Epithelial hypoxia-inducible factor-1 is protective in murine experimental colitis

The hypoxia-inducible factors (HIFs) 1alpha and 2alpha are key mammalian transcription factors that exhibit dramatic increases in both protein stability and intrinsic transcriptional potency during low-oxygen stress. This increased stability is due to the absence of proline hydroxylation, which in normoxia promotes binding of HIF to the von Hippel-Lindau (VHL tumor suppressor) ubiquitin ligase. We now show that hypoxic induction of the COOH-terminal transactivation domain (CAD) of HIF occurs through abrogation of hydroxylation of a conserved asparagine in the CAD. Inhibitors of Fe(II)- and 2-oxoglutarate-dependent dioxygenases prevented hydroxylation of the Asn, thus allowing the CAD to interact with the p300 transcription coactivator. Replacement of the conserved Asn by Ala resulted in constitutive p300 interaction and strong transcriptional activity. Full induction of HIF-1alpha and -2alpha, therefore, relies on the abrogation of both Pro and Asn hydroxylation, which during normoxia occur at the degradation and COOH-terminal transactivation domains, respectively.

We have developed a simple and reproducible rat model of chronic colonic inflammation by the intraluminal instillation of a solution containing a "barrier breaker" and a hapten. Administration of the hapten 2,4,6-trinitrobenzenesulfonic acid (5-30 mg) in 0.25 ml of 50% ethanol as the "barrier breaker" produced dose-dependent colonic ulceration and inflammation. At a dose of 30 mg, trinitrobenzenesulfonic acid/ethanol-induced ulceration and marked thickening of the bowel wall persisted for at least 8 wk. Histologically, the inflammatory response included mucosal and submucosal infiltration by polymorphonuclear leukocytes, macrophages, lymphocytes, connective tissue mast cells, and fibroblasts. Granulomas were observed in 57% of the rats killed 3 wk after induction of inflammation. Langhan's-type giant cells were also observed. Segmental ulceration and inflammation were common. The characteristics and relatively long duration of inflammation and ulceration induced in this model afford an opportunity to study the pathophysiology of colonic inflammatory disease in a specifically controlled fashion, and to evaluate new treatments potentially applicable to inflammatory bowel disease in humans.

The transcriptional response to lowered oxygen levels is mediated by the hypoxia-inducible transcription factor (HIF-1), a heterodimer consisting of the constitutively expressed aryl hydrocarbon receptor nuclear translocator (ARNT) and the hypoxic response factor HIF-1alpha. To study the role of the transcriptional hypoxic response in vivo we have targeted the murine HIF-1alpha gene. Loss of HIF-1alpha in embryonic stem (ES) cells dramatically retards solid tumor growth; this is correlated with a reduced capacity to release the angiogenic factor vascular endothelial growth factor (VEGF) during hypoxia. HIF-1alpha null mutant embryos exhibit clear morphological differences by embryonic day (E) 8.0, and by E8.5 there is a complete lack of cephalic vascularization, a reduction in the number of somites, abnormal neural fold formation and a greatly increased degree of hypoxia (measured by the nitroimidazole EF5). These data demonstrate the essential role of HIF-1alpha in controlling both embryonic and tumorigenic responses to variations in microenvironmental oxygenation.