Cell Protein Tyrosine Phosphatase Research Articles

ETHE1 dampens colorectal cancer angiogenesis by promoting TC45 Dephosphorylation of STAT3 to inhibit VEGF-A expression

Angiogenesis is critical for colorectal cancer (CRC) progression, but its mechanisms remain unclear. Here, we reveal that ethylmalonic encephalopathy protein 1 (ETHE1), an essential enzyme in hydrogen sulfide catabolism, inhibits VEGF-A expression and tumor angiogenesis in vitro and in vivo. Moreover, we find that this biological function of ETHE1 depends on the STAT3/VEGF-A pathway. Further investigation demonstrates that ETHE1 promotes the interaction between T cell protein tyrosine phosphatase (TC45) and STAT3, resulting in decreased STAT3 phosphorylation and inhibition of the STAT3 signaling pathway. In clinical samples, we find that ETHE1 is downregulated in CRC and positively correlates with survival outcomes of CRC patients. Meanwhile, the negative correlation of ETHE1 and VEGF-A expression is verified in CRC specimens, and the patients with low ETHE1 and high VEGF-A expression exhibits poorer prognosis. Collectively, our study identifies ETHE1 as a novel regulator of tumor angiogenesis, implying its potential as a prognostic biomarker and promising antiangiogenic target for CRC patients.

Alteration of circulating ACE2-network related microRNAs in patients with COVID-19

Angiotensin converting enzyme 2 (ACE2) serves as the primary receptor for the SARS-CoV-2 virus and has implications for the functioning of the cardiovascular system. Based on our previously published bioinformatic analysis, in this study we aimed to analyze the diagnostic and predictive utility of miRNAs (miR-10b-5p, miR-124-3p, miR-200b-3p, miR-26b-5p, miR-302c-5p) identified as top regulators of ACE2 network with potential to affect cardiomyocytes and cardiovascular system in patients with COVID-19. The expression of miRNAs was determined through qRT-PCR in a cohort of 79 hospitalized COVID-19 patients as well as 32 healthy volunteers. Blood samples and clinical data of COVID-19 patients were collected at admission, 7-days and 21-days after admission. We also performed SHAP analysis of clinical data and miRNAs target predictions and advanced enrichment analyses. Low expression of miR-200b-3p at the seventh day of admission is indicative of predictive value in determining the length of hospital stay and/or the likelihood of mortality, as shown in ROC curve analysis with an AUC of 0.730 and a p-value of 0.002. MiR-26b-5p expression levels in COVID-19 patients were lower at the baseline, 7 and 21-days of admission compared to the healthy controls (P < 0.0001). Similarly, miR-10b-5p expression levels were lower at the baseline and 21-days post admission (P = 0.001). The opposite situation was observed in miR-124-3p and miR-302c-5p. Enrichment analysis showed influence of analyzed miRNAs on IL-2 signaling pathway and multiple cardiovascular diseases through COVID-19-related targets. Moreover, the COVID-19-related genes regulated by miR-200b-3p were linked to T cell protein tyrosine phosphatase and the HIF-1 transcriptional activity in hypoxia. Analysis focused on COVID-19 associated genes showed that all analyzed miRNAs are strongly affecting disease pathways related to CVDs which could be explained by their strong interaction with the ACE2 network.

