Katrina M. Comerford

Prolyl hydroxylase-1 negatively regulates IκB kinase-β, giving insight into hypoxia-induced NFκB activity

Hypoxia is a feature of the microenvironment of a growing tumor. The transcription factor NFκB is activated in hypoxia, an event that has significant implications for tumor progression. Here, we demonstrate that hypoxia activates NFκB through a pathway involving activation of IκB kinase-β (IKKβ) leading to phosphorylation-dependent degradation of IκBα and liberation of NFκB. Furthermore, through increasing the pool and/or activation potential of IKKβ, hypoxia amplifies cellular sensitivity to stimulation with TNFα. Within its activation loop, IKKβ contains an evolutionarily conserved LxxLAP consensus motif for hydroxylation by prolyl hydroxylases (PHDs). Mimicking hypoxia by treatment of cells with siRNA against PHD-1 or PHD-2 or the pan-prolyl hydroxylase inhibitor DMOG results in NFκB activation. Conversely, overexpression of PHD-1 decreases cytokine-stimulated NFκB reporter activity, further suggesting a repressive role for PHD-1 in controlling the activity of NFκB. Hypoxia increases both the expression and activity of IKKβ, and site-directed mutagenesis of the proline residue (P191A) of the putative IKKβ hydroxylation site results in a loss of hypoxic inducibility. Thus, we hypothesize that hypoxia releases repression of NFκB activity through decreased PHD-dependent hydroxylation of IKKβ, an event that may contribute to tumor development and progression through amplification of tumorigenic signaling pathways.

Role of vasodilator-stimulated phosphoprotein in PKA-induced changes in endothelial junctional permeability

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.

Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia

Phosphorylation-dependent ubiquitination combined with proteasomal degradation of transcriptional regulators is a recently appreciated mechanism for control of a number of inflammatory genes. Far less is known about the counterregulatory mechanisms that repress transcriptional activity in these pathways during resolution. Here, we investigated the transient nature of hypoxia-induced tumor necrosis factor (TNF)α in T84 cells, a process we have previously shown to involve phosphorylation-dependent degradation of the cAMP-response element-binding protein (CREB). Initial studies indicate hypoxia-induced TNFα to be a transient event, the resolution of which is associated with the appearance of a higher molecular weight modified form of CREB. Gene array analysis of mRNA derived from hypoxic cells identified a time-dependent induction of small ubiquitin-related modifier (SUMO)-1 mRNA. In prolonged hypoxia, CREB is posttranslationally modified by SUMO-1. Furthermore, SUMO-1 overexpression stabilizes CREB in hypoxia and enhances CREB-dependent reporter gene activity. Site-directed mutagenesis of lysine residues K285 and K304 identifies them as SUMO acceptors in vivo and in vitro. Mutation of K304 also results in loss of CREB nuclear localization, implying a role for SUMO-1 modification at this site in the subcellular localization of CREB. Thus, in prolonged hypoxia, CREB is modified by association with SUMO-1. Furthermore, we hypothesize that such an event stabilizes and promotes nuclear localization of CREB and thus complements an endogenous resolution phase for hypoxia-induced inflammatory processes.

c-Jun NH2-Terminal Kinase Activation Contributes to Hypoxia-Inducible Factor 1α–Dependent P-Glycoprotein Expression in Hypoxia

We previously have shown that hypoxia increases the expression of P-glycoprotein, which in turn increases tumor cell capacity to actively extrude chemotherapeutic agents and may contribute to tumor drug resistance. This event is mediated through the hypoxia-inducible factor (HIF-1). Here, we investigated the role of the stress-activated protein kinase c-Jun NH2-terminal kinase (JNK) in the signaling mechanisms underlying these events. Hypoxia activates JNK activity in vitro and in vivo. Overexpression of mitogen-activated protein kinase (MAPK) kinase kinase (MEKK-1), which preferentially activates JNK, mimics, in a nonadditive way, hypoxia-induced activity of the MDR1 promoter and expression of MDR1 mRNA and P-glycoprotein. Furthermore, the JNK inhibitor SP600125 selectively and specifically inhibits hypoxia- and MEKK-1–induced MDR1 promoter activity in a dose-dependent manner. JNK inhibition also reversed hypoxia- and MEKK-1–induced activity of an HIF-1–dependent reporter gene. MEKK-1–induced MDR1 expression depends on a functional HIF-1 binding site (hypoxia-responsive element). Hypoxia- but not cobalt chloride–dependent HIF-1–DNA binding and transcriptional activation was inhibited by SP600125, indicating that hypoxia-induced signaling to HIF-1 depends on JNK activation. Because it has been reported that reactive oxygen species are increased in hypoxia and related to JNK activation, we investigated their role in signaling this response. Whereas exogenous addition of H2O2 was sufficient to activate JNK, reactive oxygen species scavengers were without effect on hypoxia-induced JNK or HIF-1 activation. Thus, hypoxia-elicited MDR1 expression, which depends on HIF-1 activation, depends at least in part on signaling via activation of JNK. Furthermore, these events are independent of the generation of reactive oxygen intermediates. Thus, JNK may represent a therapeutic target in the prevention of tumor resistance to chemotherapeutic treatment.

