Figure 3
From: A FKBP5 mutation is associated with Paget’s disease of bone and enhances osteoclastogenesis
The FKBP51V55L mutation promotes osteoclastogenesis and enhances osteoclast bone resorption activity. (a) Genotyping strategy for the generation of FKBP51V55L KI mice. The upper panel shows the structure of the murine FKBP5 gene; the location of codon 55 in exon 3 is indicated. The second panel shows the structure of the initial targeted FKBP5 locus retaining the neo minigene. The primers used to identify the priming sites (3′ PCR_F, 3′ PCR_R) used for Southern blot analysis of ES cells and the AflII and HindIII restriction sites are shown. The bottom panel shows the structure of the final targeted FKBP5 locus after Cre recombination; the locations of the V55L codon substitution in exon 3 and the single remaining LoxP site are shown. The primers used to confirm excision of the neo minigene are also shown (F1, R1). (b) TRAP staining of osteoclasts. BMMs from FKBP51V55L mutant mice and wild-type mice were cultured with varying concentrations of RANKL as indicated and M-CSF (50 ng ml−1) for 3 days. The cells were then stained by TRAP staining, and photomicrographs were obtained using a Nikon Eclipse Ti inverted microscope. Original magnification, × 100. (c) Toluidine blue staining of resorption pits. The photomicrographs were obtained using a Nikon Eclipse Ti inverted microscope. Original magnification, × 100. (d) Numbers of osteoclasts derived from FKBP51V55L mutant and wild-type mice in vitro. When the concentration of RANKL was low (30 ng ml−1), the number of osteoclasts derived from wild-type and FKBP51V55L mutant BMMs did not significantly differ. When the concentration of RANKL was increased (70 ng ml−1, 150 ng ml−1), the number of osteoclasts derived from both wild-type and FKBP51V55L mutant BMMs increased, and the number of osteoclasts derived from FKBP51V55L mutant BMMs was significantly greater than the number obtained from wild-type BMMs. The data shown are representative of three independent experiments. *P<0.05. (e) The areas of resorption pits were calculated using Image Pro Plus. The results show that the mean area of the resorption pits in each field is larger in the mutant group than in the wild-type group. The data shown are representative of three independent experiments. **P<0.01. BMM, bone marrow-derived monocyte/macrophage cells; TRAP, tartrate-resistant acid phosphatase.
