title: Critical role of cAMP response element binding protein expression in hypoxia-elicited induction of epithelial tumor necrosis factor-alpha.
abstract: Tissue hypoxia is intimately associated with a number of chronic inflammatory conditions of the intestine. In this study, we investigated the impact of hypoxia on the expression of a panel of inflammatory mediators by intestinal epithelia. Initial experiments revealed that epithelial (T84 cell) exposure to ambient hypoxia evoked a time-dependent induction of the proinflammatory markers tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), and major histocompatibility complex (MHC) class II (37 +/- 6.1-, 7 +/- 0.8-, and 9 +/- 0.9-fold increase over normoxia, respectively, each p < 0.01). Since the gene regulatory elements for each of these molecules contains an NF-kappaB binding domain, we investigated the influence of hypoxia on NF-kappaB activation. Cellular hypoxia induced a time-dependent increase in nuclear p65, suggesting a dominant role for NF-kappaB in hypoxia-elicited induction of proinflammatory gene products. Further work, however, revealed that hypoxia does not influence epithelial intercellular adhesion molecule 1 (ICAM-1) or MHC class I, the promoters of which also contain NF-kappaB binding domains, suggesting differential responses to hypoxia. Importantly, the genes for TNF-alpha, IL-8, and MHC class II, but not ICAM-1 or MHC class I, contain cyclic AMP response element (CRE) consensus motifs. Thus, we examined the role of cAMP in the hypoxia-elicited phenotype. Hypoxia diminished CRE binding protein (CREB) expression. In parallel, T84 cell cAMP was diminished by hypoxia (83 +/- 13.2% decrease, p < 0.001), and pharmacologic inhibition of protein kinase A induced TNF-alpha and protein release (9 +/- 3.9-fold increase). Addback of cAMP resulted in reversal of hypoxia-elicited TNF-alpha release (86 +/- 3.2% inhibition with 3 mM 8-bromo-cAMP). Furthermore, overexpression of CREB but not mutated CREB by retroviral-mediated gene transfer reversed hypoxia-elicited induction of TNF-alpha defining a causal relationship between hypoxia-elicited CREB reduction and TNF-alpha induction. Such data indicate a prominent role for CREB in the hypoxia-elicited epithelial phenotype and implicate intracellular cAMP as an important second messenger in differential induction of proinflammatory mediators.
authors.affiliation: Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: C T
authors.initials: CT
authors.lastname: Taylor
authors.forename: N
authors.initials: N
authors.lastname: Fueki
authors.forename: A
authors.initials: A
authors.lastname: Agah
authors.forename: R M
authors.initials: RM
authors.lastname: Hershberg
authors.forename: S P
authors.initials: SP
authors.lastname: Colgan
mesh_terms: D015687:Cell Hypoxia; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000096:biosynthesis* / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases