Bone strength and mineralization are regulated independently of bone mass by ephrinB2-dependent autophagic processes in osteocytes

Abstract

Bone formation has two phases: a rapid initiation (primary mineralization), followed by slower accrual of mineral (secondary mineralization) that continues until that portion of bone is renewed by remodeling. Initiation of bone mineralization, and late stage osteoblast differentiation, requires expression of EphrinB2 (Efnb2) in the osteoblast lineage, but the function of EphrinB2 in matrix-embedded osteocytes is not known.We previously reported at this meeting that osteocyte-specific EphrinB2 null mice (Dmp1Cre.Efnb2f/f) exhibit an intrinsic defect in material strength. Their brittle bone phenotype was not associated with any change in moment of inertia, osteoblast numbers, nor any defect in the initiation of osteoid mineralization. However, secondary mineralization was significantly accelerated: after initiation of mineralization, mineral:matrix ratio, and carbonate:matrix ratio increased more rapidly in EphrinB2-deficient bones than controls (detected by synchrotron-based Fourier-transform infrared microspectroscopy). This indicated that osteocytic ephrinB2 suppresses mineral accumulation in bone.To identify novel mechanisms by which osteocytes regulate secondary mineralization bone mass, we carried out RNA sequencing of marrow-flushed cortical bone from Dmp1Cre.Efnb2f/f brittle-bone mice and Dmp1Cre control littermates. This revealed 782 significantly up-regulated genes and 1024 down-regulated genes (FDR < 0.05). Genes previously known to regulate mineralization (e.g. Dmp1, Mepe, Sost, Phospho1, Enpp1, Enpp2) were not significantly modified by EphrinB2 deletion, and no regulation of the collagen type I genes (Col1a1 or Col1a2) was detected. By a literature search, we identified that >30% of the top 30 differentially expressed genes are associated with autophagy, a mechanism that mediates intracellular recycling and exocytic secretion. We therefore generated stable ephrinB2-deficient osteocyte cell lines (Ocy454) using shRNA. EphrinB2-deficient osteocytes deposited greater amounts of mineral in vitro than controls. They also showed a significantly greater increase in autophagic flux than control cells (the increase in LC3-II:I ratio in response to chloroquine treatment was increased by 30% compared to controls). This suggests that secondary mineralization of the bone matrix is controlled by autophagic processes in osteocytes, in a manner that is limited by ephrinB2. Such processes may be disrupted in conditions of bone fragility that are independent of bone mass.