PTPN2 Regulates Interactions Between Macrophages and Intestinal Epithelial Cells to Promote Intestinal Barrier Function.

BACKGROUND & AIMS: The mechanisms by which macrophages regulate intestinal epithelial cell (IEC) barrier properties are poorly understood. Protein tyrosine phosphatase non-receptor type 2 (PTPN2) protects the IEC barrier from inflammation-induced disruption and regulates macrophage functions. We investigated whether PTPN2 controls interactions between IECs and macrophages to maintain intestinal barrier function. METHODS: Human IEC (Caco-2BBe/HT-29.cl19a cells) and mouse enteroid monolayers were cocultured with human macrophages (THP-1, U937, primary monocyte-derived macrophages from patients with inflammatory bowel disease [IBD]) or mouse macrophages, respectively. We assessed barrier function (transepithelial electrical resistance [TEER] and permeability to 4-kDa fluorescently labeled dextran or 70-kDa rhodamine B-dextran) and macrophage polarization. We analyzed intestinal tissues from mice with myeloid cell-specific deletion of PTPN2 (Ptpn2-LysMCre mice) and mice without disruption of Ptpn2 (controls); some mice were given injections of a neutralizing antibody against interleukin (IL) 6. Proteins were knocked down in macrophages and/or IECs with small hairpin RNAs. RESULTS: Knockdown of PTPN2 in either macrophages and/or IECs increased the permeability of IEC monolayers, had a synergistic effect when knocked down from both cell types, and increased the development of inflammatory macrophages in macrophage-IEC cocultures. Colon lamina propria from Ptpn2-LysMCre mice had significant increases in inflammatory macrophages; these mice had increased in vivo and ex vivo colon permeability to 4-kDa fluorescently labeled dextran and reduced ex vivo colon TEER. Nanostring analysis showed significant increases in the expression of IL6 in colon macrophages from Ptpn2-LysMCre mice. An IL6-blocking antibody reversed the effects of PTPN2-deficient macrophages, reducing the permeability of IEC monolayers in culture and in Ptpn2-LysMCre mice. Macrophages from patients with IBD carrying a single-nucleotide polymorphism associated with the disease (PTPN2 rs1893217) had the same features of PTPN2-deficient macrophages from mice, including reduced TEER and increased permeability in cocultures with human IEC or mouse enteroid monolayers, which were restored by anti-IL6. CONCLUSIONS: PTPN2 is required for interactions between macrophages and IECs; loss of PTPN2 from either cell type results in intestinal barrier defects, and loss from both cell types has a synergistic effect. We provide a mechanism by which the PTPN2 gene variants compromise intestinal epithelial barrier function and increase the risk of inflammatory disorders such as IBD.

Claudin-1 regulates intestinal epithelial homeostasis through the modulation of Notch-signalling.

OBJECTIVE: Claudin-1 expression is increased and dysregulated in colorectal cancer and causally associates with the dedifferentiation of colonic epithelial cells, cancer progression and metastasis. Here, we have sought to determine the role claudin-1 plays in the regulation of intestinal epithelial homeostasis. DESIGN: We have used a novel villin-claudin-1 transgenic (Cl-1Tg) mouse as model (with intestinal claudin-1 overexpression). The effect of claudin-1 expression upon colonic epithelial differentiation, lineage commitment and Notch-signalling was determined using immunohistochemical, immunoblot and real-time PCR analysis. The frequently used mouse model of dextran sodium sulfate (DSS)-colitis was used to model inflammation, injury and repair. RESULTS: In Cl-1Tg mice, normal colonocyte differentiation programme was disrupted and goblet cell number and mucin-2 (muc-2) expressions were significantly downregulated while Notch- and ERK1/2-signalling were upregulated, compared with the wild type-littermates. Cl-1Tg mice were also susceptible to colonic inflammation and demonstrated impaired recovery and hyperproliferation following the DSS-colitis. Our data further show that claudin-1 regulates Notch-signalling through the regulation of matrix metalloproteinase-9 (MMP-9) and p-ERK signalling to regulate proliferation and differentiation. CONCLUSIONS: Claudin-1 helps regulate intestinal epithelial homeostasis through the regulation of Notch-signalling. An upregulated claudin-1 expression induces MMP-9 and p-ERK signalling to activate Notch-signalling, which in turn inhibits the goblet cell differentiation. Decreased goblet cell number decreases muc-2 expression and thus enhances susceptibility to mucosal inflammation. Claudin-1 expression also induces colonic epithelial proliferation in a Notch-dependent manner. Our findings may help understand the role of claudin-1 in the regulation of inflammatory bowel diseases and CRC.

Spermidine stimulates T cell protein-tyrosine phosphatase-mediated protection of intestinal epithelial barrier function.

The gene locus encoding protein-tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with inflammatory bowel disease. Expression of the PTPN2 gene product, T cell protein-tyrosine phosphatase (TCPTP), in intestinal epithelial cells has been shown to play an important role in the protection of epithelial barrier function during periods of inflammation by acting as a negative regulator of the proinflammatory cytokine IFN-γ. Therefore, agents that increase the activity of TCPTP are of general interest as modifiers of inflammatory signaling events. A previous study demonstrated that the small molecule spermidine is a selective activator of TCPTP in vitro. The aim of this study was to investigate whether activation of TCPTP by spermidine was capable of alleviating IFN-γ-induced, proinflammatory signaling and barrier dysfunction in human intestinal epithelial cells. Studies revealed that treatment of T84 and HT29/cl.19A colonocytes with spermidine increased both TCPTP protein levels and enzymatic activity, correlating with a decrease in the phosphorylation of the signal transducers and activators of transcription 1 and 3, downstream mediators of IFN-γ signaling, upon coadministration of spermidine to IFN-γ-treated cells. On a functional level, spermidine protected barrier function in the setting of inflammation, restricting the decrease in transepithelial electrical resistance and the increase in epithelial permeability induced by IFN-γ in coincubation experiments. These data implicate spermidine as a potential therapeutic agent to treat conditions associated with elevated IFN-γ signaling and a faulty mucosal barrier.

Trichostatin-A modulates claudin-1 mRNA stability through the modulation of Hu antigen R and tristetraprolin in colon cancer cells.

Expression of claudin-1, a tight junction protein, is highly upregulated in colon cancer. We have reported that claudin-1 expression in colon cancer cells is epigenetically regulated as histone deacetylase (HDAC) inhibitors decrease claudin-1 messenger RNA (mRNA) stability and thus expression. In this regard, our data suggested a role of the 3'-untranslated region (UTR) in the regulation of HDAC-dependent regulation of claudin-1 mRNA stability. In the current study, we demonstrate, based on our continued investigation, that the ELAV-like RNA-binding proteins (RBPs), human antigen R (HuR) and tristetraprolin (TTP) associate with the 3'-UTR of claudin-1 mRNA to modulate the latter's stability. Ribonomic and site-directed mutagenesis approaches were used to confirm the binding of HuR and TTP to the 3'-UTR of claudin-1. We further confirmed their roles in the stabilization of claudin-1 mRNA, under conditions of HDAC inhibition. In summary, we report that HuR and TTP are the critical regulators of the posttranscriptional regulation of claudin-1 expression in colon cancer cells. We also demonstrate that inhibition of HDACs by trichostatin treatment decreased the binding of HuR while increasing the binding of TTP to the 3'-UTR of claudin-1. Additionally, we provide data showing transcriptional regulation of claudin-1 expression, through the regulation of transcription factor Sp1. Taken together, we demonstrate epigenetic regulation of claudin-1 expression in colon cancer cells at the transcriptional and posttranscriptional levels.

Claudin-1 up-regulates the repressor ZEB-1 to inhibit E-cadherin expression in colon cancer cells.

BACKGROUND & AIMS: Expression of the tight junction protein claudin-1 is dysregulated in colon tumors and associates with their progression. Up-regulation of claudin-1 reduces expression of E-cadherin. We investigated the mechanisms by which claudin-1 regulates E-cadherin expression and its effects in colon cancer cells. MATERIALS AND METHODS: We used gene expression analysis, immunoblotting, and reverse transcription polymerase chain reaction to associate expression of the repressor of transcription Zinc Finger E-box binding homeobox-box1 (ZEB-1) with claudin-1. We analyzed SW480 colon cancer cells that overexpressed claudin-1, or SW620 cells in which claudin-1 expression was repressed, to determine the effects on ZEB-1 and E-cadherin expression, invasive activity, and resistance to anoikis. We studied cells that expressed constitutively active or dominant negative forms of factors in the Wnt or phosphotidylinositol-3-kinase signaling pathways and used pharmacologic inhibitors of these pathways to study their role in claudin-1-dependent regulation of ZEB-1. We used microarray analysis to examine gene expression patterns in 260 colorectal tumor and normal colon samples. RESULTS: Claudin-1 down-regulates E-cadherin expression by up-regulating expression of ZEB-1. Claudin-1 activates Wnt and phosphotidylinositol-3-kinase/Akt signaling. ZEB-1 mediates claudin-1-regulated changes in cell invasion and anoikis. Expression of claudin-1 correlated with that of ZEB-1 in human colon tumor samples. In the progression from normal colonic epithelium to colon adenocarcinoma, levels of E-cadherin decreased, whereas levels of claudin-1 and ZEB-1 increased. Down-regulation of E-cadherin and up-regulation of ZEB-1 in colon tumors were associated with shorter survival times. CONCLUSIONS: Claudin-1 up-regulates the repressor ZEB-1 to reduce expression of E-cadherin in colon cancer cells, increasing their invasive activity and reducing anoikis. This pathway is associated with colorectal cancer progression and patient survival.

The JAK Inhibitor Tofacitinib Rescues Intestinal Barrier Defects Caused by Disrupted Epithelial-macrophage Interactions.

BACKGROUND AND AIMS: Loss-of-function variants in protein tyrosine phosphatase non-receptor type-2 [PTPN2] promote susceptibility to inflammatory bowel diseases [IBD]. PTPN2 regulates Janus-kinase [JAK] and signal transducer and activator of transcription [STAT] signalling, while protecting the intestinal epithelium from inflammation-induced barrier disruption. The pan-JAK inhibitor tofacitinib is approved to treat ulcerative colitis, but its effects on intestinal epithelial cell-macrophage interactions and on barrier properties are unknown. We aimed to determine if tofacitinib can rescue disrupted epithelial-macrophage interaction and barrier function upon loss of PTPN2. METHODS: Human Caco-2BBe intestinal epithelial cells [IECs] and THP-1 macrophages expressing control or PTPN2-specific shRNA were co-cultured with tofacitinib or vehicle. Transepithelial electrical resistance and 4 kDa fluorescein-dextran flux were measured to assess barrier function. Ptpn2fl/fl and Ptpn2-LysMCre mice, which lack Ptpn2 in myeloid cells, were treated orally with tofacitinib citrate twice daily to assess the in vivo effect on the intestinal epithelial barrier. Colitis was induced via administration of 1.5% dextran sulphate sodium [DSS] in drinking water. RESULTS: Tofacitinib corrected compromised barrier function upon PTPN2 loss in macrophages and/or IECs via normalisation of: [i] tight junction protein expression; [ii] excessive STAT3 signalling; and [iii] IL-6 and IL-22 secretion. In Ptpn2-LysMCre mice, tofacitinib reduced colonic pro-inflammatory macrophages, corrected underlying permeability defects, and prevented the increased susceptibility to DSS colitis. CONCLUSIONS: PTPN2 loss in IECs or macrophages compromises IEC-macrophage interactions and reduces epithelial barrier integrity. Both of these events were corrected by tofacitinib in vitro and in vivo. Tofacitinib may have greater therapeutic efficacy in IBD patients harbouring PTPN2 loss-of-function mutations.

