Comprehensive split TEV based protein-protein interaction screening reveals TAOK2 as a key modulator of Hippo signalling to limit growth.

The conserved Hippo signalling pathway plays a crucial role in tumour formation by limiting tissue growth and proliferation. At the core of this pathway are tumour suppressor kinases STK3/4 and LATS1/2, which limit the activity of the oncogene YAP1, the primary downstream effector. Here, we employed a split TEV-based protein-protein interaction screen to assess the physical interactions among 28 key Hippo pathway components and potential upstream modulators. This screen led us to the discovery of TAOK2 as pivotal modulator of Hippo signalling, as it binds to the pathway's core kinases, STK3/4 and LATS1/2, and leads to their phosphorylation. Specifically, our findings revealed that TAOK2 binds to and phosphorylates LATS1, resulting in the reduction of YAP1 phosphorylation and subsequent transcription of oncogenes. Consequently, this decrease led to a decrease in cell proliferation and migration. Interestingly, a correlation was observed between reduced TAOK2 expression and decreased patient survival time in certain types of human cancers, including lung and kidney cancer as well as glioma. Moreover, in cellular models corresponding to these cancer types the downregulation of TAOK2 by CRISPR inhibition led to reduced phosphorylation of LATS1 and increased proliferation rates, supporting TAOK2's role as tumour suppressor gene. By contrast, overexpression of TAOK2 in these cellular models lead to increased phospho-LATS1 but reduced cell proliferation. As TAOK2 is a druggable kinase, targeting TAOK2 could serve as an attractive pharmacological approach to modulate cell growth and potentially offer strategies for combating cancer.

Pro-Angiogenic Macrophage Phenotype to Promote Myocardial Repair.

BACKGROUND: Heart failure following myocardial infarction (MI) remains one of the major causes of death worldwide, and its treatment is a crucial challenge of cardiovascular medicine. An attractive therapeutic strategy is to stimulate endogenous mechanisms of myocardial regeneration. OBJECTIVES: This study evaluates the potential therapeutic treatment with annexin A1 (AnxA1) to induce cardiac repair after MI. METHODS: AnxA1 knockout (AnxA1-/-) and wild-type mice underwent MI induced by ligation of the left anterior descending coronary artery. Cardiac functionality was assessed by longitudinal echocardiographic measurements. Histological, fluorescence-activated cell sorting, dot blot analysis, and in vitro/ex vivo studies were used to assess the myocardial neovascularization, macrophage content, and activity in response to AnxA1. RESULTS: AnxA1-/- mice showed a reduced cardiac functionality and an expansion of proinflammatory macrophages in the ischemic area. Cardiac macrophages from AnxA1-/- mice exhibited a dramatically reduced ability to release the proangiogenic mediator vascular endothelial growth factor (VEGF)-A. However, AnxA1 treatment enhanced VEGF-A release from cardiac macrophages, and its delivery in vivo markedly improved cardiac performance. The positive effect of AnxA1 treatment on cardiac performance was abolished in wild-type mice transplanted with bone marrow derived from Cx3cr1creERT2Vegfflox/flox or in mice depleted of macrophages. Similarly, cardioprotective effects of AnxA1 were obtained in pigs in which full-length AnxA1 was overexpressed by use of a cardiotropic adeno-associated virus. CONCLUSIONS: AnxA1 has a direct action on cardiac macrophage polarization toward a pro-angiogenic, reparative phenotype. AnxA1 stimulated cardiac macrophages to release high amounts of VEGF-A, thus inducing neovascularization and cardiac repair.

Neutrophil elastase plays a non-redundant role in remodeling the venular basement membrane and neutrophil diapedesis post-ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury is a severe inflammatory insult associated with numerous pathologies, such as myocardial infarction, stroke and acute kidney injury. I/R injury is characterized by a rapid influx of activated neutrophils secreting toxic free radical species and degrading enzymes that can irreversibly damage the tissue, thus impairing organ functions. Significant efforts have been invested in identifying therapeutic targets to suppress neutrophil recruitment and activation post-I/R injury. In this context, pharmacological targeting of neutrophil elastase (NE) has shown promising anti-inflammatory efficacy in a number of experimental and clinical settings of I/R injury and is considered a plausible clinical strategy for organ care. However, the mechanisms of action of NE, and hence its inhibitors, in this process are not fully understood. Here we conducted a comprehensive analysis of the impact of NE genetic deletion on neutrophil infiltration in four murine models of I/R injury as induced in the heart, kidneys, intestine and cremaster muscle. In all models, neutrophil migration into ischemic regions was significantly suppressed in NE-/- mice as compared with wild-type controls. Analysis of inflamed cremaster muscle and mesenteric microvessels by intravital and confocal microscopy revealed a selective entrapment of neutrophils within venular walls, most notably at the level of the venular basement membrane (BM) following NE deletion/pharmacological blockade. This effect was associated with the suppression of NE-mediated remodeling of the low matrix protein expressing regions within the venular BM used by transmigrating neutrophils as exit portals. Furthermore, whilst NE deficiency led to reduced neutrophil activation and vascular leakage, levels of monocytes and prohealing M2 macrophages were reduced in tissues of NE-/- mice subjected to I/R. Collectively our results identify a vital and non-redundant role for NE in supporting neutrophil breaching of the venular BM post-I/R injury but also suggest a protective role for NE in promoting tissue repair. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

