Dissecting Hypoxia Inducible Factor-Driven High Bone Mass Phenotype in Mice Lacking Osteocytic Von Hippel-Lindau

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 2 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. Micro-CT 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.