Critical role of cAMP response element binding protein expression in hypoxia-elicited induction of epithelial tumor necrosis factor-alpha

Tissue hypoxia is intimately associated with a number of chronic inflammato ry conditions of the intestine. In this study, we investigated the impact o f hypoxia on the expression of a panel of inflammatory mediators by intesti nal epithelia, Initial experiments revealed that epithelial (T84 cell) expo sure to ambient hypoxia evoked a time-dependent induction of the proinflamm atory 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-kappa B binding domain, we investigated the influence of hypoxia on N F-kappa B activation. Cellular hypoxia induced a time dependent increase in nuclear p65, suggesting a dominant role for NF-kappa B in hypoxia-elicited induction of proinflammatory gene products. Further work, however, reveale d that hypoxia does not influence epithelial intercellular adhesion molecul e 1 (ICAM-1) or MHC class I, the promoters of which also contain NF-kappa B 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 cl ass I, contain cyclic AMP response element (CRE) consensus motifs, Thus, we examined the role of cAMP in the hypoxia-elicited phenotype, Hypoxia dimin ished CRE binding protein (CREB) expression. In parallel, T84 cell cAMP was diminished by hypoxia (83 +/- 13.2% decrease, p < 0.001), and pharmacologi c inhibition of protein kinase A induced TNF-alpha and protein release (9 /- 3.9-fold increase). Addback of cAMP resulted in reversal of hypoxia-elic ited TNF-alpha release (86 +/- 3.2% inhibition with 3 mM 8-bromo-cAMP). Fur thermore, overexpression of CREB but not mutated CREB by retroviral-mediate d 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-elic ited epithelial phenotype and implicate intracellular cAMP as an important second messenger in differential induction of proinflammatory mediators.