Eric A. Severson

Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis

Glioblastomas, like other solid tumors, have extensive areas of hypoxia and necrosis. The importance of hypoxia in driving tumor growth is receiving increased attention. Hypoxia-inducible factor 1 (HIF-1) is one of the master regulators that orchestrate the cellular responses to hypoxia. It is a heterodimeric transcription factor composed of alpha and beta subunits. The alpha subunit is stable in hypoxic conditions but is rapidly degraded in normoxia. The function of HIF-1 is also modulated by several molecular mechanisms that regulate its synthesis, degradation, and transcriptional activity. Upon stabilization or activation, HIF-1 translocates to the nucleus and induces transcription of its downstream target genes. Most important to gliomagenesis, HIF-1 is a potent activator of angiogenesis and invasion through its upregulation of target genes critical for these functions. Activation of the HIF-1 pathway is a common feature of gliomas and may explain the intense vascular hyperplasia often seen in glioblastoma multiforme. Activation of HIF results in the activation of vascular endothelial growth factors, vascular endothelial growth factor receptors, matrix metalloproteinases, plasminogen activator inhibitor, transforming growth factors alpha and beta, angiopoietin and Tie receptors, endothelin-1, inducible nitric oxide synthase, adrenomedullin, and erythropoietin, which all affect glioma angiogenesis. In conclusion, HIF is a critical regulatory factor in the tumor microenvironment because of its central role in promoting proangiogenic and invasive properties. While HIF activation strongly promotes angiogenesis, the emerging vasculature is often abnormal, leading to a vicious cycle that causes further hypoxia and HIF upregulation.

JAM-A regulates permeability and inflammation in the intestine in vivo

Recent evidence has linked intestinal permeability to mucosal inflammation, but molecular studies are lacking. Candidate regulatory molecules localized within the tight junction (TJ) include Junctional Adhesion Molecule (JAM-A), which has been implicated in the regulation of barrier function and leukocyte migration. Thus, we analyzed the intestinal mucosa of JAM-A–deficient (JAM-A−/−) mice for evidence of enhanced permeability and inflammation. Colonic mucosa from JAM-A−/− mice had normal epithelial architecture but increased polymorphonuclear leukocyte infiltration and large lymphoid aggregates not seen in wild-type controls. Barrier function experiments revealed increased mucosal permeability, as indicated by enhanced dextran flux, and decreased transepithelial electrical resistance in JAM-A−/− mice. The in vivo observations were epithelial specific, because monolayers of JAM-A−/− epithelial cells also demonstrated increased permeability. Analyses of other TJ components revealed increased expression of claudin-10 and -15 in the colonic mucosa of JAM-A−/− mice and in JAM-A small interfering RNA–treated epithelial cells. Given the observed increase in colonic inflammation and permeability, we assessed the susceptibility of JAM-A−/− mice to the induction of colitis with dextran sulfate sodium (DSS). Although DSS-treated JAM-A−/− animals had increased clinical disease compared with controls, colonic mucosa showed less injury and increased epithelial proliferation. These findings demonstrate a complex role of JAM-A in intestinal homeostasis by regulating epithelial permeability, inflammation, and proliferation.

Junctional Adhesion Molecule A Interacts with Afadin and PDZ-GEF2 to Activate Rap1A, Regulate β1 Integrin Levels, and Enhance Cell Migration

Junctional adhesion molecule-A (JAM-A) is a transmembrane tight junction protein that has been shown to regulate barrier function and cell migration through incompletely understood mechanisms. We have previously demonstrated that JAM-A regulates cell migration by dimerization of the membrane-distal immunoglobulin-like loop and a C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding motif. Disruption of dimerization resulted in decreased epithelial cell migration secondary to diminished levels of beta1 integrin and active Rap1. Here, we report that JAM-A is physically and functionally associated with the PDZ domain-containing molecules Afadin and PDZ-guanine nucleotide exchange factor (GEF) 2, but not zonula occludens (ZO)-1, in epithelial cells, and these interactions mediate outside-in signaling events. Both Afadin and PDZ-GEF2 colocalized and coimmunoprecipitated with JAM-A. Furthermore, association of PDZ-GEF2 with Afadin was dependent on the expression of JAM-A. Loss of JAM-A, Afadin, or PDZ-GEF2, but not ZO-1 or PDZ-GEF1, similarly decreased cellular levels of activated Rap1, beta1 integrin protein, and epithelial cell migration. The functional effects observed were secondary to decreased levels of Rap1A because knockdown of Rap1A, but not Rap1B, resulted in decreased beta1 integrin levels and reduced cell migration. These findings suggest that JAM-A dimerization facilitates formation of a complex with Afadin and PDZ-GEF2 that activates Rap1A, which regulates beta1 integrin levels and cell migration.

Annexin A1 Regulates Intestinal Mucosal Injury, Inflammation, and Repair

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (−/−) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (−/−) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.

Structural determinants of Junctional Adhesion Molecule A (JAM-A) function and mechanisms of intracellular signaling

Junctional Adhesion Molecule A (JAM-A) is a multifunctional cell surface protein that has multiple evolutionarily conserved structural features. There is now conclusive evidence that discrete structural elements on JAM-A mediate intracellular signaling events that alter cell migration and paracellular permeability. Specifically, self-dimerization between extracellular Ig-like loops and close apposition of PDZ-dependent, JAM-A-associated intracellular scaffold proteins such as Afadin and guanine-nucleotide exchange factors mediate activation of Rap1 and modulation of epithelial cell migration by effects on beta1 integrin. While the same JAM-A structural features also modulate migration of other cell types and paracellular permeability in epithelia/endothelia, additional signaling proteins/mechanisms are probably involved. Recent insights into JAM-A outside-in signaling events that regulate these cellular functions are discussed.