Alteration of circulating ACE2-network related miRNAs in patients with COVID-19

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Medical University of Warsaw and São Paulo Research Foundation (FAPESP). Introduction Coronavirus disease 2019 (COVID-19) is associated with an increased risk of mortality and adverse cardiovascular (CV) occurrences, especially in a group of patients with CV diseases (CVD). SARS-CoV-2 virus enters cells through angiotensin converting enzyme 2 (ACE2) cell membrane enzymes, leading to the downregulation in the ACE2-related pathways. Based on our previous in silico [1] results we hypothesized that alteration in the ACE2 interaction network caused by virus infection can affect the expression of miRNAs and influence severity of the disease. Purpose In this study we aimed to analyse the diagnostic and predictive utility of ACE2-related miRNAs, which were identified in our previous in silico analysis (miR-10b-5p, miR-124-3p, miR-200b-3p, miR-26b-5p, miR-302c-5p) in patients with COVID-19. We also aimed to unravel the functions of analyzed miRNAs, by using machine learning based tools, including SHAP for clinical data analysis, and bioinformatic tools for data mining, target predictions and enrichment analyses. Methods Blood samples and clinical data of 79 COVID-19 patients were collected at three different time points, including the day of admission, 7-days and 21-days after admission, as well as one time-point of 32 healthy volunteers. The composite endpoint was hospitalisation length of stay (&amp;gt;21 days) and/or death in follow-up. Results Delta low miR-200b-3p expression (after 7-days of admission) presents predictive utility in assessment of the hospital length of stay and/or death in ROC curve analysis (AUC:0.730, p=0.002) (Fig. 1). Delta low miR-200b-3p expression, together with diabetes mellitus (DM) are independent predictors of increased hospital length of stay and/or death (OR= 5.775; CI= 1.572- 21.214; p= 0.008, OR= 4.888; CI= 1.001- 23.858; p= 0.050, respectively). The expression levels of miR-26b-5p and miR-10b-5p in COVID-19 patients were found lower at the baseline, 7 and 21-days after admission compared to the healthy controls (p&amp;lt;0.0001 for all time points). SHAP analysis indicated levels of miR 200b-3p (day 7th), miR-302c-5p (day 7th), CRP (day 7th), neutrophils (day 0), and D-Dimer (day 0) as the most promising predictors of long hospitalisation for the COVID-19 patients. Pathway enrichment analysis showed that among top shared pathways between targets of analyzed miRNAs were IL-2 signaling pathway, and Pathways in cancer. miR-200b-3p showed regulation of COVID-19-related targets associated with T cell protein tyrosine phosphatase and HIF-1 transcriptional activity in hypoxia, key pathways in COVID-19. Bioinformatics analysis pointed out the role of those miRNAs in multiple CVD phenotypes associated with COVID-19 (Fig. 2). Conclusions In this study we validated and characterized miRNAs which could serve as novel, predictive biomarkers of the COVID-19 long term hospitalisation and can be used for early stratification of patients and prediction of severity of infection development in an individual.

The IBD-candidate gene, PTPN2, is a Key Regulator of Ileal Paneth Cell Function in Mice

Loss-of-function variants in the Protein Tyrosine Phosphatase non-receptor Type 2 ( PTPN2) gene are associated with increased risk of Inflammatory Bowel Disease (IBD). PTPN2 encodes T Cell Protein Tyrosine Phosphatase (TCPTP), a negative regulator of intracellular signaling pathways including JAK-STAT. PTPN2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage crosstalk, and modulating the gut microbiome in mice, although the mechanism in which PTPN2 loss affects gut microbiome is unknown. IBD patient biopsies and studies in transgenic mice show that IBD-associated genes can modify IEC subtypes and their functional roles in intestinal homeostasis. Hypothesis: PTPN2 loss compromises modulation of gut microbiome by impairing number and function of IEC subtypes. Methods: To identify how loss of PTPN2 activity in vivo affects ileal IEC subtypes and their function, RNA and protein were isolated from ileal IECs of whole-body Ptpn2-wild-type (WT), heterozygous (Het) and KO mice. RNA was assayed by two separate Nanostring ® panels: PanCancer and AutoImmune totaling &gt;1500 targets. Dysregulated targets associated with IEC function were validated by Western blot, immunostaining, and transmission electron microscopy (TEM). Altered genes were then investigated in tamoxifen-inducible intestinal epithelial-specific Ptpn2 knockout mice ( Ptpn2 Δ IEC ) and Ptpn2 fl/fl controls. Results: 1398 targets had detectable expression. Notably, Paneth cell (PC)-associated genes Lyz1, Defa6, Pla2g2a, and Fgfr3 were markedly downregulated (p&lt;0.05 and FDR&lt;0.1; n=4). Reduced PC numbers in whole-body Ptpn2-KO mice were confirmed by decreased Lysozyme and Cryptdin-1 staining (p&lt;0.001; n=5), reduced Lysozyme protein levels by western blotting (p=0.004; n=5), and loss of PC phenotype by TEM (n=3). Ki-67 staining revealed elevated ileal crypt cell proliferation (p=0.02; n=3) and longer villi (p=0.03; n=9) in Ptpn2-KO mice vs. WT. However, unchanged cleaved caspase 3 and TUNEL staining indicated no increase in IEC apoptosis. Further, TEM analysis showed disrupted endoplasmic reticulum (ER) architecture in PCs of Ptpn2-KO mice (n=3) accompanied by higher protein levels of the ER stress marker CHOP (p=0.01; n=8). Conversely, Ptpn2 Δ IEC mice showed no change in ileal villus length (n=6), number of PCs (n=7), and TEM showed abundant cytosolic granules compared to Ptpn2 fl/fl (n=4). However, immunostaining (n=6) and Western blotting (p&lt;0.05; n=6) showed reduced Lysozyme protein accompanied by elevated levels of unfolded protein response Xbp-1s in Ptpn2 Δ IEC mice (p&lt;0.05; n=6). Conclusion: Ptpn2-deficiency affects Paneth cell viability by decreasing cell number, promoting ER stress, and impairing production of PC-associated antimicrobial peptides. This likely contributes to the increased susceptibility to infection and the dysbiotic microbiota in Ptpn2-deficient mice and may explain the association of PTPN2 variants with dysbiosis in clinical IBD. Supported by NIH 2R01DK091281, 1R01AI153314-01, R21AI152017 and R01AI165490 (DFM) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The IBD-candidate gene, PTPN2, Regulates Goblet Cell Number and Expression of Intestinal Epithelial Cell Markers in a Regional-specific Manner in Mice

