Tianqing Kong

HIF-dependent induction of adenosine A2B receptor in hypoxia

Adenosine has been widely associated with hypoxia of many origins, including those associated with inflammation and tumorogenesis. A number of recent studies have implicated metabolic control of adenosine generation at sites of tissue hypoxia. Here, we examine adenosine receptor control and amplification of signaling through transcriptional regulation of endothelial and epithelial adenosine receptors. Initial studies confirmed previous findings indicating selective induction of human adenosine A2B receptor (A2BR) by hypoxia. Analysis of the cloned human A2BR promoter identified a functional hypoxia‐responsive region, including a functional binding site for hypoxia‐inducible factor (HIF) within the A2BR promoter. Further studies examining HIF‐1α DNA binding and HIF‐1α gain and loss of function confirmed strong dependence of A2BR induction by HIF‐1α in vitro and in vivo mouse models. Additional studies in endothelia overexpressing full‐length A2BR revealed functional phenotypes of increased barrier function and enhanced angiogenesis. Taken together, these results demonstrate transcriptional coordination of A2BR by HIF‐1α and amplified adenosine signaling during hypoxia. These findings may provide an important link between hypoxia and metabolic conditions associated with inflammation and angiogenesis.—Kong, T., Westerman, K. A., Faigle, M., Eltzschig, H. K., Colgan, S. P. HIF‐dependent induction of adenosine A2B receptor in hypoxia. FASEB J. 20, 2242–2250 (2006)

Central role of Sp1-regulated CD39 in hypoxia/ischemia protection

Hypoxia is common to several inflammatory diseases, where multiple cell types release adenine-nucleotides (particularly adenosine triphosphate/adenosine diphosphate). Adenosine triphosphate/adenosine diphosphate is metabolized to adenosine through a 2-step enzymatic reaction initiated by CD39 (ectonucleoside-triphosphate-diphosphohydrolase-1). Thus, extracellular adenosine becomes available to regulate multiple inflammatory endpoints. Here, we hypothesized that hypoxia transcriptionally up-regulates CD39 expression. Initial studies revealed hypoxia-dependent increases in CD39 mRNA and immunoreactivity on endothelia. Examination of the human CD39 gene promoter identified a region important in hypoxia inducibility. Multiple levels of analysis, including site-directed mutagenesis, chromatin immunoprecipitation, and inhibition by antisense, revealed a critical role for transcription-factor Sp1 in hypoxia-induction of CD39. Using a combination of cd39(-/-) mice and Sp1 small interfering RNA in in vivo cardiac ischemia models revealed Sp1-mediated induction of cardiac CD39 during myocardial ischemia. In summary, these results identify a novel Sp1-dependent regulatory pathway for CD39 and indicate the likelihood that CD39 is central to protective responses to hypoxia/ischemia.

HIF-dependent induction of apical CD55 coordinates epithelial clearance of neutrophils

Sites of inflammation are associated with dramatic shifts in tissue metabolism. Inflammation can result in significant tissue hypoxia, with resultant induction of hypoxia‐responsive genes. Given this association, we hypothesized that neutrophil (PMN) ligands expressed on epithelial cells may be regulated by hypoxia. Initial studies confirmed earlier results that epithelial hypoxia enhances PMN transepithelial migration and promotes apical clearance of PMN from the epithelial surface. A screen of known PMN ligands revealed a surprisingly stable expression pattern in hypoxia. However, this screen identified one gene, CD55, as a highly hypoxia‐inducible molecule expressed on the apical membrane of mucosal epithelia. Subsequent studies verified the induction of CD55 mRNA and protein expression by hypoxia. Overexpression of CD55 by transfection in nonhypoxic epithelia resulted in a similar pattern of apical PMN clearance, and peptide mimetics corresponding to the PMN binding site on DAF blocked such apical clearance of PMN. Studies directed at understanding molecular pathways of hypoxia inducibility revealed that a ∼200 bp region of the CD55 gene conferred hypoxia inducibility for CD55. These studies identified a functional binding site for the transcriptional regulator hypoxia‐inducible factor (HIF). Taken together, these results identify HIF‐dependent induction of epithelial CD55 in the resolution of ongoing inflammation through clearance of apical PMN. Louis, N. A., Hamilton, K E., Kong, T., Colgan, S. P. HIF‐dependent induction of apical CD55 coordinates epithelial clearance of neutrophils. FASEB J. 19, 950–959 (2005)

Identification of vasodilator-stimulated phosphoprotein (VASP) as an HIF-regulated tissue permeability factor during hypoxia

