A mouse model for human osteogenesis imperfecta type VI

Abstract

Osteogenesis imperfecta type VI (OI type VI) has recently be linked to a mutation in the SERPINF1 gene, which encodes pigment epithelium-derived factor (PEDF), a ubiquitously expressed protein originally described for its neurotrophic and antiangiogenic properties. In this study, we characterized the skeletal phenotype of a mouse with targeted disruption of Pedf. In normal mouse bone, Pedf was localized to osteoblasts and osteocytes. Micro-computed tomography (µCT) and quantitative bone histomorphometry in femurs of mature Pedf null mutants revealed reduced trabecular bone volume and the accumulation of unmineralized bone matrix. Fourier transform infrared microscopy (FTIR) indicated an increased mineral:matrix ratio in mutant bones, which were more brittle than controls. In vitro, osteoblasts from Pedf null mice exhibited enhanced mineral deposition as assessed by Alizarin Red staining and an increased mineral:matrix determined by FTIR analysis of calcified nodules. The findings in this mouse model mimic the principal structural and biochemical features of bone observed in humans with OI type VI and consequently provide a useful model with which to further investigate the role of PEDF in this bone disorder.

Figure 1. PEDF expression and loss of function in vivo

(A) Immunohistochemical staining of PEDF in osteoblasts (arrows) in trabecular bone from a wild type mouse. (B) CD31 staining in bone from wild type (a) and Pedf^-/- mice (b). (C-D) Quantitative micro-CT analysis of the distal femur in wild-type (WT) and Pedf^-/- mice at 12-weeks of age (n=4–5mice). (C) Representative images. (D) Bone volume/tissue volume, BV/TV (%). (E-I) Histomorphometric analysis of the distal femoral metaphysis in male wild-type and Pedf^-/- mice at 6-weeks of age (n=5–7mice). (E) Osteoblast Number per bone perimeter, Ob. N/BPM (no./100mm) at 6 weeks of age. (F) Osteoclast Number per bone perimeter at 6 weeks of age. (G) Bone formation rate per bone surface, BFR/BS, mm³/cm²/yr. (H) Osteoid thickness (uM). (I) Osteoid maturation time (days). (J) FTIR measures of mineral:matrix ratios in cortical and trabecular bone. (K-M) Three-point bending analysis of femurs in wild-type and PEDF^-/- mice (n = 5–8 mice). (K) Ultimate force (N). (L) Ultimate displacement (mm). (M) Energy to Failure (mJ). All error bars represent SEM. *p < 0.05

Figure 2. PEDF expression and loss of function in primary cultultured osteoblasts

(A) Relative expression levels of mRNA in samples collected in freshly plated osteoblasts (day 0) and at 7 and 14 days of culture in differentiation medium. (B) Western blot of Pedf protein levels in osteoblasts from wild type mice at the indicated times in culture. Actin used as loading control. (C) Examination of osteoblast differentiation in wild-type and Pedf^-/- osteoblasts. Alkaline phosphatase (ALP) and Alizarin Red (ARS) staining after 7 and 14 days of differentiation, respectively. (D) Mineral:matrix ratio of cultured osteoblasts. (E) RT-PCR analysis of Ank, NPP1, Annexins 2, 5 and 6 expression performed at 14 days of differentiation. (F) RT-PCR analysis of Dmp1, Phex and Bsp expression at day 14. All error bars represent SEM. *p < 0.05