Disruption of Prolyl hydroxylase domain protein-2 (PHD2) activity impairs TGFβ-dependent sphingolipid metabolism in murine placenta

Objectives: Impairment of O2-dependent TGFβ3 signaling characterizes placental pathologies such as preeclampsia and IUGR and causes altered sphingolipid metabolism leading to increased trophoblast cell death. Under normoxic condition, degradation of the alpha subunit of hypoxia-inducible factor (HIF1A), a regulator of oxygen homeostasis, is tightly controlled by prolyl hydroxylase enzymes (PHD1-3). Ample evidence indicates that PHD2 is the key hydroxylase involved in regulating HIF1A stability in the human placenta. Our objective was to characterize the biological relevance of the O2/TGFβ3 axis in controlling sphingolipid metabolism in the murine placenta. Methods: We generated a pharmacological hypoxic mouse model by inhibiting PHD2 activity using FG-4592, a small-molecule PHD inhibitor. Pregnant mice injected daily between E7.5 and E13.5 with FG-4592 (0.5 mg/kg) were sacrificed at E13.5. Control mice were injected with an equivalent volume of DMSO. Placentae were collected and processed for protein and sphingolipidomic analyses using immunohistochemistry, Western Blotting and liquid chromatography linked to tandem mass spectrometry (LC-MS/MS), respectively. Results: PHD2 inhibition increased placental HIF1A, decreased its hydroxylation at the proline P564 residue, and stimulated TGFβ signaling. Administration of FG-4592 resulted in reduced placental and pup weights. Striking placental morphological defects including compaction of labyrinth zone and reduced vascularization were found. In addition, PHD2 inhibition resulted in decreased levels of sphingolipid regulatory enzymes, acid ceramidase (ASAH1) and sphingosine kinase (SPHK1), and this associated with elevated ceramide content as well as increased autophagy markers. Conclusion: These results highlight the importance of the O2/TGFβ axis on murine placental development and function. A simulated condition of hypoxia with FG-4592 triggers TGFβ signaling, thereby impacting on sphingolipid metabolism and affecting placental cell homeostasis.