At sites of ongoing inflammation, polymorphonuclear leukocytes (PMN, neutrophils) migrate across vascular endothelia, and such transmigration has the potential to disturb barrier properties and can result in intravascular fluid loss and edema. It was recently appreciated that endogenous pathways exist to dampen barrier disruption during such episodes and may provide an important anti-inflammatory link. For example, during transmigration, PMN-derived adenosine activates endothelial adenosine receptors and induces a cAMP-dependent resealing of endothelial barrier function. In our study reported here, we sought to understand the link between cyclic nucleotide elevation and increased endothelial barrier function. Initial studies revealed that adenosine-induced barrier function is tightly linked to activation of protein kinase A (PKA). Because PKA selectively phosphorylates serine and threonine residues, we screened zonula occludens-1 (ZO-1) immunoprecipitates for the existence of such phosphorylated proteins as targets for barrier regulation. This analysis revealed a dominantly phosphorylated band at 50 kDa. Microsequencing identified this protein as vasodilator-stimulated phosphoprotein (VASP), an actin binding protein with multiple serine/threonine phosphorylation sites. Immunofluorescent microscopy revealed that VASP localizes to endothelial junctional complexes and colocalizes with ZO-1, occludin, and junctional adhesion molecule-1 (JAM-1). To address the role of phospho-VASP in regulation of barrier function, we generated a phosphospecific VASP antibody targeting the Ser157 residue phosphorylation site, the site preferred by PKA. Immunolocalization studies with this antibody revealed that upon PKA activation, phospho-VASP appears at cell-cell junctions. Transient transfection of truncated VASP fragments revealed a parallel increase in barrier function. Taken together, these studies reveal a central role for phospho-VASP in the coordination of PKA-regulated barrier function, such as occurs during episodes of inflammation. E ndothelial cells that line blood vessels are the primary determinants of vascular permeability [1], and structural and functional integrity of the endothelium is crucial in determining overall vascular permeability. Endothelial injury, such as occurs during leukocyte-endothelial interactions, may result in increased paracellular permeability, decreased barrier function, and subsequent intravascular loss of fluid and local tissue edema [2,3]. During transendothelial migration (TEM), polymorphonuclear leukocyte (PMN)-derived adenosine activates endothelial adenosine receptors and induces a cAMP-dependent sealing of endothelial monolayers [4,5]. Elevated levels of cAMP could enhance barrier function by activating protein kinase A (PKA) and subsequent phosphorylation of key complex-associated proteins [6,7]. At present, the intracellular signals that link cAMP elevation and vascular barrier function have not been identified.Regulation of junctional permeability is coupled to actin-based systems, a...
Neutrophil migration across mucosal epithelium during inflammatory episodes involves the precise orchestration of a number a cell surface molecules and signaling pathways. After successful migration to the apical epithelial surface, apically localized epithelial proteins may serve to retain PMN at the lumenal surface. At present, identification of apical epithelial ligands and their PMN counter-receptors remain elusive. Therefore, to define the existence of apical epithelial cell surface proteins involved in PMN–epithelial interactions, we screened a panel of antibodies directed against epithelial plasma membranes. This strategy identified one antibody (OE-1) that both localized to the apical cell membrane and significantly inhibited PMN transmigration across epithelial monolayers. Microsequence analysis revealed that OE-1 recognized human decay-accelerating factor (DAF, CD55). DAF is a highly glycosylated, 70–80-kD, glycosyl-phosphatidyinositol–linked protein that functions predominantly as an inhibitor of autologous complement lysis. DAF suppression experiments using antisense oligonucleotides or RNA interference revealed that DAF may function as an antiadhesive molecule promoting the release of PMN from the lumenal surface after transmigration. Similarly, peptides corresponding to the antigen recognition domain of OE-1 resulted in accumulation of PMN on the apical epithelial surface. The elucidation of DAF as an apical epithelial ligand for PMN provides a target for novel anti-inflammatory therapies directed at quelling unwanted inflammatory episodes.
