Spontaneous mutation of Dock7 results in lower trabecular bone mass and impaired periosteal expansion in aged female Misty mice

Abstract

Misty mice (m/m) have a loss of function mutation in Dock7 gene, a guanine nucleotide exchange factor, resulting in low bone mineral density, uncoupled bone remodeling and reduced bone formation. Dock7 has been identified as a modulator of osteoblast number and in vitro osteogenic differentiation in calvarial osteoblast culture. In addition, m/m exhibit reduced preformed brown adipose tissue innervation and temperature as well as compensatory increase in beige adipocyte markers. While the low bone mineral density phenotype is in part due to higher sympathetic nervous system (SNS) drive in young mice, it is unclear what effect aging would have in mice homozygous for the mutation in the Dock7 gene. We hypothesized that age-related trabecular bone loss and periosteal envelope expansion would be altered in m/m. To test this hypothesis, we comprehensively characterized the skeletal phenotype of m/m at 16, 32, 52, and 78wks of age. When compared to age-matched wild-type control mice (+/+), m/m had lower areal bone mineral density (aBMD) and areal bone mineral content (aBMC). Similarly, both femoral and vertebral BV/TV, Tb.N, and Conn.D were decreased in m/m while there was also an increase in Tb.Sp. As low bone mineral density and decreased trabecular bone were already present at 16wks of age in m/m and persisted throughout life, changes in age-related trabecular bone loss were not observed highlighting the role of Dock7 in controlling trabecular bone acquisition or bone loss prior to 16wks of age. Cortical thickness was also lower in the m/m across all ages. Periosteal and endosteal circumferences were higher in m/m compared to +/+ at 16wks. However, endosteal and periosteal expansion were attenuated in m/m, resulting in m/m having lower periosteal and endosteal circumferences by 78wks of age compared to +/+, highlighting the critical role of Dock7 in appositional bone expansion. Histomorphometry revealed that osteoblasts were nearly undetectable in m/m and marrow adipocytes were elevated 3.5 fold over +/+ (p=0.014). Consistent with reduced bone formation, osteoblast gene expression of Alp, Col1a1, Runx-2, Sp7, and Bglap was significantly decreased in m/m whole bone. Furthermore, markers of osteoclasts were either unchanged or suppressed. Bone marrow stromal cell migration and motility were inhibited in culture and changes in senescence markers suggest that osteoblast function may also be inhibited with loss of Dock7 expression in m/m. Finally, increased Oil Red O staining in m/m ear mesenchymal stem cells during adipogenesis highlights a potential shift of cells from the osteogenic to adipogenic lineages. In summary, loss of Dock7 in the aging m/m resulted in an impairment of periosteal and endocortical envelope expansion, but did not alter age-related trabecular bone loss. These studies establish Dock7 as a critical regulator of both cortical and trabecular bone mass, and demonstrate for the first time a novel role of Dock7 in modulating compensatory changes in the periosteum with aging.

Keywords: Aging; Bone; Dock7; Misty; Osteoporosis; Periosteal.

Figure 1. m/m show severe age-related bone loss

Female m/m (open squares) and +/+ (closed circles) were weighted and screened using Lunar PIXImus Densitometer for body composition including (A) body mass, (B) fat-free mass, (C) fat mass, (D) total aBMD, (E) total aBMC, (F) femoral aBMD, and (G) femoral aBMC. Points represent mean ± SEM of n=13–15 per group. 2-way ANOVA: P <0.0001 for genotype in all measurements except fat mass (not significant). Post hoc: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to +/+.

Figure 2. +/+ and m/m have similar trabecular bone loss with age

(A) Representative μCT images of distal femur trabecular bone, and (B) femoral microarchitecture examined in +/+ and m/m from 16 to 78 wks of age. Trabecular microarchitecture was measured by μCT and femur length, trabecular bone volume/total volume (BV/TV), trabecular number (Tb.N.), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp.), connectivity density (Conn.D.), and structural modeling index (SMI) were calculated. Closed circles (+/+) and open squares (m/m) represent mean ± SEM of n=9–10 per group. 2-way ANOVA: P <0.0001 for genotype in all measurements except Tb.Th. and SMI (not significant). Post hoc: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to +/+.

Figure 3. +/+ and m/m have similar trabecular bone loss in vertebrae with age

(A) Representative images of L5 vertebrae, and (B) vertebral microarchitecture examined in +/+ and m/m from 16 to 78 wks of age. Trabecular microarchitecture was measured by μCT and bone volume/total volume (BV/TV), trabecular number (Tb.N.), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp.), connectivity density (Conn.D.), and structural modeling index (SMI) were calculated. Closed circles (+/+) and open squares (m/m) represent mean ± SEM of n=8–10 per group. 2-way ANOVA: P <0.0001 for genotype in all measurements except Tb.Th. (not significant). Post hoc: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to +/+.

Figure 4. m/m have decreased cortical expansion

(A) Cortical thickness, (B) periosteal (Ps) circumference, and (C) endosteal (Ec) circumference were measured by μCT. Closed circles (+/+) and open squares (m/m) represent mean ± SEM of n=9–10 per group. 2-way ANOVA: P <0.0001 for genotype in all measurements. Post hoc: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to +/+.

Figure 5. Serum markers of bone formation and bone resorption were unchanged in m/m

P1NP, CTx, and RANKL protein levels were measured by enzyme immunoassay in +/+ and m/m at 16 and 52 wks of age (n=10). 2-way ANOVA: All measures had non-significant P values for genotype. Post hoc: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to +/+.

Figure 6. m/m have extreme age-related increase in marrow adiposity

Femurs were isolated from 16, 32, 52, and 78 wks, decalcified, paraffin embedded, and stained with H & E. Images show accumulation of white marrow adipocyte ghosts in the distal femur epiphysis.

Figure 7. Decreased osteoblast gene expression persists in the aged m/m

Gene expression of whole tibia was analyzed by qRT-PCR for expression of (A) osteoblast genes, (B) osteoclast genes, and (C) inflammatory cytokines. n=8–10 per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to +/+.

Figure 8. Gene expression of senescence markers is modestly altered in m/m

Gene expression of p16 and p21 senescence markers was analyzed by qRT-PCR in (A) whole tibia of 16 and 52 wks old mice and (B) BMSCs from +/+ and m/m mice. Values represent mean ± SEM of n=6–10 per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to +/+.

Figure 9. BMSCs motility is impaired in BMSCs on a Collagen I matrix

(A) BMSC migration was assessed in a transwell assay on membranes coated with collagen I. (B) Adhesion of BMSCs was analyzed in collagen I coated wells. The number of cells on the membrane (A) or well (B) were quantified. Closed circles (+/+) and open squares (m/m) represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to +/+.

Figure 10. Adipogenic differentiation is increased in m/m

(A) Tyrosine hyroxylase (TH) positive sympathetic nerve fibers were quantified in histological BAT sections from 52 wks old +/+ and m/m. Representative images of staining are shown on right. Black arrows mark examples of TH positive nerve fibers. (B) UCP-1 protein expression was detected in histological BAT sections from 52 wks old +/+ and m/m with an anti-UCP-1 antibody. (C) Adipogenic differentiation was analyzed in eMSC by Oil Red O staining after 7 days in adipogenic media. Proliferation was measured in undifferentiated eMSCs with BrdU straining.