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.

The JAK-Inhibitor Tofacitinib Rescues Human Intestinal Epithelial Cells and Colonoids from Cytokine-Induced Barrier Dysfunction.

BACKGROUND: Alterations to epithelial tight junctions can compromise the ability of the epithelium to act as a barrier between luminal contents and the underlying tissues, thereby increasing intestinal permeability, an early critical event in inflammatory bowel disease (IBD). Tofacitinib (Xeljanz), an orally administered pan-Janus kinase (JAK) inhibitor, was recently approved for the treatment of moderate to severe ulcerative colitis. Nevertheless, the effects of tofacitinib on intestinal epithelial cell functions are largely unknown. The aim of this study was to determine if JAK inhibition by tofacitinib can rescue cytokine-induced barrier dysfunction in intestinal epithelial cells (IECs). METHODS: T84 IECs were used to evaluate the effects of tofacitinib on JAK-signal transducer and activator of transcription (STAT) activation, barrier permeability, and expression and localization of tight junction proteins. The impact of tofacitinib on claudin-2 promoter activity was assessed in HT-29 IECs. Tofacitinib rescue of barrier function was also tested in human colonic stem cell-derived organoids. RESULTS: Pretreatment with tofacitinib prevented IFN-γ-induced decreases in transepithelial electrical resistance (TER) and increases in 4 kDa FITC-dextran permeability (FD4), partly due to claudin-2 transcriptional regulation and restriction of ZO-1 rearrangement at tight junctions. Although tofacitinib administered after IFN-γ challenge only partially normalized TER and claudin-2 levels, FD4 permeability and ZO-1 localization were fully recovered. The IFN-γ-induced FD4 permeability in primary human colonoids was fully rescued by tofacitinib. CONCLUSIONS: These data suggest differential therapeutic efficacy of tofacitinib in the rescue of pore vs leak-tight junction barrier defects and indicate a potential contribution of improved epithelial barrier function to the beneficial effects of tofacitinib in IBD patients.

T cell protein tyrosine phosphatase prevents STAT1 induction of claudin-2 expression in intestinal epithelial cells.

T cell protein tyrosine phosphatase (TCPTP) dephosphorylates a number of substrates, including JAK-STAT (signal transducer and activator of transcription) signaling proteins, which are activated by interferon (IFN)-γ, a major proinflammatory cytokine involved in conditions such as inflammatory bowel disease. A critical function of the intestinal epithelium is formation of a selective barrier to luminal contents. The structural units of the epithelium that regulate barrier function are the tight junctions (TJs), and the protein composition of the TJ determines the tightness of the barrier. Claudin-2 is a TJ protein that increases permeability to cations and reduces transepithelial electrical resistance (TER). We previously showed that transient knockdown (KD) of TCPTP permits increased expression of claudin-2 by IFN-γ. Here, we demonstrate that the decreased TER in TCPTP-deficient epithelial cells is alleviated by STAT1 KD. Moreover, increased claudin-2 in TCPTP-deficient cells requires enhanced STAT1 activation and STAT1 binding to the CLDN2 promoter. We also show that mutation of this STAT-binding site prevents elevated CLDN2 promoter activity in TCPTP-deficient epithelial cells. In summary, we demonstrate that TCPTP protects the intestinal epithelial barrier by restricting STAT-induced claudin-2 expression. This is a potential mechanism by which loss-of-function mutations in the gene encoding TCPTP may contribute to barrier defects in chronic intestinal inflammatory disease.

p-Coumaric acid, a dietary polyphenol ameliorates inflammation and curtails cartilage and bone erosion in the rheumatoid arthritis rat model.

