These studies provide the first molecular link between hypoxia and intestinal epithelial barrier protection. Here, we identify a previously unappreciated HIF-1α regulated pathway for induction of ITF. ITF is a small, protease-resistant peptide that is abundant throughout the mammalian intestinal tract 2728
. Increased ITF expression has been observed in proximity to sites of injury in the gastrointestinal tract, including peptic ulcers and active inflammatory bowel disease 272829
. ITF can promote epithelial barrier function, protecting against injury, and facilitating repair after damage occurs irrespective of the nature of the initial wound. However, specific transcriptional pathways for ITF induction are not well understood 30
The hypoxia-dependent production of ITF represents a novel, innate protective mechanism that may guard the immunologic components of the lamina propria from exposure to pathogenic luminal bacteria, antigens, and toxins during episodes of diminished oxygen delivery. Accumulating evidence suggests a variety of physiological roles for ITF 28
, however, molecular mechanisms regulating ITF expression have not been studied in detail. Rather, the signaling pathways activated by the addition of exogenous ITF have been more closely examined and include the inactivation of extracellular signal regulatory kinase, regulated expression of α- and β-catenin, and transactivation of the epidermal growth factor receptor 313233
. Recent work has delineated that ITF can elicit distinct signaling pathways for epithelial restitution and inhibition of apoptosis, the latter of which involves activation of mitogen-activated protein kinase pathways independent of epidermal growth factor receptor phosphorylation 33
. Important for the present studies, these same extracellular signal regulatory kinase pathways were recently demonstrated to critically function in the development of intestinal epithelial barrier function through regulated expression of claudin-2 and organization of membrane proteins within the tight junction 34
. Thus, it is likely that ITF-specific, extracellular signaling pathways regulate epithelial barrier under these defined conditions.
At the tissue and cellular level, an array of genes pivotal to survival in low oxygen states are activated 4353637
. Original studies of hypoxia-induced EPO production concentrated on the regulation of gene transcription. Sequences from the EPO promoter were isolated and identified as a heterodimer with independently regulated subunits termed HIF-1, a member of the rapidly growing per-ARNT-sim family of basic helix-loop-helix transcription factors 56
. HIF-1 exists as an αβ heterodimer, the activation of which is dependent on stabilization of an O2
-dependent degradation domain of the α subunit by the ubiquitin–proteasome pathway 7
. Although not certain, HIF-1 appears to reside in the cytoplasm of normoxic cells, and like a number of other transcription factors (e.g., nuclear factor κB, β-catenin), HIF-1 translocates to the nucleus to form a functional complex 3839
. Binding of HIF-1 to consensus domains in the EPO enhancer results in the transcriptional induction of HIF-1–bearing gene promoters 4
. Subsequently, it was determined that HIF-1 is widely expressed and that consensus HIF-1–binding sequences exist in a number of genes other than EPO and are termed HRE 4
. Of note on this accord, we did not precisely map the ITF HRE. Attempts to define this site in exact terms were hampered by the complexity of the flanking region around the HIF-1 consensus sequence (data not shown). For example, the immediate region of the HIF-1 consensus site also contains consensus binding sites for the transcription factors myeloid zinc finger-1 (MZF1, consensus 5′-AGGGGGA-3′), GATA-1 (consensus 5′-GGGGATTCTG-3′), GATA-2 (consensus 5′-CAGGATAAGG-3′), Sp1 (consensus 5′-GTGGCAGGGT-3′), MyoD (consensus 3′-GCTGTCCACCGG-5′), and upstream stimulatory factor (USF, consensus 5′-CCACCTGT-3′). Important in this regard, at least three of these transcription factors (GATA-1, GATA-2, and MyoD) have been recently implicated in either induction or repression of genes in hypoxia 40414243
. Therefore, it is possible that regulation of the ITF gene at this site could be an interplay of positive and negative signaling pathways, and given this complexity, more work will be necessary to define the exact details of this HRE.
Our results suggest that ITF may represent a potential novel therapeutic approach for a variety of diseases associated with mucosal hypoxia and altered epithelial barrier function, including inflammatory bowel disease, necrotizing enterocolitis, and ischemic colitis 44
. In addition, these results suggest that a receptor/binding site for ITF may be present at sites other than the gastrointestinal tract, such as the endothelium. Thus, the protective effect of ITF on the hypoxia-induced decline in endothelial permeability suggests that ITF may have even broader therapeutic relevance in conditions of vascular leakiness such as septic shock. Although it is unknown whether ITF would function alone in these responses, it is possible, as previously proposed, that ITF acts in concert with and complementary to other protective growth factors and cytokines resident at sites of tissue damage 29
. Efforts to better understand ITF signaling pathways and to identify other hypoxia-elicited protective elements could provide future focus for the development of novel treatments.