Loss-of-function variants in the Protein Tyrosine Phosphatase non-receptor Type 2 ( PTPN2) gene are associated with increased risk of Inflammatory Bowel Disease (IBD). PTPN2 encodes T Cell Protein Tyrosine Phosphatase (TCPTP), a negative regulator of intracellular signaling pathways including JAK-STAT. PTPN2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage crosstalk, and modulating the gut microbiota in mice and IBD patients. Aim: To determine if Ptpn2 loss compromises IEC subtypes that restrict gut dysbiosis. Methods: Ileal and colonic IEC subtypes and their function were assessed by PAS staining of goblet cells and immunostaining. RNA isolated from colonic IECs of whole-body Ptpn2-wild-type (WT), heterozygous (Het) and KO mice was assayed by two separate Nanostring ® panels: PanCancer and AutoImmune totaling &gt;1500 targets. Flow cytometry was performed to evaluate abundance of mucosal-associated immune cells. Results: Goblet cells were reduced in the ileal and cecal crypts compared with WT mice (p&lt;0.05; n=5) while numbers in the proximal and distal colon were variable. Ki-67 immunostaining revealed increased proliferating cells in the proximal and distal colon (p&lt;0.01; n=3). Nanostring analysis had 1399 targets with detectable expression. Notably, expression of growth factors Egf and Fgfr3, differentiation factors Atoh1 and Mycn, tuft cell marker Dclk1, and intestinal stem cell marker Lgr5, were all downregulated in colonic IECs from Ptpn2-KO mice compared with WT (p&lt;0.05; FDR&lt;0.1; n=4). Moreover, abundance of mucosal neutrophils, macrophages, CD4 + expressing IFN-γ, CD8 + expressing TNF/IFN-γ, and CD4 + expressing IL-22 T-cells were higher in colon and cecum of Ptpn2-KO mice compared with WT and HET mice (p&lt;0.01; n=5), whereas abundance of IL-4 expressing CD4 + cells, and Tregs expressing IL-10 were decreased in all intestinal segments of Ptpn2-KO mice (p&lt;0.01; n=5). Conclusion: Ptpn2 loss negatively impacts abundance of IEC subtypes in a region-specific manner, affecting epithelial proliferation and expression of critical differentiation/cell markers. The elevated abundance of immune cells expressing pro-inflammatory signals accompanied by decreased levels of cells expressing IL-4 and IL-10, could contribute to a skewed abundance of IEC subtypes and their function. The observed effects may contribute to the increased susceptibility to infection and the dysbiotic microbiota in Ptpn2-deficient mice, and partly explain association of PTPN2 variants with dysbiosis in clinical IBD. Supported by NIH 2R01DK091281, 1R01AI153314-01, R21AI152017 and R01AI165490 (DFM). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Epigenetic modification of CSDE1 locus dictates immune recognition of nascent tumorigenic cells.

Weak immunogenicity of tumor cells is a root cause for the ultimate failure of immunosurveillance and immunotherapy. Although tumor evolution can be shaped by immunoediting toward a less immunogenic phenotype, mechanisms governing the initial immunogenicity of primordial tumor cells or original cancer stem cells remain obscure. Here, using a single tumor-repopulating cell (TRC) to form tumors in immunodeficient or immunocompetent mice, we demonstrated that immunogenic heterogeneity is an inherent trait of tumorigenic cells defined by the activation status of signal transducer and activator of transcription 1 (STAT1) protein in the absence of immune pressure. Subsequent investigation identified that the RNA binding protein cold shock domain-containing protein E1 (CSDE1) can promote STAT1 dephosphorylation by stabilizing T cell protein tyrosine phosphatase (TCPTP). A methyltransferase SET and MYN domain-containing 3 (SMYD3) was further identified to mediate H3K4 trimethylation of CSDE1 locus, which was under the regulation of mechanotransduction by cell-matrix and cell-cell contacts. Thus, owing to the differential epigenetic modification and subsequent differential expression of CSDE1, nascent tumorigenic cells may exhibit either a high or low immunogenicity. This identified SMYD3-CSDE1 pathway represents a potential prognostic marker for cancer immunotherapy effectiveness that requires further investigation.

PTPN2 in the Immunity and Tumor Immunotherapy: A Concise Review.

PTPN2 (protein tyrosine phosphatase non-receptor 2), also called TCPTP (T cell protein tyrosine phosphatase), is a member of the PTP family signaling proteins. Phosphotyrosine-based signaling of this non-transmembrane protein is essential for regulating cell growth, development, differentiation, survival, and migration. In particular, PTPN2 received researchers’ attention when Manguso et al. identified PTPN2 as a cancer immunotherapy target using in vivo CRISPR library screening. In this review, we attempt to summarize the important functions of PTPN2 in terms of its structural and functional properties, inflammatory reactions, immunomodulatory properties, and tumor immunity. PTPN2 exerts synergistic anti-inflammatory effects in various inflammatory cells and regulates the developmental differentiation of immune cells. The diversity of PTPN2 effects in different types of tumors makes it a potential target for tumor immunotherapy.

Valeriaquinone A, a unique anthraquinone–coumarin hybrid with selective inhibition of PTP1B from Knoxia valerianoides

Valeriaquinone A (1), an unprecedented anthraquinone–coumarin hybrid, was isolated from the roots of Knoxia valerianoides. Its structure was determined by extensive spectroscopic analyses and X-ray diffraction. The plausible biosynthetic pathways for 1 were proposed. Compound 1 exhibited strong protein tyrosine phosphatase 1B (PTP1B) inhibition with high selectivity (> 30 fold) over homologous T cell protein tyrosine phosphatase (TCPTP) potentially by binding to an allosteric site predicted by kinetic analysis and molecular docking. Moreover, compound 1 showed significant cytotoxic activities against three human hepatoma cell lines (HepG2, QGY-7703, and SMMC-7721) with half maximal inhibitory concentration (IC50) values of 1.39 ± 0.2, 10.34 ± 2.09, and 5.56 ± 0.47 µmol/L, respectively.

Discovery of novel and selective inhibitors targeting protein tyrosine phosphatase 1B (PTP1B): Virtual screening and molecular dynamic simulation

Protein tyrosine phosphatase 1B (PTP1B) is a promising target for Type II diabetes, obesity, and cancer therapeutics. However, capturing selectivity over T cell protein tyrosine phosphatase (TCPTP) is key to PTP1B inhibitor discovery. Current studies demonstrate that the phosphotyrosine (pTyr) binding site confers selectivity to inhibitors. To identify novel selective inhibitors of PTP1B, drugs in the DrugBank were docked into the active and pTyr site using virtual docking tools. The most suitable drugs were selected based on their docking scores, similarity, and visual results before molecular dynamic simulations were performed. A combination of virtual screening and molecular dynamic simulation approaches indicated that five drugs (DB03558, DB05123, DB03310, DB05446, DB03530) targeting the active and second pTyr binding site of PTP1B could be potential selective inhibitors. This study showed that the hit drugs (experimental, research, and approved) could serve as potential selectivity PTP1B inhibitors and as useful treatments for diabetes and cancer. The hit drugs can be experimentally validated via in vitro molecular testing and in vivo animal testing; alternatively, they can be included in ongoing clinical trials. In addition, more effective molecules can be designed by derivatizing these drugs.

PTPN2 mutations cause epithelium-intrinsic barrier loss that synergizes with mucosal immune hyperactivation.

It is clear that excessive mucosal immune activation and intestinal barrier dysfunction both contribute to inflammatory bowel disease (IBD) pathogenesis. T cell protein tyrosine phosphatase (TCPTP), which extinguishes signaling in immune cells, is linked to IBD and other immune-mediated diseases. In this issue of the JCI, Marchelletta and Krishnan et al. demonstrate that, in intestinal epithelial cells, TCPTP regulates tight junction permeability in vivo. Intestinal epithelial TCPTP loss potentiated cytokine-induced barrier loss, and this synergized with effects of TCPTP loss in immune cells. This work implicates a single mutation as the cause of distinct functional aberrations in diverse cell types and demonstrates how one genetic defect can drive multihit disease pathogenesis.

