Critical Role of cAMP Response Element Binding Protein Expression in Hypoxia-elicited Induction of Epithelial Tumor Necrosis Factor-α*

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-α (TNF-α), 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-κB binding domain, we investigated the influence of hypoxia on NF-κB activation. Cellular hypoxia induced a time-dependent increase in nuclear p65, suggesting a dominant role for NF-κB 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-κB binding domains, suggesting differential responses to hypoxia. Importantly, the genes for TNF-α, 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-α and protein release (9 ± 3.9-fold increase). Addback of cAMP resulted in reversal of hypoxia-elicited TNF-α 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-α defining a causal relationship between hypoxia-elicited CREB reduction and TNF-α 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.