CXADR-Like Membrane Protein Regulates Colonic Epithelial Cell Proliferation and Prevents Tumor Growth.

BACKGROUND & AIMS: CXADR-like membrane protein (CLMP) is structurally related to coxsackie and adenovirus receptor. Pathogenic variants in CLMP gene have been associated with congenital short bowel syndrome, implying a role for CLMP in intestinal development. However, the contribution of CLMP to regulating gut development and homeostasis is unknown. METHODS: In this study, we investigated CLMP function in the colonic epithelium using complementary in vivo and in vitro approaches, including mice with inducible intestinal epithelial cell (IEC)-specific deletion of CLMP (ClmpΔIEC), intestinal organoids, IECs with overexpression, or loss of CLMP and RNA sequencing data from individuals with colorectal cancer. RESULTS: Loss of CLMP enhanced IEC proliferation and, conversely, CLMP overexpression reduced proliferation. Xenograft experiments revealed increased tumor growth in mice implanted with CLMP-deficient colonic tumor cells, and poor engraftment was observed with CLMP-overexpressing cells. ClmpΔIEC mice showed exacerbated tumor burden in an azoxymethane and dextran sulfate sodium-induced colonic tumorigenesis model, and CLMP expression was reduced in human colorectal cancer samples. Mechanistic studies revealed that CLMP-dependent regulation of IEC proliferation is linked to signaling through mTOR-Akt-ß-catenin pathways. CONCLUSIONS: These results reveal novel insights into CLMP function in the colonic epithelium, highlighting an important role in regulating IEC proliferation, suggesting tumor suppressive function in colon cancer.

Development and validation of a reporter cell line for rapid AAV quality control assessment.

Adenovirus-associated virus is a powerful vector system for transducing cells in vivo. It is widely used in animal systems due to high transduction efficiency of non-dividing cells with more than a dozen serotypes that have preferential tissue tropism. The viral genome remains episomal in the nucleus but maintains sustained expression in terminally differentiated cells for several weeks to months. Despite the popularity of recombinant AAV (rAAV) vectors, quality control testing of the virus after production is largely limited to physical characteristics such as viral genomes/ml determinations and silver staining acrylamide gels to determine purity. Functional testing, in vivo, is not practical due to high cost and restricted access of animal care and long duration of the assay (2-3 weeks). Some functional testing can be accomplished in cultured cells such as HEK293 cells, but HEK293 cells limit the types of rAAV constructs that can be tested. Many rAAV constructs are designed to study neurons in the brain with neural-specific promoters and many are floxed with loxp sites to be "activated" only in Cre-expressing neurons in transgenic animals. To develop a reporter cell line for rapid rAAV quality control assessment of these neural-specific, floxed rAAV constructs, we used the lentiviral system to stably express Cre recombinase in the SH-SY5Y neuroblastoma cell line. •A simple and economic method to evaluate recombinant AAV in vitro.•Allows functional validation of rAAV across a wide range of serotypes and promoters.•Allows functional validation of Cre-dependent rAAV constructs.

TNFα promotes mucosal wound repair through enhanced platelet activating factor receptor signaling in the epithelium.

Pathobiology of several chronic inflammatory disorders, including ulcerative colitis and Crohn's disease is related to intermittent, spontaneous injury/ulceration of mucosal surfaces. Disease morbidity has been associated with pathologic release of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). In this report, we show that TNFα promotes intestinal mucosal repair through upregulation of the GPCR platelet activating factor receptor (PAFR) in the intestinal epithelium. Platelet activating factor (PAF) was increased in healing mucosal wounds and its engagement with epithelial PAFR leads to activation of epidermal growth factor receptor, Src and Rac1 signaling to promote wound closure. Consistent with these findings, delayed colonic mucosal repair was observed after administration of a neutralizing TNFα antibody and in mice lacking PAFR. These findings suggest that in the injured mucosa, the pro-inflammatory milieu containing TNFα and PAF sets the stage for reparative events mediated by PAFR signaling.

Macrophage-dependent neutrophil recruitment is impaired under conditions of increased intestinal permeability in JAM-A-deficient mice.