Epithelial permeability is tightly regulated by intracellular messengers. Critical to maintaining barrier integrity is the formation of tight junction complexes. A number of signaling pathways have been implicated in tight junction biogenesis; however, the precise molecular mechanisms are not fully understood. A growing body of evidence suggests a role for intracellular cAMP in tight junction assembly. Using an epithelial model, we investigated the role of cAMP signal transduction in barrier recovery after Ca2+ switch. Our data demonstrate that elevation of intracellular cAMP levels significantly enhanced barrier recovery after Ca2+ switch. Parallel experiments revealed that epithelial barrier recovery is diminished by H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (protein kinase A) activity. Of the possible PKA effector proteins, the vasodilator-stimulated phosphoprotein (VASP) is an attractive candidate, since it has been implicated in actin-binding and cross-linking functions. We therefore hypothesized that VASP may play a role in the cAMP-mediated regulation of epithelial junctional reassembly after Ca2+ switch. We demonstrate here that VASP is phosphorylated via a PKA-dependent process under conditions that enhance barrier recovery. Confocal laser scanning microscopy studies revealed that VASP localizes with ZO-1 at the tight junction and at cell-cell borders and that phospho-VASP appears at the junction after Ca2+ switch. Subsequent transfection studies utilizing epithelial cells expressing truncated forms of VASP abnormal in oligomerization or actin-binding activity revealed a functional diminution of barrier recovery after Ca2+ chelation. Our present studies suggest that VASP may provide a link between cAMP signal transduction and epithelial permeability.
During episodes of inflammation, neutrophils (polymorphonuclear leukocytes[PMNs]) encounter subendothelial matrix substrates that may require additional signaling pathways as directives for movement through the extracellular space. Using an in vitro endothelial and epithelial model, inhibitors of phosphoinositide 3-kinase (PI3K) were observed to promote chemoattractant-stimulated migration by as much as 8 ؎ 0.3-fold. Subsequent studies indicated that PMNs respond in a similar manner to RGD-containing matrix substrates and that PMN-matrix interactions are potently inhibited by antibodies directed against  3 -but not  1 -integrin antibodies, and that PI3K inhibitors block  3 -integrin dependence. Biochemical analysis of intracellular  3 -integrin uncoupling by PI3K inhibitors revealed diminished  3 -integrin tyrosine phosphorylation and decreased association with p72 syk . Similarly, the p72 syk inhibitor piceatannol promoted PMN transmatrix migration, whereas HIV-tat peptide-facilitated loading of peptides corresponding to the  3 -integrin cytoplasmic tail identified the functional tyrosine residues for this activity. These data indicate that PI3K-regulated  3 -integrin represents a natural "braking" mechanism for PMNs during transit through the extracellular matrix. IntroductionMigration of neutrophils (polymorphonuclear leukocytes [PMNs ]) to sites of inflammation requires the coordinated interplay of soluble mediators, extracellular matrix ligands, and cell surface adhesion molecules. To subserve this function, PMNs must traverse endothelial cells lining the inner lumen of blood vessels. This process of transendothelial migration requires engagement and disengagement of a number of surface molecules and has been extensively studied. 1 After successful transendothelial migration, PMNs encounter subendothelial extracellular matrices in transit to inflammatory sites. Anchorage of cells to the extracellular matrix is mediated in part by integrins, a large family of heterodimeric cell surface proteins that mediate numerous cell functions, including motility, differentiation, and proliferation. 2 Integrin engagement of matrix ligand results in highly regulated signal transduction processes that cooperatively involve both "outside-in" and "insideout" pathways. 3 Importantly, integrin signaling can vary depending on the stimulus and on the cell type, 4 the molecular details of which are not fully understood at the present time.Recent studies have identified an important role for phosphoinositide-3-OH kinase (PI3K) in leukocyte migration. [5][6][7] The 4 known isoforms of PI3K are ␣, , ␥, and ␦, and extracellular ligand coupling through membrane-localized G proteins generates the signaling molecule phosphatidylinositol 3,4,5-triphosphate. 8 PI3K coordinates a number of leukocyte effector functions, including leukocyte migration. [5][6][7]9 Although not fully understood at present, it appears that PI3K is central to stimulated chemotaxis. For instance, mice lacking the catalytic domain of leukocyte-specific PI3...