T cell protein tyrosine phosphatase protects intestinal barrier function by restricting epithelial tight junction remodeling.

Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.

Abstract 2824: SHP2-PDHA metabolic axis is critical for adipocyte function by mitigating ER stress-induced adipocytes dysfunction: Implication for obesity-driven pancreatic ductal adenocarcinoma progression

Abstract Obesity remains a major risk factor for the development of pancreatic cancer. Animal studies demonstrated that obese mice develop larger and more metastatic pancreatic cancer compared to their lean counterpart. Within the tumor environment are adipocytes that secrete adipokines, which promote inflammation and directly stimulate pancreatic cancer cells. Understanding the mechanisms that alter adipocyte function and adipokine secretion is critical for circumventing adipocyte-mediated influence on pancreatic cancer progression. Targeting of the Src homology region 2 containing protein tyrosine phosphatase (SHP-2) in the cancer cells has recently shown promising potential, yet the effect of SHP2 inhibition on stromal components has not been addressed. Here we demonstrate that SHP2 and the mitochondrial protein pyruvate dehydrogenase (PDHA) are critical mediators of adipocyte function, through mitigation of ER stress in adipocytes. In order to understand the role of SHP2 and PDHA in adipocytes, we performed Oil Red O staining and western blot assays on cultured adipocytes. We found that the transition from pre-adipocytes to fully developed adipocytes is marked by significant accumulation of phospho-SHP2, PDHA and the ER stress marker Bip. Chemical inhibition of SHP2 or PDHA augmented ER stress and impaired lipid production in the adipocytes; these effects were reversed by the ER stress inhibitor, 4-Phenylbyturic acid (4-PBA). Moreover, pancreatic cancer cells exposed to conditioned media from differentiated adipocytes, in which either SHP2 or PDHA was inhibited, are less aggressive and invasive compared to PDAC cells grown in conditioned media from control adipocytes with no inhibitor treatment. Therefore, our studies indicate that SHP2 and PDHA, in adipocytes, are key mediators of obesity-driven pancreatic cancer progression. Future studies will focus on understanding the SHP2-PDHA axis in adipocytes and further to elucidate how cancer cell extrinsic effects of SHP2 inhibition could alter PDAC progression. Citation Format: Appolinaire A. Olou, Jarrid Jack, Sharon Manley, Joseph Ambrose, McKinnon Walsh, Austin Eades, Michael N. VanSaun. SHP2-PDHA metabolic axis is critical for adipocyte function by mitigating ER stress-induced adipocytes dysfunction: Implication for obesity-driven pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2824.

T Cell Protein Tyrosine Phosphatase in Glucose Metabolism.

T cell protein tyrosine phosphatase (TCPTP), a vital regulator in glucose metabolism, inflammatory responses, and tumor processes, is increasingly considered a promising target for disease treatments and illness control. This review discusses the structure, substrates and main biological functions of TCPTP, as well as its regulatory effect in glucose metabolism, as an attempt to be referenced for formulating treatment strategies of metabolic disorders. Given the complicated regulation functions in different tissues and organs of TCPTP, the development of drugs inhibiting TCPTP with a higher specificity and a better biocompatibility is recognized as a promising therapeutic strategy for diabetes or obesity. Besides, treatments targeting TCPTP in a specific tissue or organ are suggested to be considerably promising.

T Cell Protein Tyrosine Phosphatase in Osteoimmunology.

Osteoimmunology highlights the two-way communication between bone and immune cells. T cell protein tyrosine phosphatase (TCPTP), also known as protein-tyrosine phosphatase non-receptor 2 (PTPN2), is an intracellular protein tyrosine phosphatase (PTP) essential in regulating immune responses and bone metabolism via dephosphorylating target proteins. Tcptp knockout in systemic or specific immune cells can seriously damage the immune function, resulting in bone metabolism disorders. This review provided fresh insights into the potential role of TCPTP in osteoimmunology. Overall, the regulation of osteoimmunology by TCPTP is extremely complicated. TCPTP negatively regulates macrophages activation and inflammatory factors secretion to inhibit bone resorption. TCPTP regulates T lymphocytes differentiation and T lymphocytes-related cytokines signaling to maintain bone homeostasis. TCPTP is also expected to regulate bone metabolism by targeting B lymphocytes under certain time and conditions. This review offers a comprehensive update on the roles of TCPTP in osteoimmunology, which can be a promising target for the prevention and treatment of inflammatory bone loss.

