Overexpression of secreted frizzled-related protein 1 inhibits bone formation and attenuates parathyroid hormone bone anabolic effects

Abstract

Secreted frizzled-related protein 1 (sFRP1) is an antagonist of Wnt signaling, an important pathway in maintaining bone homeostasis. In this study we evaluated the skeletal phenotype of mice overexpressing sFRP1 (sFRP1 Tg) and the interaction of parathyroid hormone (PTH) treatment and sFRP1 (over)expression. Bone mass and microarchitecture were measured by micro-computed tomography (microCT). Osteoblastic and osteoclastic cell maturation and function were assessed in primary bone marrow cell cultures. Bone turnover was assessed by biochemical markers and dynamic bone histomorphometry. Real-time PCR was used to monitor the expression of several genes that regulate osteoblast maturation and function in whole bone. We found that trabecular bone mass measurements in distal femurs and lumbar vertebral bodies were 22% and 51% lower in female and 9% and 33% lower in male sFRP1 Tg mice, respectively, compared with wild-type (WT) controls at 3 months of age. Genes associated with osteoblast maturation and function, serum bone formation markers, and surface based bone formation were significantly decreased in sFRP1 Tg mice of both sexes. Bone resorption was similar between sFRP1 Tg and WT females and was higher in sFRP1 Tg male mice. Treatment with hPTH(1-34) (40 microg/kg/d) for 2 weeks increased trabecular bone volume in WT mice (females: +30% to 50%; males: +35% to 150%) compared with sFRP1 Tg mice (females: +5%; males: +18% to 54%). Percentage increases in bone formation also were lower in PTH-treated sFRP1 Tg mice compared with PTH-treated WT mice. In conclusion, overexpression of sFRP1 inhibited bone formation as well as attenuated PTH anabolic action on bone. The gender differences in the bone phenotype of the sFRP1 Tg animal warrants further investigation.

Fig. 1

(A) Representative genotyping for sFRP1. The sFRP1 transgenic mice (Tg) PCR product gives two bands at 1 kb (Actin) and 300 bp (EGFP), whereas wild-type (WT) mice have one band at 1 kb. (B) Proteins were extracted and analyzed by immunoblotting with antibodies against sFRP1 in selected tissues. GAPDH was used as a control. (C) Relative quantitative measurement of sFRP1 levels in selected tissues from both WT and sFRP1 Tg female and male mice. ^ap < .05 from the WT mice.

Fig. 2

Representative µCT images of the distal femoral trabecular bone from WT and sFRP1 Tg mice at the age of 14 weeks. Scale bars: 1.0 mm.

Fig. 3

Bone stromal cell culture from 14-week-old WT or sFRP1 Tg mice. (A) Cell proliferation in WT or sFRP1 Tg mice. Bone marrow cells derived from either WT or sFRP1 Tg mice were cultured on a 96-well plate for 4 days, and then the cells were incubated with BrdU for 2 hours. Absorbance at 370 to 392 nm was measured with a microplate reader. (B–D) Bone marrow cells were extracted and cultured with ascorbic acid and β-glycerophosphate for 14 days. After ALP levels were obtained (B), the cells were stained with ALP staining to monitor ALP-positive colony formation (C). Alizarin red staining (AR) was performed at day 21 to monitor mineralization nodule formation (D).

Fig. 4

Expression of several genes that regulate osteoblast maturation and function in whole bone from 14-week-old WT or sFRP1 Tg mice. (A) RNA was extracted from bone and bone marrow of the tibiae and real-time PCR was performed to monitor gene expression that was associated with osteoblast differentiation and maturation (Runx2, Osterix, Osteocalcin, RANKL, and OPG) or Wnt signaling (Lrp5 and Lrp6). (B) Protein was extracted from bone and bone marrow from WT or sFRP1 Tg mice and subjected to Western blotting of the active dephosphorylated β-catenin. ^ap < .05 from the WT.

Fig. 5

Expression of several genes that regulate osteoblast maturation and function in whole bone from 12-week-old WT or sFRP1 Tg mice treated with PTH for 2 weeks. (A) RNA was extracted from bone and bone marrow from the tibiae of WT or sFRP1 Tg mice treated with PTH. Real-time PCR was performed to monitor gene expression that was associated with osteoblast differentiation and maturation (Runx2, Osterix, Osteocalcin, RANKL, and OPG) or Wnt signaling (Lrp5 and Lrp6). (B) Protein was extract from bone and bone marrow of WT or sFRP1 Tg mice treated with PTH and subjected to Western blotting of the active dephosphorylated β-catenin. ^bp < 0.05 from vehicle-treated WT or sFRP1 Tg mice.