Increased tissue permeability is commonly associated with hypoxia of many origins. Since hypoxia‐inducible factor (HIF) represents a predominant hypoxia signaling mechanism, we compared hyp‐oxia‐elicited changes in tissue barrier function in mice conditionally lacking intestinal epithelial hypoxia‐inducible factor‐1α (hifla). Somewhat surprisingly, these studies revealed that mutant hifla mice were protected from hypoxia‐induced increases in intestinal permeability in vivo. Guided by microarray analysis of tissues derived from these mutant hifla mice, we identified HIF‐1‐dependent repression of vasodilator‐stimulated phosphoprotein (VASP), a molecule known to be important in the control of cytoskeletal dynamics, including barrier function. Studies at the mRNA and protein level confirmed hypoxia‐elicited repression of VASP in murine tissue, cultured epithelia and endothelia, as well as human saphenous vein ex vivo. Targeted repression of VASP by siRNA recapitulated our findings with hypoxia and directed overexpression of VASP abolished hypoxia‐induced barrier dysfunction. Studies in the cloned human VASP promoter revealed hypoxia‐dependent transcriptional repression, and functional studies by chromatin immunoprecipitation (ChIP) and site‐directed mutagenesis revealed hypoxia‐dependent binding of HIF‐1α to the human VASP promoter. These studies identify HIF‐1‐dependent repression of VASP as a control point for hypoxia‐regulated barrier dysfunction.—Rosenberger, P., Khoury, J., Kong, T., Weissmuller, T., Robinson, A. M., Colgan, S. P. Identification of vasodilator‐stimulated phosphoprotein (VASP) as an HIF‐regulated tissue permeability factor during hypoxia. FASEB J. 21, 2613–2621 (2007)

Gα12 Stimulates Apoptosis in Epithelial Cells through JNK1-mediated Bcl-2 Degradation and Up-regulation of IκBα

Apoptosis is an essential mechanism for the maintenance of somatic tissues, and when dysregulated can lead to numerous pathological conditions. G proteins regulate apoptosis in addition to other cellular functions, but the roles of specific G proteins in apoptosis signaling are not well characterized. Gα12 stimulates protein phosphatase 2A (PP2A), a serine/threonine phosphatase that modulates essential signaling pathways, including apoptosis. Herein, we examined whether Gα12 regulates apoptosis in epithelial cells. Inducible expression of Gα12 or constitutively active (QL)α12 in Madin-Darby canine kidney cells led to increased apoptosis with expression of QLα12, but not Gα12. Inducing QLα12 led to degradation of the anti-apoptotic protein Bcl-2 (via the proteasome pathway), increased JNK activity, and up-regulated IκBα protein levels, a potent stimulator of apoptosis. Furthermore, the QLα12-stimulated activation of JNK was blocked by inhibiting PP2A. To characterize endogenous Gα12 signaling pathways, non-transfected MDCK-II and HEK293 cells were stimulated with thrombin. Thrombin activated endogenous Gα12 (confirmed by GST-tetratricopeptide repeat (TPR) pull-downs) and stimulated apoptosis in both cell types. The mechanisms of thrombin-stimulated apoptosis through endogenous Gα12 were nearly identical to the mechanisms identified in QLα12-MDCK cells and included loss of Bcl-2, JNK activation, and up-regulation of IκBα. Knockdown of the PP2A catalytic subunit in HEK293 cells inhibited thrombin-stimulated apoptosis, prevented JNK activation, and blocked Bcl-2 degradation. In summary, Gα12 has a major role in regulating epithelial cell apoptosis through PP2A and JNK activation leading to loss of Bcl-2 protein expression. Targeting these pathways in vivo may lead to new therapeutic strategies for a variety of disease processes.

H2O2 activates G protein, α 12 to disrupt the junctional complex and enhance ischemia reperfusion injury

The epithelial cell tight junction separates apical and basolateral domains and is essential for barrier function. Disruption of the tight junction is a hallmark of epithelial cell damage and can lead to end organ damage including renal failure. Herein, we identify Gα12 activation by H2O2 leading to tight junction disruption and demonstrate a critical role for Gα12 activation during bilateral renal ischemia/reperfusion injury. Madin–Darby canine kidney (MDCK) cells with inducible Gα12 (Gα12-MDCK) and silenced Gα12 (shGα12-MDCK) were subjected to ATP depletion/repletion and H2O2/catalase as models of tight junction disruption and recovery by monitoring transepithelial resistance. In ATP depleted cells, barrier disruption and recovery was not affected by Gα12, but reassembly was accelerated by Gα12 depletion. In contrast, silencing of Gα12 completely protected cells from H2O2-stimulated barrier disruption, a response that rapidly occurred in control cells. H2O2 activated Src and Rho, and Src inhibition (by PP2), but not Rho (by Y27632), protected cells from H2O2-mediated barrier disruption. Immunofluorescent and biochemical analysis showed that H2O2 led to increased tyrosine phosphorylation of numerous proteins and altered membrane localization of tight junction proteins through Gα12/Src signaling pathway. Gα12 and Src were activated in vivo during ischemia/reperfusion injury, and transgenic mice with renal tubular QLα12 (activated mutant) expression were delayed in recovery and showed more extensive injury. Conversely, Gα12 knockout mice were nearly completely protected from ischemia/reperfusion injury. Taken together, these studies reveal that ROS stimulates Gα12 to activate injury pathways and identifies a therapeutic target for ameliorating ROS mediated injury.

