Inducible Wnt16 inactivation: WNT16 regulates cortical bone thickness in adult mice

Abstract

Substantial progress has been made in the therapeutic reduction of vertebral fracture risk in patients with osteoporosis, but non-vertebral fracture risk has been improved only marginally. Human genetic studies demonstrate that the WNT16 locus is a major determinant of cortical bone thickness and non-vertebral fracture risk and mouse models with life-long Wnt16 inactivation revealed that WNT16 is a key regulator of cortical thickness. These studies, however, could not exclude that the effect of Wnt16 inactivation on cortical thickness might be caused by early developmental and/or growth effects. To determine the effect of WNT16 specifically on adult cortical bone homeostasis, Wnt16 was conditionally ablated in young adult and old mice through tamoxifen-inducible Cre-mediated recombination using CAG-Cre-ER; Wnt16^flox/flox (Cre-Wnt16^flox/flox) mice. First, 10-week-old Cre-Wnt16^flox/flox and Wnt16^flox/flox littermate control mice were treated with tamoxifen. Four weeks later, Wnt16 mRNA levels in cortical bone were reduced and cortical thickness in femur was decreased in Cre-Wnt16^flox/flox mice compared to Wnt16^flox/flox mice. Then, inactivation of Wnt16 in 47-week-old mice (evaluated four weeks later) resulted in a reduction of Wnt16 mRNA levels, cortical thickness and cortical bone strength with no effect on trabecular bone volume fraction. Mechanistic studies demonstrated that the reduced cortical bone thickness was caused by a combination of increased bone resorption and reduced periosteal bone formation. In conclusion, WNT16 is a crucial regulator of cortical bone thickness in young adult and old mice. We propose that new treatment strategies targeting the adult regulation of WNT16 might be useful to reduce fracture risk at cortical bone sites.

Keywords: WNT16; cortical thickness; tamoxifen; transgenic.

Figure 1

Inducible inactivation of Wnt16 in young adult male mice reduces cortical bone thickness. Fourteen-week-old male Cre-Wnt16^flox/flox (n = 7) and littermate Wnt16^flox/flox (n = 6) control mice treated for four consecutive days with either low-dose (0.25 mg/mouse/day) or high-dose (1 mg/mouse/day) tamoxifen at the age of ten weeks. (A) Expression level of Wnt16 mRNA in cortical bone of femur, gonadal fat and liver. (B) Cortical bone thickness of femur as analyzed using μCT. ND, not detectable. Values are given as mean ± s.e.m. (Wnt16^flox/flox low-dose, n = 6, high-dose n = 7; Cre-Wnt16^flox/flox low and high dose, n = 7). *P < 0.05, **P < 0.01, Student’s t test, Cre-Wnt16^flox/flox vs Wnt16^flox/flox control mice. (C) Correlation between Wnt16 mRNA levels in cortical bone of femur and cortical bone thickness in the femur diaphysis. Pearson’s correlation coefficient is given.

Figure 2

Inducible inactivation of Wnt16 in older female mice reduces cortical bone thickness. 51-week-old female Cre-Wnt16^flox/flox and littermate Wnt16^flox/flox control mice treated with tamoxifen (1 mg/mouse/day) for four consecutive days at 47 weeks of age. (A) Wnt16 mRNA levels in cortical diaphyseal bone of femur. (B) Cortical thickness of the mid-diaphysis of femur as analyzed using µCT and (C) maximal (max.) load at failure determined by three-point bending of humerus. (D) mRNA levels of CatK, Opg and Rankl in cortical diaphyseal bone of femur. Values are given as mean ± s.e.m. (Wnt16^flox/flox n = 11; Cre-Wnt16^flox/flox n = 9). **P < 0.01, Student’s t test, Cre-Wnt16^flox/flox vs Wnt16^flox/flox control mice.