The role of the hypoxia-inducible factor (HIF) subunits 1α and 2α in response to hypoxia is well established in lung epithelial cells, whereas little is known about HIF-3α with respect to transcriptional and translational regulation by hypoxia. HIF-3α and HIF-1α are two similar but distinct basic helix-loop-helix-PAS proteins, which have been postulated to activate hypoxia responsive genes in response to hypoxia. Here, we used quantitative real time RT-PCR and immunoblotting to determine the activation of HIF-3α vs. HIF-1α by hypoxia. HIF-3α was strongly induced by hypoxia (1% O 2 ) both at the level of protein and mRNA due to an increase in protein stability and transcriptional activation, whereas HIF-1α protein and mRNA levels enhanced transiently and then decreased because of a reduction in its mRNA stability in A549 cells, as measured on mRNA and protein levels. Interestingly, HIF-3α and HIF-1α exhibited strikingly similar responses to a variety of activating or inhibitory pharmacological agents. These results demonstrate that HIF-3α is expressed abundantly in lung epithelial cells, and that the transcriptional induction of HIF-3α plays an important role in the response to hypoxia in vitro. Our findings suggest that HIF-3α, as a member of the HIF system, is complementary rather than redundant to HIF-1α induction in protection against hypoxic damage in alveolar epithelial cells.

Isoflurane can up-regulate HIF-1alpha and enhance HIF-1-responsive genes heme oxygenase 1, inducible nitric oxide synthase, and VEGF mRNA expression in Hep3B cells. The induction of HIF-1alpha by isoflurane does not involve protein degradation but depends on translation pathway.

The transcription factor hypoxia-inducible factor (HIF)-1 plays a central physiological role in oxygen and energy homeostasis, and is activated during hypoxia by stabilization of the subunit HIF-1α. Recent studies have demonstrated that non-hypoxic stimuli can also activate HIF-1α in a cell-specific manner. Here, we demonstrate that stimulation of BEAS-2B cells and primary human bronchial epithelial cells by proinflammatory cytokines TNFα/IL-4 strongly induced expression and transcriptional activity of HIF-1α under normoxic conditions and amplified hypoxic HIF-1α activation. TNFα/IL-4 stimulated de novo HIF-1α gene transcription and translation rather than affected HIF-1α protein degradation and mRNA decay process. The activation of HIF-1α by TNFα/IL-4 was countered by the phosphoinositol 3-kinase (PI3K) inhibitor LY-294002 and rapamycin, an antagonist of mammalian target of rapamycin (mTOR), but not by inhibition of the MAPK pathway. In line, TNFα/IL-4 also activated NF-κB, whereas blocking of NF-κB by an inhibitor or silencing NF-κB subunit p65 attenuated HIF-1α activation by TNFα/IL-4. We also found the collaborative induction of VEGF, a potent angiogenic factor required for airway remodeling, by TNFα/IL-4 and hypoxia partially via HIF-1α pathway in BEAS-2B cells. This study reports the previously unsuspected collaborative regulation of HIF-1α by TNFα/IL-4 and hypoxia in bronchial epithelial cells partially via PI3K-mTOR and NF-κB pathway, and thereby will lead to the elucidation of the importance of HIF-1 in integrating inflammatory and hypoxic response in the pathogenesis of airway diseases.

Isoflurane preconditioning can attenuate pulmonary proinflammatory cytokine release and decrease the mortality induced by severe sepsis. Early protection seems to be mediated partly through inhibition of iNOS-NO pathway activation.