Secreted Frizzled-Related Protein 4 Is a Negative Regulator of Peak BMD in SAMP6 Mice

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Abstract
We segregated a QTL for peak BMD on Chr 13 by generating congenic sublines of the senescence‐accelerated mouse SAMP6. Sfrp 4 within this locus was responsible for lower BMD of SAMP6. Introduction: Our genome‐wide linkage study using SAMP6 and SAMP2 showed a significant quantitative trait locus (QTL) for peak BMD on chromosome (Chr) 13. To verify the gene that regulates peak BMD, we generated a congenic strain, P6.P2‐Pbd2b, which carried a 15‐cM SAMP2 interval on an osteoporotic SAMP6 background, and showed that this Pbd2 locus increased peak BMD in SAMP6. Materials and Methods: To narrow down this interval, we generated a new congenic subline P6.P2–13. We studied the effect of this locus on morphological and histomorphological features in vivo and on osteoblasts in vitro. The levels of expression of all genes in the segregated interval were examined, and we clarified the effect of the candidate gene, secreted frizzled‐related protein (Sfrp4), on osteoblasts in vitro. Results: The new congenic strain, P6.P2–13, retained the 2.4‐Mb SAMP2 interval on the SAMP6 background, and 11 genes existed in this interval. In morphometrical analysis, P6.P2–13 increased the bone area fraction (BA/TA) by 6.6% at the diaphysial cortex (p < 0.001) and increased the trabecular bone volume (BV/TV) by 54.2% at the distal metaphysis (p < 0.05) in the femora compared with those of SAMP6. The bone formation rate of P6.P2–13 was markedly increased at the periosteal surface of femoral cortex and that was caused by a higher proliferation rate of osteoblasts in P6.P2–13 compared with those in SAMP6. Quantitative RT‐PCR analysis of calvaria tissue showed ∼40‐fold higher levels of expression of Sfrp4 in SAMP6 than in P6.P2–13. Taken together with the result that recombinant Sfrp4 suppressed the proliferation of osteoblasts, we hypothesized that Sfrp4 inhibited the proliferation of osteoblasts through its antagonistic effect on Wnt signaling. TCF/β‐catenin–dependent reporter activity in osteoblasts derived from SAMP6 showed lower responsiveness for the Wnt ligand, Wnt3A, than that in osteoblasts from P6.P2–13. Conclusions: In SAMP6 mice, Sfrp4 negatively regulates bone formation and decreases BMD through the inhibition of Wnt signaling.