Long non-coding RNA TINCR promotes hepatocellular carcinoma proliferation and invasion via STAT3 signaling by direct interacting with T-cell protein tyrosine phosphatase (TCPTP)

ABSTRACT The long non-coding RNAs (lncRNAs) participate in modulating numerous important cancer phenotypes via formation of RNA-protein complex. TINCR (terminal differentiation-induced lncRNA) modulates cancer cell behavior in many human malignancies, such as hepatocellular carcinoma (HCC). Herein, we proposed to investigate the underlying mechanism by which TINCR regulates HCC progression via formation of RNA-protein. RNA pulldown, LC-MS/MS, bioinformatics analysis, and RNA immunoprecipitation (RIP) assays were employed to identify TINCR-interacting protein TCPTP in HCC cells. The siRNAs for TINCR and TCPTP were transfected into HCC cells. The plasmids encoding full length or the 1–360 nt deletion of TINCR were generated and applied to cell transfection. The CCK-8, colony formation, EdU, wound healing along with transwell assays were employed to examine cell proliferation, apoptosis, migration, and infiltration. Real-time PCR, as well as western blot assays were employed to assess the levels of STAT3 phosphorylation and its target genes. We identified 1–360 nt region of TINCR, which directly bound with the phosphatase domain of TCPTP to inhibit its tyrosine phosphatase activity. Then, the results showed that the increasing of cell growth, migration, infiltration, and the reducing of apoptosis in TINCR-knockdown HCC cells was remarkably reversed with TCPTP silence. Additionally, Δ1-360 TINCR overexpression did not affect HCC cell growth, apoptosis, migration, infiltration, and STAT3 target genes expression. Our data revealed that TINCR directly bound TCPTP and suppressed the dephosphorylation of STAT3, thus promoting STAT3 activation and its downstream target genes in HCC progression and tumorigenicity. Highlights LncRNA TINCR interacted with protein TCPTP LncRNA TINCR maintained STAT3 phosphorylation LncRNA TINCR affected STAT3 signaling in HCC Abbreviations: lncRNAs: long non-coding RNAs; TINCR: terminal differentiation-induced lncRNA; TCPTP: T cell protein tyrosine phosphatase; siRNA: small-interfering RNA; HCC: hepatocellular carcinoma; nt: nucleotide; LC-MS/MS: Liquid Chromatography - Tandem Mass Spectrometry; RIP: RNA immunoprecipitation; ANOVA: analysis of variance; EdU: 5-ethynyl-2’-deoxyuridine; real-time PCR: real-time polymerase chain reaction; CCK-8: cell counting kit-8; aa: amino acids; STAT3: signal transducer and activator of transcription 3

In Vitro Activity Assays to Quantitatively Assess the Endogenous Reversible Oxidation State of Protein Tyrosine Phosphatases in Cells.

The reversible oxidation of protein tyrosine phosphatases (PTPs) impairs their ability to dephosphorylate substrates in vivo. This transient inactivation of PTPs occurs as their conserved catalytic cysteine residue reacts with cellular oxidants thereby abolishing the ability of this reactive cysteine to attack the phosphate of the target substrate. Hence, in vivo, the inhibition of specific PTPs in response to regulated and localized rises in cellular oxidants enables phospho-dependent signaling. We present assays that measure the endogenous activity of specific PTPs that become transiently inactivated in cells exposed to growth factors. Here, we describe the methods and highlight the pitfalls to avoid post-lysis oxidation of PTPs in order to assess the inactivation and the reactivation of PTPs targeted by cellular oxidants in signal transduction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Cell transfection (optional) Support Protocol: Preparation of degassed lysis buffers Basic Protocol 2: Cellular extraction in anaerobic conditions Basic Protocol 3: Enrichment and activity assay of specific PTPs Alternate Protocol: Measurement of active PTPs via direct cysteinyl labeling.