This study was designed to explore the underlying mechanism of p-coumaric acid (CA), a dietary polyphenol in adjuvant-induced arthritis (AIA) rat model with reference to synovitis and osteoclastogenesis. Celecoxib (COX-2 selective inhibitor) (5 mg/kg b.wt) was used as a reference drug. CA remarkably suppressed the paw edema, body weight loss and inflammatory cytokine and chemokine levels (TNF-α, IL-1ß, IL-6, and MCP-1) in serum and ankle joint of arthritic rats. Consistently, CA reduced the expression of osteoclastogenic factors (RANKL and TRAP), pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-17), and inflammatory enzymes (iNOS and COX-2) in arthritic rats. However, OPG expression was found elevated. Besides, the abundance of transcription factors (NF-κB-p65, and p-NF-κB-p65, NFATc-1, and c-Fos) and MAP kinases (JNK, p-JNK, and ERK1/2) expression was alleviated in CA administered arthritic rats. In addition, CA truncated osteoclastogenesis by regulating the RANKL/OPG imbalance in arthritic rats and suppressing the RANKL-induced NFATc-1 and c-Fos expression in vitro. Radiological (CT and DEXA scan) and histological assessments authenticated that CA inhibited TRAP, bone destruction and cartilage degradation in association with enhanced bone mineral density. Taken together, our findings suggest that CA demonstrated promising anti-arthritic effect and could prove useful as an alternative drug in RA therapeutics. © 2017 BioFactors, 43(5):698-717, 2017.

VSL#3 Probiotic Stimulates T-cell Protein Tyrosine Phosphatase-mediated Recovery of IFN-γ-induced Intestinal Epithelial Barrier Defects.

BACKGROUND: VSL#3 is a probiotic compound that has been used in the treatment of inflammatory bowel disease. T-cell protein tyrosine phosphatase (TCPTP) is the protein product of the inflammatory bowel disease candidate gene, PTPN2, and we have previously shown that it protects epithelial barrier function. The aim of this study was to investigate whether VSL#3 improves intestinal epithelial barrier function against the effects of the inflammatory bowel disease-associated proinflammatory cytokine, interferon-gamma (IFN-γ) through activation of TCPTP. METHODS: Polarized monolayers of T84 intestinal epithelial cells were treated with increasing concentrations of VSL#3 to determine effects on TCPTP expression and enzymatic activity. Therapeutic effects of VSL#3 against barrier disruption by IFN-γ were measured by transepithelial electrical resistance and fluorescein isothiocyanate-dextran permeability. A novel TCPTP-deficient HT-29 intestinal epithelial cell line was generated to study the role of TCPTP in mediating the effects of VSL#3. Tight junction protein distribution was assessed with confocal microscopy. RESULTS: VSL#3 increased TCPTP protein levels and enzymatic activity, correlating with a VSL#3-induced decrease in IFN-γ signaling. VSL#3 corrected the decrease in transepithelial electrical resistance and the increase in epithelial permeability induced by IFN-γ. Moreover, the restorative effect of VSL#3 against IFN-γ signaling, epithelial permeability defects, altered expression and localization of the tight junction proteins claudin-2, occludin, and zonula occludens-1, were not realized in stable TCPTP/(PTPN2)-deficient HT-29 intestinal epithelial cells. CONCLUSIONS: VSL#3 reduces IFN-γ signaling and IFN-γ-induced epithelial barrier defects in a TCPTP-dependent manner. These data point to a key role for TCPTP as a therapeutic target for restoration of barrier function using probiotics.

Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a severe form of congenital diarrhea that arises from inactivating mutations in the gene encoding myosin Vb (MYO5B). We have examined the association of mutations in MYO5B and disruption of microvillar assembly and polarity in enterocytes. Stable MYO5B knockdown (MYO5B-KD) in CaCo2-BBE cells elicited loss of microvilli, alterations in junctional claudins, and disruption of apical and basolateral trafficking; however, no microvillus inclusions were observed in MYO5B-KD cells. Expression of WT MYO5B in MYO5B-KD cells restored microvilli; however, expression of MYO5B-P660L, a MVID-associated mutation found within Navajo populations, did not rescue the MYO5B-KD phenotype but induced formation of microvillus inclusions. Microvilli establishment required interaction between RAB8A and MYO5B, while loss of the interaction between RAB11A and MYO5B induced microvillus inclusions. Using surface biotinylation and dual immunofluorescence staining in MYO5B-KD cells expressing mutant forms of MYO5B, we observed that early microvillus inclusions were positive for the sorting marker SNX18 and derived from apical membrane internalization. In patients with MVID, MYO5B-P660L results in global changes in polarity at the villus tips that could account for deficits in apical absorption, loss of microvilli, aberrant junctions, and losses in transcellular ion transport pathways, likely leading to the MVID clinical phenotype of neonatal secretory diarrhea.

Rab25 regulates integrin expression in polarized colonic epithelial cells.

Rab25 is a tumor suppressor for colon cancer in humans and mice. To identify elements of intestinal polarity regulated by Rab25, we developed Caco2-BBE cell lines stably expressing short hairpin RNA for Rab25 and lines rescuing Rab25 knockdown with reexpression of rabbit Rab25. Rab25 knockdown decreased α2-, α5-, and ß1-integrin expression. We observed colocalization and direct association of Rab25 with α5ß1-integrins. Rab25 knockdown also up-regulated claudin-1 expression, increased transepithelial resistance, and increased invasive behavior. Rab25-knockdown cells showed disorganized brush border microvilli with decreases in villin expression. All of these changes were reversed by reintroduction of rabbit Rab25. Rab25 knockdown altered the expression of 29 gene transcripts, including the loss of α5-integrin transcripts. Rab25 loss decreased expression of one transcription factor, ETV4, and overexpression of ETV4 in Rab25-knockdown cells reversed losses of α5ß1-integrin. The results suggest that Rab25 controls intestinal cell polarity through the regulation of gene expression.

Caudal homeobox protein Cdx-2 cooperates with Wnt pathway to regulate claudin-1 expression in colon cancer cells.

Dysregulation of tight junctions (TJs) is often associated with human diseases including carcinogenesis and recent studies support role of TJ integral proteins in the regulation of Epithelial-to-Mesenchymal Transition (EMT). In this regard, expression of claudin-1, a key constituent of TJs, is highly increased in colon cancer and is causally associated with the tumor growth and progression. However, mechanism/s underlying regulation of claudin-1 expression in intestinal epithelial cells remains poorly understood. In our studies, we have identified putative binding sites for intestinal transcription factors Cdx1, -2 and GATA4 in the 5'-flanking region of the claudin-1 gene. Our further studies using full length and/or deletion mutant constructs in two different human colon cancer cell lines, SW480 and HCT116, showed key role of Cdx1, Cdx2 and GATA4 in the regulation of claudin-1 mRNA expression. However, overexpression of Cdx2 had the most potent effect upon claudin-1 mRNA expression and promoter activity. Also, in colon cancer patient samples, we observed a significant and parallel correlation between claudin-1 and Cdx2 expressions. Chromatin immunoprecipitation (ChIP) assay confirmed the Cdx2 binding with claudin-1 promoter in vivo. Using Cdx2 deletion mutant constructs, we further mapped the Cdx2 C-terminus domain to be important in the regulation of claudin-1 promoter activity. Interestingly, co-expression of activated ß-catenin further induced the Cdx2-dependent upregulation of claudin-1 promoter activity while expression of the dominant negative (dn)-TCF-4 abrogated this activation. Taken together, we conclude that homeodomain transcription factors Cdx1, Cdx2 and GATA4 regulate claudin-1 gene expression in human colon cancer cells. Moreover, a functional crosstalk between Wnt-signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins and GATA-proteins is demonstrated in the regulation of claudin-1 promoter-activation.