Junctional adhesion molecule-A (JAM-A) is a transmembrane glycoprotein expressed on leukocytes, endothelia, and epithelia that regulates biological processes including barrier function and immune responses. While JAM-A has been reported to facilitate tissue infiltration of leukocytes under inflammatory conditions, the contributions of leukocyte-expressed JAM-A in vivo remain unresolved. We investigated the role of leukocyte-expressed JAM-A in acute peritonitis induced by zymosan, lipopolysaccharide (LPS), or TNFα using mice with selective loss of JAM-A in myelomonocytic cells (LysM-Cre;Jam-afl/fl). Surprisingly, in LysM-Cre;Jam-afl/fl mice, loss of JAM-A did not affect neutrophil (PMN) recruitment into the peritoneum in response to zymosan, LPS, or TNFα although it was significantly reduced in Jam-aKO mice. In parallel, Jam-aKO peritoneal macrophages exhibited diminished CXCL1 chemokine production and decreased activation of NF-kB, whereas those from LysM-Cre;Jam-afl/fl mice were unaffected. Using Villin-Cre;Jam-afl/fl mice, targeted loss of JAM-A on intestinal epithelial cells resulted in increased intestinal permeability along with reduced peritoneal PMN migration as well as lower levels of CXCL1 and active NF-kB similar to that observed in Jam-aKO animals. Interestingly, in germ-free Villin-Cre;Jam-afl/fl mice, PMN recruitment was unaffected suggesting dependence on gut microbiota. Such observations highlight the functional link between a leaky gut and regulation of innate immune responses.

Role of JAM-A tyrosine phosphorylation in epithelial barrier dysfunction during intestinal inflammation.

Junctional adhesion molecule-A (JAM-A), an epithelial tight junction protein, plays an important role in regulating intestinal permeability through association with a scaffold signaling complex containing ZO-2, Afadin, and the small GTPase Rap2. Under inflammatory conditions, we report that the cytoplasmic tail of JAM-A is tyrosine phosphorylated (p-Y280) in association with loss of barrier function. While barely detectable Y280 phosphorylation was observed in confluent monolayers of human intestinal epithelial cells under basal conditions, exposure to cytokines TNFα, IFNγ, IL-22, or IL-17A, resulted in compromised barrier function in parallel with increased p-Y280. Phosphorylation was Src kinase dependent, and we identified Yes-1 and PTPN13 as a major kinase and phosphatase for p-JAM-A Y280, respectively. Moreover, cytokines IL-22 or IL-17A induced increased activity of Yes-1. Furthermore, the Src kinase inhibitor PP2 rescued cytokine-induced epithelial barrier defects and inhibited phosphorylation of JAM-A Y280 in vitro. Phosphorylation of JAM-A Y280 and increased permeability correlated with reduced JAM-A association with active Rap2. Finally, we observed increased phosphorylation of Y280 in colonic epithelium of individuals with ulcerative colitis and in mice with experimentally induced colitis. These findings support a novel mechanism by which tyrosine phosphorylation of JAM-A Y280 regulates epithelial barrier function during inflammation.

Intracellular Desmoglein-2 cleavage sensitizes epithelial cells to apoptosis in response to pro-inflammatory cytokines.

Desmosomal cadherins mediate intercellular adhesion and have also been shown to regulate homeostatic signaling in epithelial cells. We have previously reported that select pro-inflammatory cytokines induce Dsg2 ectodomain cleavage and shedding from intestinal epithelial cells (IECs). Dsg2 extracellular cleaved fragments (Dsg2 ECF) function to induce paracrine pro-proliferative signaling in epithelial cells. In this study, we show that exposure of IECs to pro-inflammatory cytokines interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) resulted in Dsg2 intracellular cleavage and generation of a ~55 kDa fragment (Dsg2 ICF). Dsg2 intracellular cleavage is mediated by caspase-8 and occurs prior to Dsg2 extracellular cleavage and the execution of apoptosis. Expression of exogenous Dsg2 ICF in model IECs resulted in increased sensitivity to apoptotic stimuli and apoptosis execution. Additionally, expression of the Dsg2 ICF repressed the anti-apoptotic Bcl-2 family member proteins Bcl-XL and Mcl1. Taken together, our findings identify a novel mechanism by which pro-inflammatory mediators induce modification of Dsg2 to activate apoptosis and eliminate damaged cells, while also promoting release of Dsg2 ECF that promotes proliferation of neighboring cells and epithelial barrier recovery.

Macrophage-derived IL-10 mediates mucosal repair by epithelial WISP-1 signaling.

