Dissecting HIF-Driven High Bone Mass Phenotype in Mice Lacking Osteocytic Vhl

Abstract Osteocytes regulate skeletal homeostasis and respond to oxygen deprivation via hypoxia inducible factors (HIFs), the principal transcriptional mediators of the cellular hypoxia response. There are two transcriptionally active HIF-α paralogs, HIF-1α and HIF-2α, that have overlapping and distinct transcriptional targets. Under normoxic conditions, the E3 ubiquitin ligase Von Hippel-Lindau (VHL) acts as the master regulator of HIFs by targeting them for proteasomal degradation, whereas hypoxia prevents this, allowing for HIF stabilization and accumulation. Osteocyte-enriched deletion of Vhl (Vhl cKO) produces a high bone mass (HBM) phenotype. Vhl loss stabilizes both HIFs, leaving their distinct contributions to this HBM phenotype undefined. To dissect their role in osteocytes, we developed compound Vhl; Hif1a cKO, Vhl; Hif2a cKO mice, mice expressing individual and combined degradation-resistant (cDR) paralogs HIF-1α (HIF-α cDR), HIF-2α (HIF-2α cDR), and HIF-1α; HIF-2α cDR and evaluated their impact on bone structure and function. Microcomputed tomography revealed that Vhl cKO mice exhibited the highest trabecular bone volume fraction, a phenotype not fully reproduced in any HIF-modified genotype. Among partial phenocopies, Vhl; Hif2a cKO produced the largest partial increase, followed by Vhl; Hif1a cKO, and HIF-2α cDR. HIF-1α; HIF-2α cDR displayed modest elevation, while cre-negative and HIF-1α cDR were indistinguishable. Cortical area expansion required Vhl deletion and was not reproduced by single-paralog HIF stabilization. While Vhl cKO femora exhibited significantly increased whole-bone mechanical properties relative to sex- and age-matched controls, all HIF-modified genotypes had similar material properties and HIF-2α activation appeared to compromise material integrity. HIF-1α and HIF-2α therefore exert non-redundant, compartment-specific roles in the Vhl cKO HBM phenotype and are each required for maximal bone integrity. Yet, no HIF-modified genotype, including dual HIF-1α/HIF-2α stabilization, reproduced the Vhl cKO phenotype, implicating the possibility of VHL functions beyond HIF stabilization in the full skeletal response.