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. 2019 Jul 31;10(1):3436.
doi: 10.1038/s41467-019-11373-9.

Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone

Christina Vrahnas  1   2   3 , Martha Blank  1   2 , Toby A Dite  2   4   3 , Liliana Tatarczuch  5 , Niloufar Ansari  1   2 , Blessing Crimeen-Irwin  1 , Huynh Nguyen  6 , Mark R Forwood  6 , Yifang Hu  7 , Mika Ikegame  8 , Keith R Bambery  9 , Cyril Petibois  10 , Eleanor J Mackie  5 , Mark J Tobin  9 , Gordon K Smyth  7   11 , Jonathan S Oakhill  2   4   12 , T John Martin  1   2 , Natalie A Sims  13   14
Affiliations

Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone

Christina Vrahnas et al. Nat Commun. .

Erratum in

Abstract

Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Parathyroid hormone (PTH) and PTH-related protein (PTHrP) stimulate Efnb2 in osteocytes and bone strength is impaired in mice lacking EphrinB2 in osteocytes. a, b Efnb2 messenger RNA (mRNA) levels, relative to Hprt1, in Ocy454 cells differentiated for 14 days and treated with 10 nM hPTH(1–34) or hPTHrP(1–141) for 6 h (a) and Ocy454 cells with stable short hairpin RNA (shRNA) knockdown of PTHrP (Pthlh) at 0, 7, and 14 days of differentiation (b). Data are mean ± SEM, n = 6 replicates; representative of three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001 compared to control by Student’s t test. c Efnb2 mRNA levels in green fluorescent protein-positive (GFP+) osteocytes isolated from Dmp1Cre.DMP1-GFP-Tg.Efnb2w/w (w/w) and Dmp1Cre.DMP1-GFP-Tg.Efnb2f/f (f/f) mice compared to GFP− cells. ND = not detected. Data mean ± SEM of two experiments, n = 6 mice/group; pooled. d Average load–deformation curve from three-point bending tests (each dot represents the average for the noted sample group; error bars excluded to highlight the shape of curves) of femora from 12-week-old female Dmp1Cre.Efnb2f/f (f/f) mice and Dmp1Cre controls (w/w). e Kaplan–Meier curve showing percentage of unbroken femora with increasing deformation (p value from Mantel–Cox log-rank test). f–i Calculated indices of bone strength: ultimate force (f), ultimate deformation (g), post-yield deformation (h), and energy absorbed to failure (i). Data are mean ± SEM, n = 10–12/group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. w/w controls by Student’s t test
Fig. 2
Fig. 2
Impaired strength in Dmp1Cre.Efnb2f/f mice is a material defect and is not caused by altered bone geometry. a Femoral moment of inertia. b–e Three-point bending test data corrected for bone cross-sectional area, including average stress–strain curve (each dot represents the average load and deformation for the noted sample group; error bars excluded to highlight the shape of curves) (b), ultimate stress (c), ultimate strain (d), and toughness (e) of 12-week old female Dmp1Cre (w/w) and Dmp1Cre.Efnb2f/f (f/f) femora. f Indentation distance increase derived from reference point indentation. Data are represented as mean ± SEM, n = 10–12/group. *p < 0.05, **p < 0.01 vs. w/w controls by Student’s t test
Fig. 3
Fig. 3
Elevated mineral accrual, carbonate deposition, and reduced amide I:II ratio in Dmp1Cre.Efnb2f/f cortical bone. a Regions of periosteal bone used for synchrotron-based Fourier-transform infrared microspectroscopy (sFTIRM) analysis. Note the double calcein label on the newly mineralized periosteum; regions 1–3 (15 μm2 in size) denote bone areas of increasing maturity with increasing distance from the periosteal edge. b Averaged sFTIRM spectra from regions 1, 2, and 3 of all 12-week old female Dmp1Cre (w/w) and Dmp1Cre.Efnb2f/f (f/f) tibiae; grey boxes show approximate regions of the amide I, amide II, phosphate, and carbonate peaks (see Method for details). c–f sFTIRM-derived mineral:matrix (c), carbonate:mineral (d), amide I:II (e), and collagen crosslinking (f) ratios in regions 1–3 of w/w and f/f tibiae. Data are represented as mean ± SEM, with individual values, n = 13/group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. region 1 of same genotype (bone maturation effect), +p < 0.05, ++p < 0.01 vs. w/w in the same region (genotype effect) by two-way analysis of variance (ANOVA)
Fig. 4
Fig. 4
Polarized Fourier-transform infrared (FTIR) imaging confirms altered collagen distribution in Dmp1Cre.Efnb2f/f mice. Representative FTIR images showing relative intensity of amide I and amide II peaks in the cortical mid-shaft from 12-week old female Dmp1Cre (w/w) and Dmp1Cre.Efnb2f/f (f/f) mice under the (a, b) 0° and (d, e) 90° polarizing filters; quantification of the amide I:II ratio under the 0° (c) and 90° (f) polarizing filters in periosteal (P), central (C) and endocortical (E) regions (shown in the top left of panel a). Data are mean ± SEM, n = 12–13/group. *p < 0.05 vs. Dmp1Cre at the same region (genotype effect), +p < 0.05, ++p < 0.01, +++p < 0.001 vs region indicated by the bracket, within the same genotype (region effect) by two-way analysis of variance (ANOVA). Scale bar = 50 μm
Fig. 5
Fig. 5