In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by dynamic crosstalk between immune cells and the epithelium; however, the mechanisms involved remain incompletely understood. Here, we report that IL-10 was rapidly induced following intestinal mucosal injury and was required for optimal intestinal mucosal wound closure. Conditional deletion of IL-10 specifically in CD11c-expressing cells in vivo implicated macrophages as a critical innate immune contributor to IL-10-induced wound closure. Consistent with these findings, wound closure in T cell- and B cell-deficient Rag1-/- mice was unimpaired, demonstrating that adaptive immune cells are not absolutely required for this process. Further, following mucosal injury, macrophage-derived IL-10 resulted in epithelial cAMP response element-binding protein (CREB) activation and subsequent synthesis and secretion of the pro-repair WNT1-inducible signaling protein 1 (WISP-1). WISP-1 induced epithelial cell proliferation and wound closure by activating epithelial pro-proliferative pathways. These findings define the involvement of macrophages in regulating an IL-10/CREB/WISP-1 signaling axis, with broad implications in linking innate immune activation to mucosal wound repair.

HNF4α regulates claudin-7 protein expression during intestinal epithelial differentiation.

The intestinal epithelium is a dynamic barrier that maintains the distinct environments of intestinal tissue and lumen. Epithelial barrier function is defined principally by tight junctions, which, in turn, depend on the regulated expression of claudin family proteins. Claudins are expressed differentially during intestinal epithelial cell (IEC) differentiation. However, regulatory mechanisms governing claudin expression during epithelial differentiation are incompletely understood. We investigated the molecular mechanisms regulating claudin-7 during IEC differentiation. Claudin-7 expression is increased as epithelial cells differentiate along the intestinal crypt-luminal axis. By using model IECs we observed increased claudin-7 mRNA and nascent heteronuclear RNA levels during differentiation. A screen for potential regulators of the CLDN7 gene during IEC differentiation was performed using a transcription factor/DNA binding array, CLDN7 luciferase reporters, and in silico promoter analysis. We identified hepatocyte nuclear factor 4α as a regulatory factor that bound endogenous CLDN7 promoter in differentiating IECs and stimulated CLDN7 promoter activity. These findings support a role of hepatocyte nuclear factor 4α in controlling claudin-7 expression during IEC differentiation.

Annexin A1-containing extracellular vesicles and polymeric nanoparticles promote epithelial wound repair.

Epithelial restitution is an essential process that is required to repair barrier function at mucosal surfaces following injury. Prolonged breaches in epithelial barrier function result in inflammation and further damage; therefore, a better understanding of the epithelial restitution process has potential for improving the development of therapeutics. In this work, we demonstrate that endogenous annexin A1 (ANXA1) is released as a component of extracellular vesicles (EVs) derived from intestinal epithelial cells, and these ANXA1-containing EVs activate wound repair circuits. Compared with healthy controls, patients with active inflammatory bowel disease had elevated levels of secreted ANXA1-containing EVs in sera, indicating that ANXA1-containing EVs are systemically distributed in response to the inflammatory process and could potentially serve as a biomarker of intestinal mucosal inflammation. Local intestinal delivery of an exogenous ANXA1 mimetic peptide (Ac2-26) encapsulated within targeted polymeric nanoparticles (Ac2-26 Col IV NPs) accelerated healing of murine colonic wounds after biopsy-induced injury. Moreover, one-time systemic administration of Ac2-26 Col IV NPs accelerated recovery following experimentally induced colitis. Together, our results suggest that local delivery of proresolving peptides encapsulated within nanoparticles may represent a potential therapeutic strategy for clinical situations characterized by chronic mucosal injury, such as is seen in patients with IBD.

IFNγ-induced suppression of ß-catenin signaling: evidence for roles of Akt and 14.3.3ζ.

The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. ß-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/ß-catenin signaling. Here we show that inhibition of Akt/ß-catenin-mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated ß-catenin 552 (pß-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits ß-catenin transactivation in response to IFNγ stimulation. IFNγ initially promotes ß-catenin transactivation through Akt-dependent C-terminal phosphorylation of ß-catenin to promote its association with 14.3.3ζ. Augmented ß-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/ß-catenin from the nucleus, thereby inhibiting ß-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.

Periostin and receptor activator of nuclear factor κ-B ligand expression in allergic fungal rhinosinusitis.