A series of novel 2-arylbenzoxazoles that upregulate the production of utrophin in murine H2K cells, as assessed using a luciferase reporter linked assay, have been identified. This compound class appears to hold considerable promise as a potential treatment for Duchenne muscular dystrophy. Following the delineation of structure-activity relationships in the series, a number of potent upregulators were identified, and preliminary ADME evaluation is described. These studies have resulted in the identification of 1, a compound that has been progressed to clinical trials.
Macrophages are a critically important component of the innate and adaptive immune systems. They are equipped with oxidative and non-oxidative mechanisms to kill ingested pathogens. Natural Killer Lytic-Associated Molecule (NKLAM) is an E3 ubiquitin ligase expressed in macrophages and natural killer cells. We show that NKLAM expression in macrophages was enhanced by Toll-like receptor agonists and pro-inflammatory cytokines. Using confocal microscopy, we found that NKLAM colocalized with ingested E. coli. In assays using IgG-opsonized latex beads as targets, we demonstrated that NKLAM translocated to the phagosome early during maturation at a time that coincided with elevated levels of ubiquitinated phagosome proteins. In killing assays with bone marrow-derived macrophages from wild type and NKLAM-deficient mice, we found that NKLAM-deficient macrophages demonstrated less killing of Escherichia coli than wild type macrophages. Collectively, our data show that NKLAM is a novel component of macrophage phagosomes and is involved in macrophage bactericidal functions.
Signal transducer and activator of transcription 1 (STAT1) is critically important for the transcription of a large number of immunologically relevant genes. In macrophages, interferon gamma (IFNγ) signal transduction occurs via the JAK/STAT pathway and ends with the transcription of a number of genes necessary for a successful host immune response. The predominant mechanism of regulation of STAT1 is phosphorylation; however, there is a growing body of evidence that demonstrates STAT1 is also regulated by ubiquitination. In this report we show that JAK1 and STAT1 in macrophages deficient in an E3 ubiquitin ligase termed Natural Killer Lytic-Associated Molecule (NKLAM) are hyperphosphorylated following IFNγ stimulation. We found NKLAM was transiently localized to the IFNγ receptor complex during stimulation with IFNγ, where it bound to and mediated K63-linked ubiquitination of STAT1. In vitro nucleofection studies demonstrated that STAT1-mediated transcription was significantly reduced in NKLAM-KO macrophages. There was no obvious defect in STAT1 nuclear translocation; however, STAT1 from NKLAM-KO macrophages had a reduced ability to bind a functional gamma activation DNA sequence. There was also less mRNA expression of STAT1-mediated genes in NKLAM-KO macrophages treated with IFNγ. Our results demonstrate for the first time that NKLAM is a positive regulator of STAT1-mediated transcriptional activity and is an important component of the innate immune response.
The expression and phosphorylation state of the vasodilator-stimulated phosphoprotein (VASP), a membrane-associated focal adhesion protein, was investigated in human neutrophils. Adhesion and spreading of neutrophils induced the rapid phosphorylation of VASP. The phosphorylation of VASP was dependent on cell spreading, as VASP was expressed as a dephosphorylated protein in round adherent cells and was phosphorylated at the onset of changes in cell shape from round to spread cells. Immunofluorescence microscopy demonstrated that VASP was localized at the cell cortex in round cells and redistributed to focal adhesions at the ventral surface of the cell body during cell spreading. Dual labeling of spread cells indicated that VASP was colocalized with F-actin in filopodia and in focal adhesions, suggesting that the phosphorylation of VASP during cell spreading may be involved in focal adhesion complex organization and actin dynamics. VASP is a prominent substrate for both cGMP-dependent protein kinase (cGK) and cAMP-dependent protein kinase. Evidence suggested that cGK regulated neutrophil spreading, as both VASP phosphorylation and neutrophil spreading were inhibited by Rp-8-pCPT-cGMPS (cGK inhibitor), but not KT5720 (cAMP-dependent protein kinase inhibitor). In contrast, neutrophil spreading was accelerated when cGMP levels were elevated with 8-Br-cGMP, a direct activator of cGK. Furthermore, the same conditions that lead to VASP phosphorylation during neutrophil adherence and spreading induced significant elevations of cGMP in neutrophils. These results indicate that cGMP/cGK signal transduction is required for neutrophil spreading, and that VASP is a target for cGK regulation.
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