(Re) Solving Repair After Myocardial Infarction.

Cardiovascular diseases, including myocardial infarction and its complications such as heart failure, are the leading cause of death worldwide. To date, basic and translational research becomes necessary to unravel the mechanisms of cardiac repair post-myocardial infarction. The local inflammatory tissue response after acute myocardial infarction determines the subsequent healing process. The diversity of leukocytes such as neutrophils, macrophages and lymphocytes contribute to the clearance of dead cells while activating reparative pathways necessary for myocardial healing. Cardiomyocyte death triggers wall thinning, ventricular dilatation, and fibrosis that can cause left ventricular dysfunction and heart failure. The ultimate goal of cardiac repair is to regenerate functionally viable myocardium after myocardial infarction to prevent cardiac death. Current therapies for heart failure after myocardial infarction are limited and non-curative. At the moment in clinic, conventional surgical interventions such as coronary artery bypass graft or percutaneous coronary interventions are only able to partially restore heart function, with a minor improvement in the left ventricular ejection fraction. The goal of this review is to provide an overview of endogenous myocardial repair mechanisms possibly transferable to future treatment strategies. Among the innovative factors identified as essential in cardiac healing, we highlight specialized pro-resolving mediators as the emerging factors that provide the key molecular signals for the activation of the reparative cells in the myocardium.

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.

Ly6Chigh Monocytes Oscillate in the Heart During Homeostasis and After Myocardial Infarction-Brief Report.

OBJECTIVE: Circadian regulation of neutrophil homeostasis affects myocardial infarction (MI) healing. It is unknown whether diurnal variations of monocyte counts exist in the heart and whether this affects their cardiac infiltration in response to MI. APPROACH AND RESULTS: Murine blood and organs were harvested at distinct times of day and analyzed by flow cytometry. Ly6Chigh monocyte surface expression levels of chemokine receptors (CCR) were ≈2-fold higher at the beginning of the active phase, Zeitgeber Time (ZT) 13 compared with ZT5. This was because of enhanced receptor surface expression at ZT13, whereas no significant changes in total cellular protein levels were found. Most blood Ly6Chigh monocytes were CCR2high, whereas only a minority was CCR1high and CCR5high. We also found diurnal changes of classical monocyte blood counts in humans, being higher in the evening, while exhibiting enhanced CCR2 surface expression in the morning. In support of monocyte oscillations between blood and tissue, murine cardiac Ly6Chigh monocyte counts were highest at ZT13, accompanied by an upregulation of cardiac CC chemokine ligand 2 mRNA. Mice subjected to MI at ZT13 had an even higher upregulation of CCR2 surface expression on circulating monocytes compared with noninfarcted mice and more elevated cardiac CC chemokine ligand 2 protein expression and more pronounced Ly6Chigh monocyte infiltration compared with ZT5-infarcted mice. Concomitantly, CCR2 antagonism only inhibited the excessive cardiac Ly6Chigh monocyte infiltration after ZT13 MI but not ZT5 MI. CONCLUSIONS: CCR2 surface expression on Ly6Chigh monocytes changes in a time-of-day-dependent manner, which crucially affects cardiac monocyte recruitment after an acute ischemic event.

The advantageous role of annexin A1 in cardiovascular disease.

