Osteoblast-Specific Overexpression of Human WNT16 Increases Both Cortical and Trabecular Bone Mass and Structure in Mice

Abstract

Previous genome-wide association studies have identified common variants in genes associated with bone mineral density (BMD) and risk of fracture. Recently, we identified single nucleotide polymorphisms (SNPs) in Wingless-type mouse mammary tumor virus integration site (WNT)16 that were associated with peak BMD in premenopausal women. To further identify the role of Wnt16 in bone mass regulation, we created transgenic (TG) mice overexpressing human WNT16 in osteoblasts. We compared bone phenotypes, serum biochemistry, gene expression, and dynamic bone histomorphometry between TG and wild-type (WT) mice. Compared with WT mice, WNT16-TG mice exhibited significantly higher whole-body areal BMD and bone mineral content (BMC) at 6 and 12 weeks of age in both male and female. Microcomputer tomography analysis of trabecular bone at distal femur revealed 3-fold (male) and 14-fold (female) higher bone volume/tissue volume (BV/TV), and significantly higher trabecular number and trabecular thickness but lower trabecular separation in TG mice compared with WT littermates in both sexes. The cortical bone at femur midshaft also displayed significantly greater bone area/total area and cortical thickness in the TG mice in both sexes. Serum biochemistry analysis showed that male TG mice had higher serum alkaline phosphatase, osteocalcin, osteoprotegerin (OPG), OPG to receptor activator of NF-kB ligand (tumor necrosis family ligand superfamily, number 11; RANKL) ratio as compared with WT mice. Also, lower carboxy-terminal collagen cross-link (CTX) to tartrate-resistant acid phosphatase 5, isoform b (TRAPc5b) ratio was observed in TG mice compared with WT littermates in both male and female. Histomorphometry data demonstrated that both male and female TG mice had significantly higher cortical and trabecular mineralizing surface/bone surface and bone formation rate compared with sex-matched WT mice. Gene expression analysis demonstrated higher expression of Alp, OC, Opg, and Opg to Rankl ratio in bone tissue in the TG mice compared with WT littermates. Our data indicate that WNT16 is critical for positive regulation of both cortical and trabecular bone mass and structure and that this molecule might be targeted for therapeutic interventions to treat osteoporosis.

Genetics of pediatric bone strength

“…46 In knockout mice missing Wnt16, the periosteal bone formation rate as well as the mineral apposition rate were reduced, 47 and mice conditionally overexpressing human WNT16 in osteoblasts showed increased cortical and trabecular bone mass. 48 Similarly, loss of function mutations in LRP5, which encodes a transmembrane WNT co-receptor that synergistically binds WNT along with the frizzled receptor, disrupt normal signaling and lead to low-bone mass (osteoporosis-pseudoglioma syndrome), whereas gain of function mutations result in high bone mass. In addition, other members of the LRP family are involved in the maintenance of bone, 49 and a variant B84 kb upstream of LRP3 has been associated with pediatric bone mass during the mid-to-late stages of puberty.…”

Enhanced Identification of Potential Pleiotropic Genetic Variants for Bone Mineral Density and Breast Cancer

“…CPED1 locates adjacent to WNT16, which is a very important gene for bone metabolism. WNT16 positively regulates bone mass and structure [42], and it’s also functional in the pathology of cancers including BC. Human mammary epithelial cells express WNT16, in most BC cell lines the expression of WNT16 is usually down-regulated [43].…”

Bone Mass and Strength are Significantly Improved in Mice Overexpressing Human WNT16 in Osteocytes

“…Measurements of gene expression were performed by real-time PCR using bone and other organs from both male and female WT and Dmp1-hWNT16 TG mice as described previously (14). All qPCR reactions were performed using the custom-made primer and probe sets from Integrated DNA Technology (IDT, USA) for human WNT16 ( hWNT16 ), endogenous mouse Wnt16 (Wnt16) , runt related transcription factor 2 ( Runx2 ), alkaline phosphatase ( Alp ), osteocalcin ( OC ), osteoprotegerin ( Opg ) and tumor necrosis factor (ligand) superfamily, member 11 ( Rankl or Tnfs11 ).…”

“…The whole body, femur and lumbar vertebrae 1 through 5 of the WT and Dmp1-hWNT16 TG mice were scanned using DXA (PIXImus II mouse densitometer; Lunar Corp., Madison, WI, USA) with ultra-high resolution (0.18 × 0.18 mm/pixel) as described previously (14). After completion of the scan of each bone, mutually exclusive region of interest (ROI) boxes were drawn around the bone from which femur aBMD (g/cm 2 ) and BMC (g) measurements were obtained.…”

“…To further identify the role of WNT16 in bone biology, recently, we created transgenic mice overexpressing human WNT16 in osteoblast, and demonstrated that the transgenic mice displayed increased cortical and trabecular bone mass and structure in both growing and adult age, with robust trabecular phenotype particularly in female mice (14). Further, using transgenic mice overexpressing mouse Wnt16 in osteoblasts, Movérare-Skrtic S. et al, showed that the transgene had a robust effect on trabecular bone phenotype in adult female mice (15).…”