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

ABSTRACT: On molecular level cells sense changes in oxygen availability through the prolyl-4-hydroxylase domain enzymes (PHDs), which regulate the protein stability of the α-subunit of the transcription factor hypoxia inducible factor (HIF). Especially PHD3 has been additionally associated with apoptotic cell death. We hypothesized that PHD3 plays a role in cell-fate decisions in macrophages. Therefore myeloid-specific PHD3 knock out mice (PHD3-/-) were created and analysed. PHD3-deficient bone marrow-derived macrophages (BMDM) showed no altered HIF-1alpha or HIF-2alpha stabilization or increased HIF target genes expression in normoxia or hypoxia. Macrophage M1 and M2-polarization was unchanged likewise. Compared to macrophages from wild type littermates PHD3-/- BMDM exhibited a significant reduction in TUNEL positive cells after serum withdrawal or treatment with staurosporine and s-nitroso-N-acetylpenicillamine (SNAP). Under the same conditions PHD3-/- BMDM also showed less Annexin V staining which is representative for membrane disruption and indicated a reduced early apoptosis. In an unbiased transcriptome screen we found that angiopoietin like protein 2 (Angptl2) expression was 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE55693 | GEO | 2014/12/31

SECONDARY ACCESSION(S): PRJNA240564

REPOSITORIES: GEO

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Project description:HIF-1a activates genes under hypoxia and was hypothesized to regulate bone regeneration. Surprisingly, HET HIF-1a fracture calluses are larger, stronger and stiffer than WT HIF-1a calluses due to decreased apoptosis. These data identify apoptosis inhibition as a means to enhance bone regeneration. Introduction: Bone regeneration subsequent to fracture involves the synergistic activation of multiple signaling pathways. Localized hypoxia following fracture activates hypoxia-inducible factor 1 alpha (HIF-1a) leading to increased expression of HIF-1 target genes. We therefore hypothesized that HIF-1a is a key regulator of bone regeneration Materials and Methods: Fixed femoral fractures were generated in mice with partial HIF-1a deficiency (HET HIF-1a) and wild type littermates (WT HIF-1a). Fracture calluses and intact contralateral femurs from post fracture day (PFD) 21 and 28 (N=5-10) were subjected to MicroCT evaluation and 4-point bending in order to assess morphometric and mechanical properties. Molecular analyses were carried out on PFD 7, 10 and 14 samples (N=3) to determine differential gene expression at both mRNA and protein levels. Finally, TUNEL staining was performed on PFD 14 samples (N=2) to elucidate differential apoptosis. Results: Surprisingly, fracture calluses from HET HIF-1a mice exhibit greater mineralization and are larger, stronger and stiffer. Microarray analyses focused on hypoxia-induced genes revealed differential expression (between genotypes) of several genes associated with the apoptotic pathway. Real-time PCR confirmed these results, demonstrating higher expression of pro-apoptotic PP2A and lower expression of anti-apoptotic BCL2 in WT HIF-1a calluses. Subsequent TUNEL staining demonstrates that WT HIF-1a calluses contain larger numbers of TUNEL positive chondrocytes and osteoblasts than HET HIF-1a calluses. Conclusions: We conclude that partial HIF-1a deficiency results in decreased chondrocytic and osteoblastic apoptosis; thereby allowing the development of larger, stiffer calluses and enhancing bone regeneration. Furthermore, apoptosis inhibition may be a promising target for developing new treatments to accelerate bone regeneration. Keywords: Bone, Fracture, Apoptosis, Hypoxia, Microarray

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

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