T‐cell Protein Tyrosine Phosphatase is a Key Regulator of Paneth Cell Function

Loss‐of‐function single nucleotide polymorphisms (SNPs) in the Protein Tyrosine Phosphatase non‐receptor Type 2 (PTPN2) gene, are associated with increased susceptibility to Inflammatory Bowel Disease (IBD). PTPN2 encodes the T Cell Protein Tyrosine Phosphatase (TCPTP) which is a negative regulator of intracellular signaling pathways including JAK‐STAT. We previously presented that Paneth and goblet cell numbers are reduced in Ptpn2 knockout (KO) mice, consistent with altered differentiation of these intestinal epithelial cell (IEC) types in IBD. Alterations in IEC populations are detrimental for intestinal homeostasis and increase susceptibility to microbial infection.GoalTo identify whether Ptpn2‐KO mice display altered IEC differentiation and Paneth cell antimicrobial peptide (AMP) production.MethodsIntestinal tissues (ileum, cecum, proximal and distal colon) from 21 day old constitutive Ptpn2‐KO mice and control littermates, and from adult tamoxifen‐inducible intestinal epithelial‐specific Ptpn2ΔIEC mice (Vil‐Cre/Ptpn2), were harvested and processed for imaging, western blot, flow cytometry and qPCR.ResultsConstitutive Ptpn2‐KO mice display reduced numbers of Paneth cells and loss of the AMP lysozyme (p=0.0158; n=5). This was confirmed by Western blots of isolated IECs (~67% reduction; n=4). Expression of Paneth cell markers and differentiation factors (Mmp7, Defa5 and Sox9) were not altered in KO mice (n=10). Interestingly, Reg3‐γ, which like lysozyme also targets gram‐(+) bacteria and is secreted by Paneth cells and enterocytes, was increased in KO mice vs. control littermates (p=0.0066; n=10). Next, we identified by immunofluorescence that loss of Ptpn2 only in intestinal epithelium (Ptpn2ΔIEC) dramatically reduced colocalization of lysozyme and another Paneth cell marker, UEA‐1 (n=6). Decreased lysozyme expression in Ptpn2ΔIEC ileum was confirmed by western blot (p=0.0315; n=6). Since Paneth cells reside in the stem cell compartment, we investigated whether Ptpn2 deficiency affects the intestinal stem cells. Gene expression of Lgr5, an intestinal stem cell marker, Ephb3, a marker of the stem cell compartment, and Ascl2, a gene that controls stem cell renewal in the crypts, was unchanged in Ptpn2‐KO mice (n=10). In addition, expression of early IEC differentiation factors, Hes1 and Math1, were not altered (n=10). Il‐22 and IFN‐γ are known to stimulate secretion of AMPs by Paneth cells. Interestingly, flow cytometry showed increased abundance of Il‐22+ T cells (p=0.0187; n=5) and IFN‐γ+ T cells (p=0.0277; n=5) in the Ileum of constitutive KO mice, indicating that the defect in AMP production is likely not due to a lack of immune cell‐derived stimuli.ConclusionPtpn2‐deficiency decreases Paneth cell number and has opposing effects on their production of specific AMPs. These effects on Paneth cell number and function may contribute to the increased susceptibility to infection and the dysbiotic microbiota in Ptpn2‐deficient mice.Support or Funding InformationSupported by NIH 2R01DK091281 (DFM) and the Crohn’s and Colitis Foundation (DFM)

Protein Tyrosine Phosphatase Nonreceptor Type 2 Expression Does Not Correlate with Viral Load or Response to Direct-Acting Antiviral Therapy in Hepatitis C Virus Infections-Infected Patients

Background/Aims: The hepatitis C virus nonstructural 3/4A protease has been shown to cleave protein tyrosine phosphatase nonreceptor type 2 (PTPN2, also known as T cell protein tyrosine phosphatase), thereby inducing a shift from a Th1 toward a nonantiviral Th2 immunity. Ribavirin therapy reverses these effects and supports direct-acting antiviral (DAA) therapy as an immunomodulatory compound and ultimately improves the response to DAA therapy. Here we aimed to assess whether intrahepatic levels of PTPN2 might be used as a clinical prognostic marker for the response to DAA therapy. Methods: Liver biopsies from hepatitis C virus-infected patients with and without cirrhosis were immunohistochemically stained for PTPN2 and scored for staining intensity as well as percentage of hepatocytes positive for nuclear PTPN2 localization. PTPN2 scores were correlated to sustained virologic response after DAA therapy, viral load, serum levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase (GGT), and the Model for End-Stage Liver Disease (MELD) score at the time of liver biopsy. Results: We did not detect a difference in intrahepatic PTPN2 levels between responders with cirrhosis, responders without cirrhosis, and nonresponders to DAA therapy. There was no correlation between intrahepatic PTPN2 levels and viral load or clinical markers such as liver transaminases, GGT, or the MELD score. Conclusion: Intrahepatic PTPN2 levels assessed via IHC staining do not represent a clinical prognostic marker for the response to DAA therapy.

The activity of the Striatal-enriched protein tyrosine phosphatase in neuronal cells is modulated by adenosine A2A receptor.

