Prolyl-4-hydroxylase domain 3 (PHD3) is a critical terminator for cell survival of macrophages under stress conditions

On molecular level, cells sense changes in oxygen availability through the prolyl-4-hydroxylase domain enzymes (PHDs), which in turn regulate the protein stability of the α-subunit of the transcription factor hypoxia-inducible factor (HIF). By using molecular oxygen PHDs 1 - 3 hydroxylate two specific proline residues thereby marking HIFα for proteasomal degradation. Among the three PHD isoforms the constitutively expressed PHD2 is the main regulator of HIFα stability and thus hypoxia-inducible gene expression in normoxia. PHD3 is highly induced under hypoxic conditions and serves as a negative feedback regulator. Especially PHD3 has been additionally associated with apoptotic cell death. I hypothesized that PHD3 plays a role in cell-fate decisions in macrophages. Therefore, myeloid-specific PHD3 knockout mice (PHD3-/-) were created and analyzed. PHD3-deficient bone marrow-derived macrophages (BMDM) showed no altered HIF-1α or HIF-2α stabilization or increased HIF target gene expression in normoxia or hypoxia. Macrophage M1 and M2-polarization was unchanged likewise. Compared to macrophages from wild type littermates PHD3-/- exhibited a significant reduction in TUNEL positive cells after serum withdrawal. Under the same conditions the PHD3-/- macrophages also showed less Annexin V staining which detects the membrane disruption indicating a reduced early apoptosis. Application of apoptotic inducers such as S-nitroso-N-acetyl penicillamine (SNAP) or staurosporine also showed PHD3-/- cells to be less apoptotic. Additionally, calcein uptake results indicate that PHD3-/- macrophages are more viable. When the supernatant (medium) from the cultured macrophages was exchanged between the genotypes after 24 hrs of culture, then PHD3-/- macrophages showed more Annexin V staining suggesting that at least in part a secreted factor is involved in the PHD3 induced apoptosis mechanism. In an unbiased transcriptome screen the expression of a secretory glycoprotein angiopoietin-like protein 2 (Angptl2) expressions was found to be reduced in PHD3-/- BMDM under stress conditions. Addition of recombinant Angptl2 rescued the anti-apoptotic phenotype demonstrating that it is involved in the PHD3-mediated response towards apoptotic stimuli in macrophages. Additionally Angptl2-/- BMDM showed decreased apoptosis compared to wild type which support the lower expression of Angptl2 in PHD3-/- BMDM followed by decreased apoptosis. My data suggests that the anti-apoptotic effects in the PHD3-/- BMDMs are at least partially mediated by an altered production and response to Angptl2.