Confirmation of increased autophagy and mineralization in EphrinB2-deficient Ocy454 osteocytes, and effect of autophagy on mineralization. a Efnb2 stable knockdown (short hairpin RNA (shRNA) 1 and shRNA 2) in Ocy454 cells differentiated for 7, 11, and 14 days, mean ± SD, 3–6 replicates, representative of three independent cultures; *p < 0.05 vs. vector by Student’s t test. b, c Autophagosomes (LC3 punctae) in Efnb2-deficient Ocy454 cells; bafilomycin (Baf) treatment (50 nM for 2 h) was used to block autophagosome degradation. Data shown are mean ± SD, five replicates, representative of three independent cultures; *p < 0.05; ***p < 0.001 as indicated by one-way analysis of variance (ANOVA). Scale bar = 10 μm d Fold change of LC3-II:I ratio in Efnb2 shRNA knockdown cells treated with chloroquine (CQ) for 4 h compared to basal levels of each shRNA construct. Data are represented as mean ± SEM, three replicates, representative of three independent cultures, *p < 0.05 vs. vector control by one-way ANOVA. e, f Alizarin Red stain for mineralization in Ocy454 cells with vector or Efnb2 shRNA 1 knockdown, grown under mineralizing conditions for 14 days. Data shown are mean ± SD, four replicates, representative of three independent cultures; ***p < 0.001 vs. vector by two-way ANOVA; f Alizarin Red-stained wells at day 14, showing three replicates. g Effect of rapamycin treatment (0.05 nM in dimethyl sulfoxide (DMSO) for 24 h) on Ocy454 cells grown under mineralizing conditions for 6 days. Data shown are mean ± SD, three replicates, representative of two independent cultures; *p < 0.05 vs. DMSO and p < 0.01 vs. untreated by one-way ANOVA
Fig. 6
Fig. 6
Transmission electron microscopy of bone-embedded osteocytes in Dmp1Cre (a, d) and Dmp1Cre.Efnb2f/f (b, c, e–i) mice. a–c Low power images, showing typical osteocyte morphology in control Dmp1Cre bone (a) and in Dmp1Cre.Efnb2f/f bone (b, c). Dmp1Cre.Efnb2f/f osteocytes are grossly abnormal, showing contraction from the lacuna, extensive ruffling, and formation of matrix vesicles (arrows), but normal appearance of dendritic processes within the canaliculi (arrowheads); boxes show regions magnified in d–i. d Cytoplasm of a control Dmp1Cre osteocyte, showing a budding matrix vesicle (mv), and endoplasmic reticulum (ER) within the cytoplasm. e–i Features of Dmp1Cre.Efnb2f/f osteocytes, including autophagomes (A) and lysosomes (L). Autophagosomes are either fully enclosed (e–h) or exhibit phagophores (p) in the process of encapsulating their cargo, which has the appearance of degraded ER (e, f, i); phagophores in the process of formation are delineated with white lollipops at the tips of the membrane with sticks facing the direction of the formed membrane. Functioning ER is also observed (f), along with unencapsulated degraded ER (dER), and many matrix vesicles (mv) in various stages of budding (f–i). Scale bars a–c = 1 μm, d–i = 250 nm
Fig. 7
Fig. 7
RhoA-ROCK inhibition increases mineralization and EphrinB2 inhibits autophagy through a RhoA-ROCK-dependent mechanism. a–d Mineralization by Kusa 4b10 cells is enhanced when RhoA-ROCK is inhibited with either H1152 or Y27632. Kusa 4b10 osteocytes were cultured under mineralizing conditions and treated with H1152 (a, b) or Y27632 (c, d) at the doses indicated for the duration of the culture. Alizarin Red staining and quantitation is shown. Data are mean ± SD of three replicates from a representative of three independent experiments; **p < 0.01 vs. untreated (0) at same day of differentiation by two-way analysis of variance (ANOVA). e Autophagy is suppressed by EphrinB2 in a RhoA-ROCK-dependent manner. LC3 punctae formation in Ocy454 cells, either untreated or treated with bafilomycin A1 (Baf) (50 nM), in the presence and absence of clustered EphrinB2-Fc (1 μg/mL) and H1152 (50 μM) for 1 h. Punctae number per cell from two independent experiments, each performed in duplicate, with 95% confidence interval (CI); ***p < 0.0001; **p < 0.001 for comparisons shown, by one-way ANOVA with correction for multiple comparisons
Fig. 8
Fig. 8
Model of osteocytic EphrinB2 regulation of bone matrix composition. a In control bone (Dmp1Cre) both osteoblasts and osteocytes express EphrinB2. Osteoblasts reside on the bone surface, and pass through a transition (dashed arrows) to become mature, matrix-embedded osteocytes. Osteoblasts deposit collagen-containing osteoid (triple helical collagen fibers, shown on the right). In region 1 of newly formed bone, mineral deposition is initiated. Mineral crystals (black stars) and carbonate (orange circles) continue to accumulate, and collagen fibers become more compact as the matrix matures in regions 2 and 3. b Dmp1Cre.Efnb2f/f mice express EphrinB2 in osteoblasts, but not osteocytes. Dmp1Cre.Efnb2f/f osteocytes have increased autophagy (green cells). Osteoid deposition occurs normally, and the initiation of mineralization commences at the same rate, leading to osteoid of the same thickness (red double-headed arrow). As soon as mineralization starts, mineral is deposited in Dmp1Cre.Efnb2f/f bone at a greater level than control in regions 1, 2, and 3 to reach a level with more mineral, more carbonate substitution, and more compact collagen fibers than controls. Ultimately this leads to more brittle bone. c Close-up of the cell membrane in osteocytes in control and Dmp1Cre.Efnb2f/f bone: we propose that, in control mice, EphrinB2 suppresses autophagic processes and limits matrix vesicle release via RhoA-ROCK signalling. In Dmp1Cre.Efnb2f/f mice loss of this inhibition leads to a high level of matrix vesicle release, elevated mineralization, and a brittle bone matrix
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