Small Ubiquitin-related Modifier (SUMO)-1 Promotes Glycolysis in Hypoxia

Under conditions of hypoxia, most eukaryotic cells undergo a shift in metabolic strategy, which involves increased flux through the glycolytic pathway. Although this is critical for bioenergetic homeostasis, the underlying mechanisms have remained incompletely understood. Here, we report that the induction of hypoxia-induced glycolysis is retained in cells when gene transcription or protein synthesis are inhibited suggesting the involvement of additional post-translational mechanisms. Post-translational protein modification by the small ubiquitin related modifier-1 (SUMO-1) is induced in hypoxia and mass spectrometric analysis using yeast cells expressing tap-tagged Smt3 (the yeast homolog of mammalian SUMO) revealed hypoxia-dependent modification of a number of key glycolytic enzymes. Overexpression of SUMO-1 in mammalian cancer cells resulted in increased hypoxia-induced glycolysis and resistance to hypoxia-dependent ATP depletion. Supporting this, non-transformed cells also demonstrated increased glucose uptake upon SUMO-1 overexpression. Conversely, cells overexpressing the de-SUMOylating enzyme SENP-2 failed to demonstrate hypoxia-induced glycolysis. SUMO-1 overexpressing cells demonstrated focal clustering of glycolytic enzymes in response to hypoxia leading us to hypothesize a role for SUMOylation in promoting spatial re-organization of the glycolytic pathway. In summary, we hypothesize that SUMO modification of key metabolic enzymes plays an important role in shifting cellular metabolic strategies toward increased flux through the glycolytic pathway during periods of hypoxic stress.

Epithelial Cell-Neutrophil Interactions in the Alimentary Tract: A Complex Dialog in Mucosal Surveillance and Inflammation

Inflammatory diseases of mucosal organs as diverse as the lung, kidney, and intestine, inevitably require the intimate interactions of neutrophils with columnar epithelia. The physiologic consequences of such interactions often determine endpoint organ function, and for this reason, much recent interest has developed in identifying mechanisms and novel targets for the treatment of mucosal inflammation. Elegant in vitro model systems incorporating purified human neutrophils and human epithelial cells grown in physiologic orientations have aided in discovery of new and insightful pathways to define basic inflammatory pathways. Here, we will review the recent literature regarding the interactions between columnar epithelial cells and neutrophils, with an emphasis on intestinal epithelial cells, structural aspects of neutrophil transepithelial migration, molecular determinants of neutrophil-epithelial cell interactions, as well as modulation of these pathways. These recent studies highlight the dynamic nature of these pathways and lend insight into the complexity of treating mucosal inflammation.

Assessing Oxygen Sensitivity of the Multidrug Resistance (MDR) Gene

Publisher Summary This chapter focuses on assessing oxygen sensitivity of the multidrug resistance (MDR) gene. A major obstacle in the development of effective cancer chemotherapy is tumor development of the multidrug resistance (MDR1) phenotype. Multiple approaches are available to study the function of MDR1/P-gp and how such functions relate to tumor hypoxia. Results of these functional analyses may have broad implications for studying MDR1-expressing tumors and overall chemotherapy resistance. MDR1 expression in patients is most prominent in solid tumors, and MDR1 correlates positively with the propensity of tumors for lymph node spread and metastases. The majority of solid tumors, particularly those with a propensity for P-glycoprotein (P-gp) expression, stain positive for nuclear HIF-1α. This chapter discusses methods for investigating the mechanisms of hypoxia-dependent regulation of MDR1 gene expression in epithelial cells. Analysis of mRNA levels by genechip expression arrays and reverse transcriptase-polymerase chain reaction (RT-PCR), the use of western blotting process, an overview of multicellular dome model, and antisense oligonucleotide treatment of epithelia are discussed.

Identification of Cyclic AMP Response Element-Binding Protein-Dependent Transcriptional Responses in Hypoxia by Microarray Analysis

Publisher Summary This chapter discusses identification of cyclic AMP response element-binding protein-dependent transcriptional responses in hypoxia by microarray analysis. A functional role for the cyclic AMP response element-binding protein (CREB) is demonstrated in controlling gene expression in hypoxia. In intestinal epithelial cells, hypoxia-elicited expression of the inflammatory cytokine tumor necrosis factor α (TNF-α) is mediated through phosphorylation-dependent ubiquitination and subsequent degradation of CREB. CREB is regulated by moderate hypoxia in PC12 cells. The gene microarray analysis is used to identify a role for CREB in the transcriptional mechanisms that underlie global gene expression in epithelial cells in response to hypoxia. This technique also allows the identification of genes previously unidentified as hypoxia responsive. Different aspects of this technology such as experimental design, total RNA isolation, double-stranded cDNA synthesis, complementary RNA synthesis, target hybridization and microarray scanning, post-translational modification of CREB and data interpretation and bioinformatic analysis are discussed.