The inflammatory response protects the human body against infection and injury. However, uncontrolled and unresolved inflammation can lead to tissue damage and chronic inflammatory diseases. Therefore, active resolution of inflammation is essential to restore tissue homeostasis. This review focuses on the pro-resolving molecule annexin A1 (ANXA1) and its derived peptides. Mechanisms instructed by ANXA1 are multidisciplinary and affect leukocytes as well as endothelial cells and tissue resident cells like macrophages and mast cells. ANXA1 has an outstanding role in limiting leukocyte recruitment and different aspects of ANXA1 as modulator of the leukocyte adhesion cascade are discussed here. Additionally, this review details the therapeutic relevance of ANXA1 and its derived peptides in cardiovascular diseases since atherosclerosis stands out as a chronic inflammatory disease with impaired resolution and continuous leukocyte recruitment.

The microenvironment of injured murine gut elicits a local pro-restitutive microbiota.

The mammalian intestine houses a complex microbial community, which influences normal epithelial growth and development, and is integral to the repair of damaged intestinal mucosa(1-3). Restitution of injured mucosa involves the recruitment of immune cells, epithelial migration and proliferation(4,5). Although microenvironmental alterations have been described in wound healing(6), a role for extrinsic influences, such as members of the microbiota, has not been reported. Here, we show that a distinct subpopulation of the normal mucosal-associated gut microbiota expands and preferentially colonizes sites of damaged murine mucosa in response to local environmental cues. Our results demonstrate that formyl peptide receptor 1 (FPR1) and neutrophilic NADPH oxidase (NOX2) are required for the rapid depletion of microenvironmental oxygen and compensatory responses, resulting in a dramatic enrichment of an anaerobic bacterial consortium. Furthermore, the dominant member of this wound-mucosa-associated microbiota, Akkermansia muciniphila (an anaerobic, mucinophilic gut symbiont(7,8)), stimulated proliferation and migration of enterocytes adjacent to the colonic wounds in a process involving FPR1 and intestinal epithelial-cell-specific NOX1-dependent redox signalling. These findings thus demonstrate how wound microenvironments induce the rapid emergence of 'probiont' species that contribute to enhanced repair of mucosal wounds. Such microorganisms could be exploited as potential therapeutics.

Annexin A1: shifting the balance towards resolution and repair.

Epithelial barriers play an important role in regulating mucosal homeostasis. Upon injury, the epithelium and immune cells orchestrate repair mechanisms that re-establish homeostasis. This process is highly regulated by protein and lipid mediators such as Annexin A1 (ANXA1). In this review, we focus on the pro-repair properties of ANXA1.

Cutting Edge: IL-36 Receptor Promotes Resolution of Intestinal Damage.

IL-1 family members are central mediators of host defense. In this article, we show that the novel IL-1 family member IL-36γ was expressed during experimental colitis and human inflammatory bowel disease. Germ-free mice failed to induce IL-36γ in response to dextran sodium sulfate (DSS)-induced damage, suggesting that gut microbiota are involved in its induction. Surprisingly, IL-36R-deficient (Il1rl2(-/-)) mice exhibited defective recovery following DSS-induced damage and impaired closure of colonic mucosal biopsy wounds, which coincided with impaired neutrophil accumulation in the wound bed. Failure of Il1rl2(-/-) mice to recover from DSS-induced damage was associated with a profound reduction in IL-22 expression, particularly by colonic neutrophils. Defective recovery of Il1rl2(-/-) mice could be rescued by an aryl hydrocarbon receptor agonist, which was sufficient to restore IL-22 expression and promote full recovery from DSS-induced damage. These findings implicate the IL-36/IL-36R axis in the resolution of intestinal mucosal wounds.

Recruitment of classical monocytes can be inhibited by disturbing heteromers of neutrophil HNP1 and platelet CCL5.

In acute and chronic inflammation, neutrophils and platelets, both of which promote monocyte recruitment, are often activated simultaneously. We investigated how secretory products of neutrophils and platelets synergize to enhance the recruitment of monocytes. We found that neutrophil-borne human neutrophil peptide 1 (HNP1, α-defensin) and platelet-derived CCL5 form heteromers. These heteromers stimulate monocyte adhesion through CCR5 ligation. We further determined structural features of HNP1-CCL5 heteromers and designed a stable peptide that could disturb proinflammatory HNP1-CCL5 interactions. This peptide attenuated monocyte and macrophage recruitment in a mouse model of myocardial infarction. These results establish the in vivo relevance of heteromers formed between proteins released from neutrophils and platelets and show the potential of targeting heteromer formation to resolve acute or chronic inflammation.

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.

Annexin A1 counteracts chemokine-induced arterial myeloid cell recruitment.

