly Drosophila melanogaster and F44G4.8/DEP-1 from the nematode Caenorhabditis elegans, participate in the regulation of cellular activities including cell growth and differentiation. Despite sharing structural and functional properties, the evolutionary relationships between vertebrate and invertebrate R3 RPTPs are not fully understood. Here we gathered R3 RPTPs from organisms covering a broad evolutionary distance, annotated their structure and analyzed their phylogenetic relationships. We show that R3 RPTPs (i) have probably originated in the common ancestor of animals (metazoans), (ii) are variants of a single ancestral gene in protostomes (arthropods, annelids and nematodes); (iii) a likely duplication of this ancestral gene in invertebrate deuterostomes (echinodermes, hemichordates and tunicates) generated the precursors of PTPRQ and PTPRB genes, and (iv) R3 RPTP groups are monophyletic in vertebrates and have specific conserved structural characteristics. These findings could have implications for the interpretation of past studies and provide a framework for future studies and functional analysis of this important family of proteins.

Angiogenesis, de novo formation of blood vessels from the pre-existing vasculature, is crucial in embryo development, and in processes in the adult such as wound healing and ovulation. Angiogenesis is also involved in pathological conditions such as cancer and chronic inflammatory diseases, which are propagated by dysregulated, excess angiogenesis. On the other hand, lack of functional vessels and poor blood flow is a major problem in myocardial and peripheral ischemia. A detailed understanding of the molecular mechanisms underlying angiogenesis is of vital importance for the development of drugs to regulate angiogenesis. The aim of this thesis has been to identify genes involved in regulation of angiogenesis. We have investigated gene expression over time in endothelial cells (ECs), using different in vitro models. We show that the proteoglycan endocan is upregulated in ECs invading a fibrin matrix in response to vascular endothelial growth factor (VEGF)-A. There was increased expression of endocan in renal tumour cells and tumour vessels compared to normal renal tissues, indicating that endocan might have a role in tumour growth and tumour angiogenesis. We also show that vascular endothelial protein tyrosine phosphatase (VE-PTP) is induced in ECs during differentiation into vessel structures in a three dimensional collagen matrix. Silencing of VE-PTP disrupts vessel formation and increases the activity of VEGF receptor-2 (VEGFR-2) and downstream signalling, leading to increased EC proliferation. This presents a possible mechanism for the failure of vessel formation, as EC morphogenesis requires growth arrest of the cells. We also show that VE-PTP and VEGFR-2 are closely associated in resting ECs. VEGF-A stimulation leads to rapid loss of association, coinciding with increased phosphorylation of VEGFR-2. The function of VE-PTP in vivo was investigated using the zebrafish model. We demonstrate specific expression of a zebrafish VE-PTP orthologue (zVE-PTP) in the developing vasculature. Silencing of zVE-PTP leads to defective vessel sprouting and branching, indicating a critical role for zVE-PTP in development of the zebrafish vasculature. In conclusion, this thesis presents gene regulation during endothelial cell morphogenesis and details the expression pattern of endocan and the function of VE-PTP in regulation of VEGFR-2-driven angiogenesis.

Protein tyrosine phosphatases, together with protein tyrosine kinases, regulate the tyrosine phosphorylation that controls cell activities and proliferation. PTPRB contains an extracellular domain, a single transmembrane segment and one intracytoplasmic catalytic domain, hence it belongs to receptor type protein tyrosine phosphatases. The extracellular region of PTPRB is composed of multiple fibronectin type-3 repeats, which was shown to interact with neuronal receptor and cell adhesion molecules, such as contactin (Rios et al., 2000) and tenascin C (Adamsky et al., 2001, 2003). Expression analysis implicate the roles of PTPRB in cell adhesion, neurite growth, and neuronal differentiation (Nagata et al., 2003). In cattle cell lines, protein tyrosine phosphatases and their substrates were suggested to regulate adherens junctional integrity, the movement of macromolecules and cells through the endothelial paracellular pathway, and capillary tube stability, thus possibly being involved in the immune response (Young et al., 2003). PTPRB was also found to interact with ATP-gated ion channels in the plasma membrane, and may regulate channels by altering their tyrosine phosphorylation status (Ratcliffe et al., 2000; Kim et al., 2001). It provides a component in cell-cell and cell-extracellular matrix signaling events (Garwood et al., 2003). In human PTPRB was mapped previously to HSA12q15→ q21 by FISH (Harder et al., 1992).