All metazoans that utilize molecular oxygen (O₂) for metabolic purposes have the capacity to adapt to hypoxia, the condition that arises when O₂ demand exceeds supply. This is mediated through activation of the hypoxia-inducible factor (HIF) pathway. At physiological oxygen levels (normoxia), HIF-prolyl hydroxylases (PHDs) hydroxylate proline residues on HIF-α subunits leading to their destabilization by promoting ubiquitination by the von-Hippel Lindau (VHL) ubiquitin ligase and subsequent proteasomal degradation. HIF-α transactivation is also repressed in an O₂-dependent way due to asparaginyl hydroxylation by the factor-inhibiting HIF (FIH). In hypoxia, the O₂-dependent hydroxylation of HIF-α subunits by PHDs and FIH is reduced, resulting in HIF-α accumulation, dimerization with HIF-β and migration into the nucleus to induce an adaptive transcriptional response. Although HIFs are the canonical substrates for PHD- and FIH-mediated protein hydroxylation, increasing evidence indicates that these hydroxylases may also have alternative targets. In addition to PHD-conferred alterations in protein stability, there is now evidence that hydroxylation can affect protein activity and protein/protein interactions for alternative substrates. PHDs can be pharmacologically inhibited by a new class of drugs termed prolyl hydroxylase inhibitors which have recently been approved for the treatment of anemia associated with chronic kidney disease. The identification of alternative targets of HIF hydroxylases is important in order to fully elucidate the pharmacology of hydroxylase inhibitors (PHI). Despite significant technical advances, screening, detection and verification of alternative functional targets for PHDs and FIH remain challenging. In this review, we discuss recently proposed non-HIF targets for PHDs and FIH and provide an overview of the techniques used to identify these.

Molecular oxygen (O₂) and carbon dioxide (CO₂) are the primary gaseous substrate and product of oxidative phosphorylation in respiring organisms, respectively. Variance in the levels of either of these gasses outside of the physiological range presents a serious threat to cell, tissue, and organism survival. Therefore, it is essential that endogenous levels are monitored and kept at appropriate concentrations to maintain a state of homeostasis. Higher organisms such as mammals have evolved mechanisms to sense O₂ and CO₂ both in the circulation and in individual cells and elicit appropriate corrective responses to promote adaptation to commonly encountered conditions such as hypoxia and hypercapnia. These can be acute and transient nontranscriptional responses, which typically occur at the level of whole animal physiology or more sustained transcriptional responses, which promote chronic adaptation. In this review, we discuss the mechanisms by which mammals sense changes in O₂ and CO₂ and elicit adaptive responses to maintain homeostasis. We also discuss crosstalk between these pathways and how they may represent targets for therapeutic intervention in a range of pathological states.

The HIF hydroxylase enzymes (PHD1‐3 and FIH) are cellular oxygen‐sensors which confer hypoxic‐sensitivity upon the hypoxia‐inducible factors HIF‐1α and HIF‐2α. Microenvironmental hypoxia has a strong influence on the epithelial and immune cell function through HIF‐dependent gene expression and consequently impacts upon the course of disease progression in ulcerative colitis (UC), with HIF‐1α being protective while HIF‐2α promotes disease. However, little is known about how inflammation regulates hypoxia‐responsive pathways in UC patients. Here we demonstrate that hypoxia is a prominent microenvironmental feature of the mucosa in UC patients with active inflammatory disease. Furthermore, we found that inflammation drives transcriptional programming of the HIF pathway including downregulation of PHD1 thereby increasing the tissue responsiveness to hypoxia and skewing this response toward protective HIF‐1 over detrimental HIF‐2 activation. We identified CEBPα as a transcriptional regulator of PHD1 mRNA expression which is downregulated in both inflamed tissue derived from patients and in cultured intestinal epithelial cells treated with inflammatory cytokines. In summary, we propose that PHD1 downregulation skews the hypoxic response toward enhanced protective HIF‐1α stabilization in the inflamed mucosa of UC patients.