We recently demonstrated that a tonic activation of adenosine A2A receptors (A2A Rs) is required for cocaine-induced synaptic depression and increase in the activity of STriatal-Enriched protein tyrosine Phosphatase (STEP). In this study, we elaborated on the relationship between A2A R and STEP using genetic, pharmacological, and cellular tools. We found that the activities of protein tyrosine phosphatases (PTPs), and in particular of STEP, are significantly increased in the striatum and hippocampus of a transgenic rat strain over-expressing the neuronal A2A R (NSEA2A ) with respect to wild-type (WT) rats. Moreover the selective A2A R agonist 4-[2-[[6-Amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride up-regulates PTPs and STEP activities in WT but not in NSEA2A rats, while the selective A2A R antagonist 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol restores the tyrosine phosphatase activities in NSEA2A , having no effects in WT rats. In addition, while cocaine induced the activation of PTP and STEP in WT rats, it failed to increase phosphatase activity in NSEA2A rats. A2A Rs modulate STEP activity also in the SH-SY5Y neuroblastoma cell line, where a calcium-dependent calcineurin/PP1 pathway was found to play a major role. In summary, the present study identified a novel interaction between A2A R and STEP that could have important clinical implications, since STEP has emerged as key regulator of signaling pathways involved in neurodegenerative and neuropsychiatric diseases and A2A Rs are considered a promising target for the development of therapeutic strategies for different diseases of the central nervous system. Read the Editorial Highlight for this article on page 270.

Constitutive Ptpn2‐Loss in Mice Decreases Number and Compromises Function of Intestinal Goblet and Paneth cells

Among the ~200 susceptibility loci associated with Inflammatory Bowel Disease (IBD), are loss‐of‐function single nucleotide polymorphisms (SNPs) in the locus (18p11) containing the Protein Tyrosine Phosphatase non‐receptor Type 2 (PTPN2) gene. PTPN2 encodes the T Cell Protein Tyrosine Phosphatase (TCPTP) that functions as a negative regulator of diverse intracellular pathways repressing inflammatory signals. In Ptpn2‐deficient mice, we observed altered intestinal homeostasis in the form of intestinal epithelial barrier defects and altered intestinal microbiome composition. Intestinal epithelial homeostasis is maintained by active‐cycling and slow cycling stem cells confined within the crypt‐based niche and subsequent differentiation into secretory and absorptive epithelial lineages as cells migrate up the crypt‐villus axis. The goal of this study was to determine whether Ptpn2‐loss affects intestinal epithelial cell populations and function that could contribute to altered host‐bacterial interactions.MethodsIntestinal tissues (ileum, cecum, proximal and distal colon) from 21 day old wild type (WT), heterozygous (HET) and constitutive Ptpn2 knockout (KO) Balb/C mice were harvested and processed for imaging to detect proliferation (Ki‐67), apoptosis (TUNEL), Paneth cell (lysozyme), and goblet cell (periodic‐acid Shiff) numbers via staining of specific markers. cDNA was generated by reverse transcriptase, and mRNA expression of key genes associated with Paneth and goblet cells function were measured by PCR.ResultsProximal and distal colon of Ptpn2‐KO mice showed clusters of apoptotic cells at the crypt base in comparison to wildtype. In addition, the number of proliferative cells was higher in the crypts of proximal and distal colon of Ptpn2‐KO mice compared to WT (p=0.0032 and p=0.0005 respectively; n=3). Lysozyme‐positive Paneth cells were significantly reduced in HET mice (p=0.0452 vs. WT; n=2–5) and further reduced in KO mice (p=0.0040; n=4). Moreover, KO mice were almost completely devoid of both lysozyme positive and negative Paneth cells, the latter characterized by large cytosolic granules at the crypt base. mRNA expression of the Paneth cell antimicrobial peptide, Lyz1, was reduced in Ptpn2‐KO mice compared to WT (p=0.017; n=2), whereas expression of antimicrobial peptides Defa5 and Defa6 were not altered. Goblet cell number was slightly reduced in the ileum and distal colon of KO mice (n=2). There was also a preliminary trend towards reduced expression of goblet cell secreted mucin proteins, Muc2, a major mucus layer constituent, and Muc3, a mucin protein homologous to MUC17 in humans shown to be protective against enteroinvasive E. coli, in the cecum of Ptpn2‐KO mice compared to wildtype (n=2).ConclusionPtpn2‐deficiency compromises intestinal epithelial cell differentiation leading to altered numbers and function of Paneth and goblet cells consistent with increased susceptibility to bacterial infection.Support or Funding InformationSupported by NIH 2R01DK091281 (DFM), the Crohn's and Colitis Foundation (DFM), and Science Without Borders Fellowship (VC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.