Vhl deficiency in osteocytes produces high bone mass and hematopoietic defects

Highlights

• Osteocytes exert a robust influence on bone mass through their capacity to perceive pericellular oxygen.

• Osteocytic Hif1a is dispensible for proper skeletal development.

• Osteocytic Vhl deletion promotes canonical Wnt signaling.

• Absence of osteocytic Vhl reduces B cell maturation in bone marrow.

Abstract

Tissue oxygen (O₂) levels vary during development and disease; adaptations to decreased O₂ (hypoxia) are mediated by hypoxia-inducible factor (HIF) transcription factors. HIFs are active in the skeleton, and stabilizing HIF-α isoforms cause high bone mass (HBM) phenotypes. A fundamental limitation of previous studies examining the obligate role for HIF-α isoforms in the skeleton involves the persistence of gene deletion as osteolineage cells differentiate into osteocytes. Because osteocytes orchestrate skeletal development and homeostasis, we evaluated the influence of Vhl or Hif1a disruption in osteocytes. Osteocytic Vhl deletion caused HBM phenotype, but Hif1a was dispensable in osteocytes. Vhl cKO mice revealed enhanced canonical Wnt signaling. B cell development was reduced while myelopoiesis increased in osteocytic Vhl cKO, revealing a novel influence of Vhl/HIF-α function in osteocytes on maintenance of bone microarchitecture via canonical Wnt signaling and effects on hematopoiesis.

Introduction

Oxygen (O₂) levels vary throughout the body. They change spatiotemporally during development, and can be acutely impacted during pathophysiological insults, such as stroke or bone fracture. Adaptive responses to decreased O₂ (hypoxia) are rectified by transcriptional induction of genes promoting angiogenesis and anaerobic glycolysis, which are mediated, in part, by the hypoxia-inducible factor (HIF) family of transcription factors [1]. Constitutively expressed HIF-β subunits bind to one of three distinct HIF-α isoforms (HIF-1α, HIF-2α, and HIF-3α) to initiate gene expression, although HIF-3α is considered an inhibitor of HIF-1α and HIF-2α. Oxygen levels regulate HIF-responsive gene expression. Under normoxic conditions, HIF-α subunits are proline hydroxylated by proline hydroxylase-domain proteins (PHDs), targeted for ubiquitination by the E3 ligase complex von Hippel-Lindau (Vhl) and are ultimately degraded by the 26S proteasome. Under hypoxic conditions, prolyl hydroxylation of HIF-α is inhibited, enabling HIF-α to accumulate, translocate into the nucleus and complex with HIF-β/ARNT, and ultimately bind to HIF-response elements (HRE) within target genes.

The VHL/HIF system is highly active in the skeleton. Multiple strategies that use osteolineage-specific cre drivers and deletion of PHD or Vhl to stabilize HIF-1α and HIF-2α increase bone mass. Individually or combinatorially deleting Phd1, Phd2, or Phd3 in Osterix-positive osteoprogenitors improves trabecular microarchitecture dependent upon the degree of HIF-α stabilization [2]. Similarly, deletion of Vhl in mature osteoblasts using an osteocalcin (Bglap) cre driver increases trabecular and cortical microarchitecture [3].

The murine models described above clearly demonstrate that manipulating HIF-α levels in bone cells influences skeletal development and homeostasis. In these models, Vhl or Phd isoform elimination persists as osteoprogenitors differentiate into osteoblasts and then transition into osteocytes. Thus, these models cannot elucidate the influence of Vhl deletion in osteocytes compared to Vhl deletion in osteoprogenitors or osteoblasts. Osteocytes are the most abundant cells within bone, comprising >90% of total cells [4]. Osteocytes exert critical roles in mechano-sensing, skeletal remodeling, mineral homeostasis and hematopoiesis [5]. Indeed, the osteocyte and the extensive osteocytic network in bone are now recognized as a central regulator of skeletal activity [5,6]. The frequency of HIF-1α-positive osteocytes increases in mice under disuse conditions [7]; in vitro, exposing osteocyte-like cells to hypoxia augments secretion of chemotactic factors [8] and GDF15 to promote osteoclastogenesis [9], and influences the osteoblast-to-osteocyte transition [10]. Other osteocytic functions attributed to HIF-α function include directing appropriate bone formation in response to mechanical loading [11], sensing microdamage [12], and development of the lacunocanalicular network in which osteocytes reside [13].

Provided the recognition that osteocytes orchestrate many aspects of skeletal development and homeostasis, we sought to evaluate the influence of Vhl or Hif1a disruption in osteocytes. We used a cre-loxP system under control of the non-inducible 10 kb-Dmp1 promoter [14] to address the function of Vhl in osteocytes. Similar to results using Bglap-cre to delete Vhl in mature osteoblasts [3], osteocytic deletion of Vhl produced mice with a high bone mass (HBM) phenotype that dramatically increased trabecular and cortical microarchitecture. Femoral osteocytes in Vhl cKO mice revealed qualitative and quantitative reductions in sclerostin immunostaining, and concomitant increases in activated β-catenin levels, suggesting that Vhl/HIF-α signaling influences canonical Wnt signaling in osteocytes. Vhl cKO mice mated to mice lacking the Wnt co-receptor Lrp5 or to transgenic mice over-expressing the human SOST gene revealed that Vhl functions epistatically to canonical Wnt signaling: cortical microarchitecture resolved to wild-type phenotype in compound mice, but not trabecular microarchitecture. Osteocytic HIF-α contributes to hematopoiesis, as B cell development is blocked in osteocytic Vhl cKO mice. These data reveal a novel influence of Vhl/HIF-α function in osteocytes on maintenance of bone microarchitecture via canonical Wnt signaling and effects on hematopoiesis.