Identification of Pur alpha as a new hypoxia response factor responsible for coordinated induction of the beta 2 integrin family.

Central to the process of inflammation are hypoxic conditions that lead to the binding of circulating leukocytes to the endothelium. We have previously shown that such binding is mediated by monocytes being able to directly sense hypoxic conditions and respond by inducing their surface expression of the β2 integrin family of adhesion molecules. In this study, we show that coordinated induction of the β2 integrins during direct hypoxia-sensing occurs through transcriptional activation of each of the genes by which they are encoded. Certain of the molecular mechanisms that mediate this activation in transcription are dependent upon hypoxia-inducible factor-1 (HIF-1), whereas others are HIF-1 independent. In search of these HIF-1-independent mechanisms, we identified Purα as a new hypoxia-response factor. Binding of Purα to the HIF-1-independent β2 integrin promoters is induced by hypoxia and mutagenesis of these Purα-binding sites almost completely abolishes the ability of the promoters to respond to hypoxic conditions. Additional studies using siRNA directed against Purα also revealed a loss in the hypoxic response of the β2 integrin promoters. Taken together, our findings demonstrate that hypoxia induces a coordinated up-regulation in β2 integrin expression that is dependent upon transcriptional mechanisms mediated by HIF-1 and Purα.

Polycystin-1 protein level determines activity of the Gα12/JNK apoptosis pathway

Mutations in PKD1 are the most common cause of autosomal dominant polycystic kidney disease (ADPKD). The protein product of PKD1 (polycystin-1 (PC1)) is a large transmembrane protein with a short intracellular C terminus that interacts with numerous signaling molecules, including Gα12. Cyst formation in ADPKD results from numerous cellular defects, including abnormal cilia, changes in polarity, and dysregulated apoptosis and proliferation. Recently, we reported increased apoptosis in Madin-Darby canine kidney (MDCK) cells through Gα12 stimulation of JNK and degradation of the anti-apoptotic protein Bcl-2 (Yanamadala, V., Negoro, H., Gunaratnam, L., Kong, T., and Denker, B. M. (2007) J. Biol. Chem. 282, 24352–24363). Herein, we confirm this pathway in Gα12-silenced MDCK cells and utilize MDCK cell lines harboring either overexpressed or silenced PC1 to demonstrate that PC1 expression levels determine activity of the JNK/Bcl-2 apoptosis pathway. PC1-overexpressing MDCK cells were resistant to thrombin/Gα12-stimulated apoptosis, JNK activation, and Bcl-2 degradation. In contrast, PC1-silenced MDCK cells displayed enhanced thrombin-induced apoptosis, JNK activity, and Bcl-2 degradation. In pulldown experiments, PC1 bound to Gα12, but not the related Gα13 subunit, and thrombin-stimulated MDCK cells led to increased interaction of Gα12 with the PC1 C terminus. In transient transfection assays, a PC1 C-terminal mutant lacking the G protein-binding domain was uncoupled from PC1-inhibited apoptosis. PC1 expression levels may be increased or decreased in ADPKD, and these findings suggest a mechanism in which levels of PC1 expression modulate Gα12/JNK-stimulated apoptosis. Taken together, these findings are consistent with a set point model in which PC1 expression levels regulate specific G protein signaling pathways important to cyst development.

Induction of the von Hippel‐Lindau tumor suppressor gene by late hypoxia limits HIF‐1 expression

Hypoxia–inducible factor (HIF) remains the central focus of oxygen sensing during hypoxia. HIF is a heterodimeric transcription factor consisting of an oxygen‐regulated alpha‐ and a constitutively expressed beta subunit. The von Hippel‐Lindau tumor suppressor (pVHL) is a component of the E3 ubiquitin ligase complex and targets HIF‐α to proteasomal degradation, but also is known to exert a significant control on HIF transactivation activity. However, the understanding of the full interaction between HIF and pVHL has been hindered by a lack in the understanding of pVHL regulation. Here, we report that pVHL itself is induced in prolonged hypoxia in a kinetic that parallels the observed downregulation of HIF‐1α protein under such conditions. In addition, we document direct HIF‐1α binding to the VHL promoter and identify a functional hypoxia response element (HRE) within the VHL promoter. Such induction of pVHL in hypoxia furthermore has functional implications for the HIF dependent hypoxic response, implicating a physiologically relevant feedback mechanism. These results provide an intriguing model, whereby HIF self‐regulates expression through VHL and highlight the role of pVHL as a unifying mechanism of HIF regulation.

Gα12 Inhibits α2β1 Integrin-Mediated MDCK Cell Attachment and Migration on Collagen-I and Blocks Tubulogenesis

Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that G alpha 12 inhibits interaction of MDCK cells with collagen-I, the major ligand for alpha2 beta1 integrin. Activating G alpha 12 (QL point mutation or stimulating endogenous G alpha 12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with G alpha 12-regulated attachment to collagen-I, G alpha 12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in G alpha 12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. G alpha 12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of alpha2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated G alpha 12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, G alpha 12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for G alpha 12-integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.