Regulation of osteoblastogenesis and bone mass by Wnt10b

Abstract

Wnts comprise a family of secreted signaling proteins that regulate diverse developmental processes. Activation of Wnt signaling by Wnt10b inhibits differentiation of preadipocytes and blocks adipose tissue development; however, the effect of Wnt10b on other mesenchymal lineages has not been defined. To explore the physiological role of Wnt signaling in bone development, we analyzed FABP4-Wnt10b mice, which express the Wnt10b transgene in marrow. Femurs from FABP4-Wnt10b mice have almost four times as much bone in the distal metaphyses and are mechanically stronger. These mice maintain elevated bone mass at least through 23 months of age. In addition, FABP4-Wnt10b mice are protected from the bone loss characteristic of estrogen deficiency. We used pharmacological and genetic approaches to demonstrate that canonical Wnt signaling stimulates osteoblastogenesis and inhibits adipogenesis of bipotential mesenchymal precursors. Wnt10b shifts cell fate toward the osteoblast lineage by induction of the osteoblastogenic transcription factors Runx2, Dlx5, and osterix and suppression of the adipogenic transcription factors C/EBPalpha and PPARgamma. One mechanism whereby Wnt10b promotes osteoblastogenesis is suppression of PPARgamma expression. Finally, Wnt10b-/- mice have decreased trabecular bone and serum osteocalcin, confirming that Wnt10b is an endogenous regulator of bone formation.

Fig. 1.

Increased bone mass and strength in FABP4-Wnt10b mice. (A) μCT of femurs from wild-type and FABP4-Wnt10b mice (Upper). Three-dimensional reconstructions of metaphyseal trabeculae from regions highlighted (Lower). Morphometric properties of 1 mm³ of distal femur from wild-type (▪) and FABP4-Wnt10b () mice at 6 months of age were analyzed for bone volume fraction (B; BV/TV, %), bone mineral density (C; BMD), trabecular number (D; Tr. N.), trabecular thickness (E; Tb. Th.), trabecular spacing (F; Tb. Sp.), ultimate load (G), stiffness (H), and yield load (I). Statistical significance for each measurement was evaluated with Student's t test: P < 0.05 (*), < 0.01 (#), < 0.001 (†), with n = 6 males for each genotype.

Fig. 2.

Wnt10b expression maintains bone in ovarectomized mice. (A) μCT of femurs from 4-month wild-type and FABP4-Wnt10b mice were either sham-operated or ovariectomized at 3 months of age. Morphometric variables analyzed include bone volume/total volume (B; BV/TV), trabecular number (C; Tb. N.), trabecular thickness (D; Tb. Th.), trabecular spacing (E; Tb.Sp.), and uterine weight (F). Statistical significance for each measurement was evaluated with Student's t test: P < 0.05 (*), < 0.01 (#), with n = 6–8 mice for each treatment.

Fig. 3.

Wnt10b promotes osteoblastogenesis of pluripotent mesenchymal precursors. ST2 cells were infected with control or Wnt10b retroviruses. Two days postconfluence, RNA was isolated for quantitative PCR analysis of osteoblast genes: alkaline phosphatase (Alk. Phos.); Twist, Runx2, Msx2, Dlx5, osterix (Osx); and type I collagen (Coll I) (A) and adipocyte genes: C/EBPβ, C/EBPα, PPARγ, and FABP4 (B). Statistical differences from controls were evaluated with Student's t test: P < 0.05 (*), < 0.01 (#), < 0.001 (†), with n = 3 mice. Results are representative of at least four independent experiments. (C) Control and Wnt10b cells were infected with control and PPARγ retroviruses. Confluent cells were incubated in osteogenic media supplemented with troglitazone (5 μM). Mineral deposition was assessed on day 6 with von Kossa stain.

Fig. 4.

Reduced trabecular bone in femurs of Wnt10b-/- mice. (A) μCT of femurs from wild-type and Wnt10b-/- mice (Upper). Three-dimensional reconstructions of metaphyseal trabeculae from regions highlighted (Lower). Morphometric properties of distal femur (1 mm³) from wild-type (▪) and Wnt10b null () mice. Properties analyzed include bone volume fraction (B;BV/TV, %), bone mineral density (C; BMD), trabecular number (D; Tr. N.), trabecular thickness (E; Tb. Th.), and trabecular spacing (F; Tb. Sp.). Serum levels of osteocalcin (G) and TRAP5b activity (H) in wild-type (▪) and Wnt10b null () mice. Statistical significance for each measurement was evaluated with Student's t test: P < 0.05 (*), < 0.01 (#), and <0.001 (†), with n = 6 mice for each genotype.