BACKGROUND: Allergic fungal rhinosinusitis (AFRS) is a disease demonstrating substantial eosinophilic inflammation and characteristic radiographic bony erosion/expansion. Periostin is an extracellular matrix protein associated with eosinophil accumulation in eosinophilic esophagitis, allergic asthma mucus production, and chronic rhinosinusitis (CRS) polyp formation. Receptor activator of nuclear factor κ-B ligand (RANKL) is an osteoclast activator present in osteoporosis and periodontal disease. We sought to evaluate periostin and RANKL expression in AFRS and correlate these levels with radiographic scales of disease severity. METHODS: Thirty sinus tissue specimens were intraoperatively collected from 3 patient groups: AFRS; CRS without nasal polyps (CRSsNP); and controls (n = 10 per group). Specimens were analyzed by semiquantitative reverse-transcription polymerase chain reaction (sq-RT-PCR) and immunofluorescence (IF) labeling/confocal microscopy for the presence of both periostin and RANKL. Immunofluorescence staining intensity was quantified by pixel density analysis. Preoperative computed tomography (CT) scans from each patient were scored using both the Lund-Mackay and CT bone erosion scoring systems. RESULTS: Periostin was significantly elevated in AFRS sinus tissue compared to CRSsNP and controls, as demonstrated by IF (p < 0.001) and PCR (p = 0.011). RANKL was not detected in sinus tissue by IF or PCR. Periostin levels positively correlated with radiographic indices of disease severity for both soft tissue and bone, using Lund-Mackay (r = 0.926 [PCR] and r = 0.581 [IF]) and CT bone erosion (r = 0.672 [PCR] and r = 0.616 [IF]) scoring systems, respectively. CONCLUSION: Periostin is increased in AFRS tissue compared to CRSsNP and controls. Periostin levels positively correlate with radiologic disease severity scores. The increased levels of periostin in AFRS are possibly tied to its intense eosinophilic inflammatory etiology.

Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins.

Tight junctions (TJs) are dynamic, multiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiological development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a biosensor of TJ remodeling, we observed increased claudin 4 fluorescence recovery after photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon γ and tumor necrosis factor α increased the proportion of mobile claudin 4 in the TJ. Up-regulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin-binding incompatibility.

Annexin A2 regulates ß1 integrin internalization and intestinal epithelial cell migration.

The gastrointestinal epithelium functions as an important barrier that separates luminal contents from the underlying tissue compartment and is vital in maintaining mucosal homeostasis. Mucosal wounds in inflammatory disorders compromise the critical epithelial barrier. In response to injury, intestinal epithelial cells (IECs) rapidly migrate to reseal wounds. We have previously observed that a membrane-associated, actin binding protein, annexin A2 (AnxA2), is up-regulated in migrating IECs and plays an important role in promoting wound closure. To identify the mechanisms by which AnxA2 promotes IEC movement and wound closure, we used a loss of function approach. AnxA2-specific shRNA was utilized to generate IECs with stable down-regulation of AnxA2. Loss of AnxA2 inhibited IEC migration while promoting enhanced cell-matrix adhesion. These functional effects were associated with increased levels of ß1 integrin protein, which is reported to play an important role in mediating the cell-matrix adhesive properties of epithelial cells. Because cell migration requires dynamic turnover of integrin-based adhesions, we tested whether AnxA2 modulates internalization of cell surface ß1 integrin required for forward cell movement. Indeed, pulse-chase biotinylation experiments in IECs lacking AnxA2 demonstrated a significant increase in cell surface ß1 integrin that was accompanied by decreased ß1 integrin internalization and degradation. These findings support an important role of AnxA2 in controlling dynamics of ß1 integrin at the cell surface that in turn is required for the active turnover of cell-matrix associations, cell migration, and wound closure.

Annexin A1, formyl peptide receptor, and NOX1 orchestrate epithelial repair.

N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1. We show that epithelial cell migration was regulated by this signaling cascade through oxidative inactivation of the regulatory phosphatases PTEN and PTP-PEST, with consequent activation of focal adhesion kinase (FAK) and paxillin. In vivo studies using intestinal epithelial specific Nox1(-/-IEC) and AnxA1(-/-) mice demonstrated defects in intestinal mucosal wound repair, while systemic administration of ANXA1 promoted wound recovery in a NOX1-dependent fashion. Additionally, increased ANXA1 expression was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of ulcerative colitis patients compared with normal intestinal mucosa. Our findings delineate a novel epithelial FPR1/NOX1-dependent redox signaling pathway that promotes mucosal wound repair.