Functional Integrity of Nuclear Factor κB, Phosphatidylinositol 3′-Kinase, and Mitogen-Activated Protein Kinase Signaling Allows Tumor Necrosis Factor α-Evoked Bcl-2 Expression to Provoke Internal Ribosome Entry Site-Dependent Translation of Hypoxia-Inducible Factor 1α

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Abstract
Hypoxia-inducible factor (HIF)-1, a heterodimeric transcription factor composed of HIF-1α and HIF-1β subunits coordinates pathophysiologic responses toward decreased oxygen availability. It is now appreciated that enhanced protein translation of HIF-1α under normoxia accounts for an alternative regulatory circuit to activate HIF-1 by hormones, growth factors, or cytokines such as tumor necrosis factor α (TNF-α). Here, we aimed at understanding molecular details of HIF-1α translation in response to TNF-α. In tubular LLC-PK1 cells, activation of nuclear factor κB (NFκB) by TNF-α resulted in HIF-1α protein synthesis as determined by [35S]methionine pulse experiments. Protein synthesis was attenuated by blocking NFκB, phosphatidylinositol 3′-kinase (PI3k), and mitogen-activated protein kinase (MAPK). Use of a dicistronic reporter with the HIF-1α 5′-untranslated region (5′UTR) between two coding regions indicated that TNF-α promoted an internal ribosome entry site (IRES) rather than a cap-dependent translation. IRES-mediated translation required the functional integrity of the NFκB, PI3k, and MAPK signaling pathways. Although no signal cross-talk was noticed between NFκB, PI3k, and MAPK signaling, these pathways are needed to up-regulate the anti-apoptotic target protein Bcl-2 by TNF-α. Expression of Bcl-2 provoked not only IRES-dependent translation but also HIF-1α protein synthesis. We conclude that Bcl-2 functions as an important determinant in facilitating HIF-1α protein expression by TNF-α via an IRES-dependent translational mechanism. These observations suggest a link between Bcl-2 and HIF-1α expression, a situation with potential relevance to cancer biology.