Cis-dimerization mediates function of junctional adhesion molecule A.

Junctional adhesion molecule-A (JAM-A) is a transmembrane component of tight junctions that has been proposed to play a role in regulating epithelial cell adhesion and migration, yet mechanistic structure-function studies are lacking. Although biochemical and structural studies indicate that JAM-A forms cis-homodimers, the functional significance of dimerization is unclear. Here, we report the effects of cis-dimerization-defective JAM-A mutants on epithelial cell migration and adhesion. Overexpression of dimerization-defective JAM-A mutants in 293T cells inhibited cell spreading and migration across permeable filters. Similar inhibition was observed with using dimerization-blocking antibodies. Analyses of cells expressing the JAM-A dimerization-defective mutant proteins revealed diminished beta1 integrin protein but not mRNA levels. Further analyses of beta1 protein localization and expression after disruption of JAM-A dimerization suggested that internalization of beta1 integrin precedes degradation. A functional link between JAM-A and beta1 integrin was confirmed by restoration of cell migration to control levels after overexpression of beta1 integrin in JAM-A dimerization-defective cells. Last, we show that the functional effects of JAM dimerization require its carboxy-terminal postsynaptic density 95/disc-large/zonula occludins-1 binding motif. These results suggest that dimerization of JAM-A regulates cell migration and adhesion through indirect mechanisms involving posttranscriptional control of beta1 integrin levels.

Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

Mechanisms of Outside‐in Signaling at the Tight Junction by Junctional Adhesion Molecule A

Junctional adhesion molecule A (JAM‐A) is a tight junction–associated, PDZ binding domain containing transmembrane protein that forms cis‐homodimers in endothelial and epithelial cells. In vivo, the function of JAM‐A in colonic mucosa has been examined using JAM‐A knockout mice, which have increased intestinal permeability, inflammation and cellular proliferation compared to wild‐type controls. In vitro studies have revealed that downregulation of JAM‐A leads to altered cell migration secondary to diminished levels of β1 integrin on the cell surface. Similar findings have been observed after transfection of epithelial cells with mutant JAM‐A, which is defective in dimerization or lacks the PDZ binding domain. The dominant‐negative effects of these mutant JAM‐A proteins are most likely secondary to the inability of mutant JAM‐A to form signaling complexes, the lack of which results in decreases in active or GTP‐bound Rap1. This review highlights findings that support a hypothetical model for JAM‐A mediated outside‐in signaling. In this model,  JAM‐A dimerization is required for close cytoplasmic apposition of complexes containing specific PDZ domain‐containing scaffold proteins that activate signaling molecules to serve as effectors for the regulation of cellular functions. The possibility of interactions of JAM‐A cis‐dimers between cells in trans is also discussed.

Novel structural determinants on SIRPα that mediate binding to CD47.

Signal regulatory proteins (SIRP-α, -β, and -γ) are important regulators of several innate immune functions that include leukocyte migration. Membrane distal (D1) domains of SIRPα and SIRPγ, but not SIRPβ, mediate binding to a cellular ligand termed CD47. Because the extracellular domains of all SIRPs are highly homologous, we hypothesized that some of the 16 residues unique to SIRPα.D1 mediate binding to CD47. By site-directed mutagenesis, we determined that SIRPα binding to CD47 is independent of N-glycosylation. We also identified three residues critical for CD47 binding by exchanging residues on SIRPα with corresponding residues from SIRPβ. Cumulative substitutions of the critical residues into SIRPβ resulted in de novo binding of the mutant protein to CD47. Homology modeling of SIRPα.D1 revealed topological relationships among critical residues and allowed the identification of critical residues common to SIRPα and SIRPβ. Mapping these critical residues onto the recently reported crystal structure of SIRPα.D1 revealed a novel region that is required for CD47 binding and is distinct and lateral to another putative CD47 binding site described on that crystal structure. The importance of this lateral region in mediating SIRPα.D1 binding to CD47 was confirmed by epitope mapping analyses of anti-SIRP Abs. These observations highlight a complex nature of the ligand binding requirements for SIRPα that appear to be dependent on two distinct but adjacent regions on the membrane distal Ig loop. A better understanding of the structural basis of SIRPα/CD47 interactions may provide insights into therapeutics targeting pathologic inflammation.

Structure and function of JAM proteins

The immunoglobulin superfamily (IgSF) is a large class of proteins that includes the junctional adhesion molecule (JAM) family of proteins. The current nomenclature for JAM members designates the first three described JAM proteins as JAM-A, JAM-B, JAM-C. Two other related proteins have been reported that have not been included in the standard nomenclature and are termed JAM-4 and JAM-L (AMICA). In earlier studies, numerical designations were used to define JAM proteins according to the timing of initial characterizations. However, this early nomenclature led to confusion in terminology for JAM-B and C due to the timing in which human and murine JAM-B and JAM-C were reported. To avoid confusion, this review uses current nomenclature exclusively, as proposed originally by Muller, regardless of the designation given in the original reports [1].