RATIONALE: Chemokine-controlled arterial leukocyte recruitment is a crucial process in atherosclerosis. Formyl peptide receptor 2 (FPR2) is a chemoattractant receptor that recognizes proinflammatory and proresolving ligands. The contribution of FPR2 and its proresolving ligand annexin A1 to atherosclerotic lesion formation is largely undefined. OBJECTIVE: Because of the ambivalence of FPR2 ligands, we here investigate the role of FPR2 and its resolving ligand annexin A1 in atherogenesis. METHODS AND RESULTS: Deletion of FPR2 or its ligand annexin A1 enhances atherosclerotic lesion formation, arterial myeloid cell adhesion, and recruitment. Mechanistically, we identify annexin A1 as an endogenous inhibitor of integrin activation evoked by the chemokines CCL5, CCL2, and CXCL1. Specifically, the annexin A1 fragment Ac2-26 counteracts conformational activation and clustering of integrins on myeloid cells evoked by CCL5, CCL2, and CXCL1 through inhibiting activation of the small GTPase Rap1. In vivo administration of Ac2-26 largely diminishes arterial recruitment of myeloid cells in a FPR2-dependent fashion. This effect is also observed in the presence of selective antagonists to CCR5, CCR2, or CXCR2, whereas Ac2-26 was without effect when all 3 chemokine receptors were antagonized simultaneously. Finally, repeated treatment with Ac2-26 reduces atherosclerotic lesion sizes and lesional macrophage accumulation. CONCLUSIONS: Instructing the annexin A1-FPR2 axis harbors a novel approach to target arterial leukocyte recruitment. With the ability of Ac2-26 to counteract integrin activation exerted by various chemokines, delivery of Ac2-26 may be superior in inhibition of arterial leukocyte recruitment when compared with blocking individual chemokine receptors.

Human neutrophil formyl peptide receptor phosphorylation and the mucosal inflammatory response.

Bacterial/mitochondrial fMLF analogs bind FPR1, driving accumulation/activation of PMN at sites of infection/injury, while promoting wound healing in epithelia. We quantified levels of UFPR1 and TFPR1 in isolated PMN by use of phosphosensitive NFPRb and phosphorylation-independent NFPRa antibodies. UFPR1 and total TFPR were assessed inflamed mucosa, observed in human IBD. In isolated PMN after fMLF stimulation, UFPR1 declined 70% ((fMLF)EC50 = 11 ± 1 nM; t1/2 = 15 s) and was stable for up to 4 h, whereas TFPR1 changed only slightly. Antagonists (tBoc-FLFLF, CsH) and metabolic inhibitor NaF prevented the fMLF-dependent UFPR1 decrease. Annexin A1 fragment Ac2-26 also induced decreases in UFPR1 ((Ac2-26)EC50 ∼ 3 µM). Proinflammatory agents (TNF-α, LPS), phosphatase inhibitor (okadaic acid), and G-protein activator (MST) modestly increased (fMLF)EC50, 2- to 4-fold, whereas PTX, Ca(2+) chelators (EGTA/BAPTA), H2O2, GM-CSF, ENA-78, IL-1RA, and LXA4 had no effect. Aggregation-inducing PAF, however, strongly inhibited fMLF-stimulated UFPR1 decreases. fMLF-driven PMN also demonstrated decreased UFPR1 after traversing monolayers of cultured intestinal epithelial cells, as did PMN in intestinal mucosal samples, demonstrating active inflammation from UC patients. Total TFPR remained high in PMN within inflamed crypts, migrating through crypt epithelium, and in the lamina propria-adjoining crypts, but UFPR1 was only observed at some peripheral sites on crypt aggregates. Loss of UFPR1 in PMN results from C-terminal S/T phosphorylation. Our results suggest G protein-insensitive, fMLF-dependent FPR1 phosphorylation in isolated suspension PMN, which may manifest in fMLF-driven transmigration and potentially, in actively inflamed tissues, except at minor discrete surface locations of PMN-containing crypt aggregates.

JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function.

