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. 2023 Aug 24;11(1):47.
doi: 10.1038/s41413-023-00278-5.

Inhibiting WNT secretion reduces high bone mass caused by Sost loss-of-function or gain-of-function mutations in Lrp5

Cassandra R Diegel  1 , Ina Kramer  2 , Charles Moes  2 , Gabrielle E Foxa  1 , Mitchell J McDonald  1 , Zachary B Madaj  3 , Sabine Guth  2 , Jun Liu  4 , Jennifer L Harris  4 , Michaela Kneissel  2 , Bart O Williams  5
Affiliations

Inhibiting WNT secretion reduces high bone mass caused by Sost loss-of-function or gain-of-function mutations in Lrp5

Cassandra R Diegel et al. Bone Res. .

Abstract

Proper regulation of Wnt signaling is critical for normal bone development and homeostasis. Mutations in several Wnt signaling components, which increase the activity of the pathway in the skeleton, cause high bone mass in human subjects and mouse models. Increased bone mass is often accompanied by severe headaches from increased intracranial pressure, which can lead to fatality and loss of vision or hearing due to the entrapment of cranial nerves. In addition, progressive forehead bossing and mandibular overgrowth occur in almost all subjects. Treatments that would provide symptomatic relief in these subjects are limited. Porcupine-mediated palmitoylation is necessary for Wnt secretion and binding to the frizzled receptor. Chemical inhibition of porcupine is a highly selective method of Wnt signaling inhibition. We treated three different mouse models of high bone mass caused by aberrant Wnt signaling, including homozygosity for loss-of-function in Sost, which models sclerosteosis, and two strains of mice carrying different point mutations in Lrp5 (equivalent to human G171V and A214V), at 3 months of age with porcupine inhibitors for 5-6 weeks. Treatment significantly reduced both trabecular and cortical bone mass in all three models. This demonstrates that porcupine inhibition is potentially therapeutic for symptomatic relief in subjects who suffer from these disorders and further establishes that the continued production of Wnts is necessary for sustaining high bone mass in these models.

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Conflict of interest statement

B.O.W. is a stockholder and member of the Scientific Advisory Board for Surrozen. The Williams Laboratory has also received recent support from Janssen Pharmaceuticals for work unrelated to the studies reported here. C.M., J.L., I.K., M.K. and S.G. are employees and shareholders of Novartis.

Figures

Fig. 1
Fig. 1
Sost treatment schematic and in vivo femoral and tibial pQCT measurements. a Schematic diagram of the experimental design. Longitudinal pQCT measurements of distal femur and proximal tibia metaphysis in 3-month-old female wild-type (WT) and Sost KO mice treated with vehicle or GNF-6231 daily for 40 days. The parameters measured include (b, c) total cross-sectional BMC (d, e) total cross-sectional BMD (f, g) cancellous BMD, and (h, i) cross-sectional cortical thickness. For all graphs, the means of each group are indicated by the shape, upper and lower lines represent standard deviations, and *P < 0.05. A minimum of six animals per genotype per condition were analyzed (see Table 1)
Fig. 2
Fig. 2
Cancellous and cortical bone analysis of GNF-6231-treated Sost KO mice via µCT. Representative images of cancellous bone (a) in the distal femur and cortical bone (g) in the midshaft of the femur, demonstrating the effects of GNF-6231 treatment on 3-month-old wild-type (WT) and Sost KO mice. The trabecular parameters measured included (b) bone mineral density (BMD), c bone volume/tissue volume (BV/TV), d trabecular thickness (Tb.Th), e trabecular separation (Tb.Sp), and f trabecular number (Tb.N). The cortical parameters measured included (h) tissue mineral density (TMD), i cortical area fraction (CAF), and j cross-sectional thickness (Ct.Th). For all graphs, the means of each group are indicated by the middle line, and the upper and lower lines represent standard deviations. *P < 0.05. A minimum of five animals per genotype per condition were analyzed (Table 1)
Fig. 3
Fig. 3
Treatment schematic and whole-body areal bone mineral density measured by DXA. a Schematic diagram of the experimental design. b Longitudinal whole-body areal bone mineral density (aBMD) measured by DXA of 3-month-old Lrp5A214V littermate wild-type (WT) and A/+ females and males treated with vehicle or LGK974 daily for 5 weeks. c Longitudinal whole-body areal bone mineral density (aBMD), measured by DXA, of Lrp5G171V littermate wild-type and G/+ females and males treated with vehicle or LGK974 daily for 5 weeks. For all graphs, the means of each group are indicated by the shape. The upper and lower lines represent standard deviations, and *P < 0.05. At least five animals per sex, genotype, and condition were analyzed (see Table 2)
Fig. 4
Fig. 4
Cancellous bone analysis of LGK974-treated mice via microCT. a Representative images of trabecular bone in distal femurs, demonstrating the effects of LGK974 treatment on 3-month-old WT and Lrp5 mutant mice. The parameters measured included (b) bone mineral density (BMD), c bone volume/tissue volume, d trabecular thickness (Tb.Th), e trabecular separation (Tb.Sp), and f trabecular number (Tb.N). For all graphs, the means of each group are indicated by the middle line. These upper and lower lines represent standard deviations, and *P < 0.05. A minimum of five animals per sex, genotype, and condition were analyzed (Table 2)
Fig. 5
Fig. 5
Cortical bone analysis of LGK974-treated mice via µCT. a Representative images of cortical bone in the midshaft of femurs, demonstrating the effects of LGK974 treatment on 3-month-old WT and Lrp5 mutant mice. Trabecular bone parameters of treated Lrp5A214V mice, Lrp5G171V mice, and their respective controls were measured. These parameters included (b) tissue mineral density (TMD), c cortical area fraction (CAF), and d cross-sectional thickness (Ct.Th). For all graphs, the means of each group are indicated by the middle line, and the upper and lower lines represent standard deviations. *P < 0.05. At least five animals per sex, genotype, and condition were analyzed (Table 2)
Fig. 6
Fig. 6
Dynamic histomorphometry of femoral cortical bone. a Representative images of cortical cross-sections from fluorochrome-labeled femurs. b The endocortical mineral apposition rate (Ec.MAR), c endocortical bone formation rate (Ec.BFR), d periosteal mineral apposition rate (Ps.MAR), and e periosteal bone formation rate (Ps.BFR). For all graphs, the means of each group are indicated by the middle line, and the upper and lower lines represent standard deviations. *P < 0.05. At least three animals per sex, genotype, and condition were analyzed (Table 2)
Fig. 7
Fig. 7
Static histomorphometry of femoral trabecular bone. a Representative images of Goldner’s trichrome-stained trabecular bone of the distal femurs of mice. b The bone volume/tissue volume (BV/TV), c adipocyte number/tissue volume (Ad. N/TV), d osteoid volume/bone volume (OV/BV), e osteoid surface/bone surface (OS/BS), and f number of osteoblasts/bone surface (N.Ob/BS). For all graphs, the means of each group are indicated by the middle line, and the upper and lower lines represent standard deviations. *P < 0.05. At least three animals per sex, genotype, and condition were analyzed (Table 2)
Fig. 8
Fig. 8
Osteoclast quantification in distal femurs of mice. a Representative images of TRAP-stained femoral sagittal sections, b osteoclast surface/bone surface (Oc.S/BS), and c number of osteoclasts/bone surface (N.Oc/BS). For all graphs, the means of each group are indicated by the middle line, and the upper and lower lines represent standard deviations. *P < 0.05. A minimum of two animals per genotype and per condition were analyzed
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