Intestinal barrier function is regulated by epithelial tight junctions (TJs), structures that control paracellular permeability. Junctional adhesion molecule-A (JAM-A) is a TJ-associated protein that regulates barrier; however, mechanisms linking JAM-A to epithelial permeability are poorly understood. Here we report that JAM-A associates directly with ZO-2 and indirectly with afadin, and this complex, along with PDZ-GEF1, activates the small GTPase Rap2c. Supporting a functional link, small interfering RNA-mediated down-regulation of the foregoing regulatory proteins results in enhanced permeability similar to that observed after JAM-A loss. JAM-A-deficient mice and cultured epithelial cells demonstrate enhanced paracellular permeability to large molecules, revealing a potential role of JAM-A in controlling perijunctional actin cytoskeleton in addition to its previously reported role in regulating claudin proteins and small-molecule permeability. Further experiments suggest that JAM-A does not regulate actin turnover but modulates activity of RhoA and phosphorylation of nonmuscle myosin, both implicated in actomyosin contraction. These results suggest that JAM-A regulates epithelial permeability via association with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and control contraction of the apical cytoskeleton.

The N-BAR domain protein, Bin3, regulates Rac1- and Cdc42-dependent processes in myogenesis.

Actin dynamics are necessary at multiple steps in the formation of multinucleated muscle cells. BAR domain proteins can regulate actin dynamics in several cell types, but have been little studied in skeletal muscle. Here, we identify novel functions for the N-BAR domain protein, Bridging integrator 3 (Bin3), during myogenesis in mice. Bin3 plays an important role in regulating myofiber size in vitro and in vivo. During early myogenesis, Bin3 promotes migration of differentiated muscle cells, where it colocalizes with F-actin in lamellipodia. In addition, Bin3 forms a complex with Rac1 and Cdc42, Rho GTPases involved in actin polymerization, which are known to be essential for myotube formation. Importantly, a Bin3-dependent pathway is a major regulator of Rac1 and Cdc42 activity in differentiated muscle cells. Overall, these data classify N-BAR domain proteins as novel regulators of actin-dependent processes in myogenesis, and further implicate BAR domain proteins in muscle growth and repair.

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.

Distinct phospholipase C-ß isozymes mediate lysophosphatidic acid receptor 1 effects on intestinal epithelial homeostasis and wound closure.

Maintenance of the epithelial barrier in the intestinal tract is necessary to protect the host from the hostile luminal environment. Phospholipase C-ß (PLC-ß) has been implicated to control myriad signaling cascades. However, the biological effects of selective PLC-ß isozymes are poorly understood. We describe novel findings that lysophosphatidic acid (LPA) regulates PLC-ß1 and PLC-ß2 via two distinct pathways to enhance intestinal epithelial cell (IEC) proliferation and migration that facilitate wound closure and recovery of the intestinal epithelial barrier. LPA acting on the LPA1 receptor promotes IEC migration by facilitating the interaction of Gαq with PLC-ß2. LPA-induced cell proliferation is PLC-ß1 dependent and involves translocation of Gαq to the nucleus, where it interacts with PLC-ß1 to induce cell cycle progression. An in vivo study using LPA1-deficient mice (Lpar1(-/-)) shows a decreased number of proliferating IECs and migration along the crypt-luminal axis. Additionally, LPA enhances migration and proliferation of IECs in an LPA1-dependent manner, and Lpar1(-/-) mice display defective mucosal wound repair that requires cell proliferation and migration. These findings delineate novel LPA1-dependent lipid signaling that facilitates mucosal wound repair via spatial targeting of distinct PLC-ßs within the cell.

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.

Antiallergic cromones inhibit neutrophil recruitment onto vascular endothelium via annexin-A1 mobilization.

OBJECTIVE: To determine whether the inhibitory action of the antiallergic cromone "mast cell stabilizing" drugs on polymorphonuclear leukocyte (PMN) trafficking is mediated through an annexin-A1 (Anx-A1) dependent mechanism. METHODS AND RESULTS: Intravital microscopy was used to monitor the actions of cromones in the inflamed microcirculation. Reperfusion injury provoked a dramatic increase in adherent and emigrated leukocytes in the mesenteric vascular bed, associated with augmented tissue levels of myeloperoxidase. Nedocromil, 2 to 20 mg/kg, significantly (P<0.05) inhibited cell adhesion and emigration, as well as myeloperoxidase release, in wild-type but not Anx-A1(-/-) mice. Short pretreatment of human PMNs with nedocromil, 10 nmol/L, inhibited cell adhesion (P<0.05) in the flow chamber assay, and this effect was reversed by specific anti-AnxA1 or a combination of antiformyl peptide receptors 1 and 2, but not irrelevant control, antibodies. Western blotting experiments revealed that cromones stimulate protein kinase C-dependent phosphorylation and release Anx-A1 in human PMNs. CONCLUSIONS: We propose a novel mechanism to explain the antiinflammatory actions of cromones on PMN trafficking, an effect that has long puzzled investigators.