Excessive osteoclast activation by osteoblast paracrine factor RANKL is a major cause of the abnormal long bone phenotype in Apert syndrome model mice

Abstract

The fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling pathway plays important roles in the development and growth of the skeleton. Apert syndrome caused by gain-of-function mutations of FGFR2 results in aberrant phenotypes of the skull, midface, and limbs. Although short limbs are representative features in patients with Apert syndrome, the causative mechanism for this limb defect has not been elucidated. Here we quantitatively confirmed decreases in the bone length, bone mineral density, and bone thickness in the Apert syndrome model of gene knock-in Fgfr2^S252W/+ (EIIA-Fgfr2^S252W/+ ) mice. Interestingly, despite these bone defects, histological analysis showed that the endochondral ossification process in the mutant mice was similar to that in wild-type mice. Tartrate-resistant acid phosphatase staining revealed that trabecular bone loss in mutant mice was associated with excessive osteoclast activity despite accelerated osteogenic differentiation. We investigated the osteoblast-osteoclast interaction and found that the increase in osteoclast activity was due to an increase in the Rankl level of osteoblasts in mutant mice and not enhanced osteoclastogenesis driven by the activation of FGFR2 signaling in bone marrow-derived macrophages. Consistently, Col1a1-Fgfr2^S252W/+ mice, which had osteoblast-specific expression of Fgfr2 S252W, showed significant bone loss with a reduction of the bone length and excessive activity of osteoclasts was observed in the mutant mice. Taken together, the present study demonstrates that the imbalance in osteoblast and osteoclast coupling by abnormally increased Rankl expression in Fgfr2^S252W/+ mutant osteoblasts is a major causative mechanism for bone loss and short long bones in Fgfr2^S252W/+ mice.

Keywords: Apert syndrome; FGFR2; Rankl; long bone; osteoblast-osteoclast interaction.

Figure 1

EIIA‐Fgfr2 ^S252W/+  mice have decreases in long bone sizes and bone mineral density. (a) Physiognomy of EIIA‐Fgfr2 ^S252W/+ and littermate wild‐type (WT) mouse at postnatal Day 21 (P21). The mutant mouse had a smaller body size and short head in the superior view (left) and lateral view (right). (b) Micro‐computed tomography (CT) images of the tibial growth plate at P21 in the arterial view of a coronal section and cross‐sectional views are shown for each genotype (n ≥ 5, scale bar: 1 mm). (c) Representative micro‐CT images of cortical (top) and trabecular (bottom) bones in the proximal tibia (n ≥ 5, scale bar: 2 mm). (d–l) Histomorphometric analyses of three‐dimensional (3D) micro‐CT data. (d) Tibial length; (e) BV/TV, bone volume/tissue volume; (f) BMD, bone mineral density; (g) C. BV, cortical bone volume; (h) C. Th, cortical bone thickness; (i) Tb. Th, trabecular thickness; (j) Tb. N, trabecular number. (k) SOC BV, secondary ossification center bone volume. (l) Regression equation that described relationship between the degree of deviation of SOC BV and the tibia length in EIIA‐Fgfr2 ^S252W/+ mice. The R ² value is the coefficient of determination of the regression equation. *p < 0.05, **p < 0.01, ***p < 0.001. NS, not significant

Figure 2

EIIA‐Fgfr2 ^S252W/+  mice show normal cartilage development and growth plate structures in long bones. (a) Representative images of hematoxylin and eosin (H&E)‐stained proximal tibiae at postnatal Day 21 (P21) from wild‐type (WT) and EIIA‐Fgfr2 ^S252W/+ mice (n = 4, scale bar: 200 μm). (b) Graph of morphometric measurements of the PZ, HZ, and MP. A normal structure of the epiphyseal growth plate was observed in EIIA‐Fgfr2 ^S252W/+ mice. (c) Representative images of H&E‐stained proximal tibiae in newborn(P0), P3, and P7 WT (top) and EIIA‐Fgfr2 ^S252W/+ (bottom) mice (scale bar: 100 μm). (d–f) Representative images of immunohistochemistry (IHC) at P21 revealed no apparent differences in expression of type 10 collagen (COL10) (d), proliferating cell nuclear antigen (PCNA) (e), and SOX9 (f) between WT and EIIA‐Fgfr2 ^S252W/+ mice (n = 4). HZ, hypertrophic zone; MP, metaphysis; PZ, proliferating zone

Figure 3

Osteoclastogenesis is enhanced in the trabecular bone of EIIA‐Fgfr2 ^S252W/+  mice. (a) Representative images of tartrate‐resistant acid phosphatase (TRAP)‐stained trabecular bone and secondary ossification center (SOC) of proximal tibiae in postnatal Day 21 (P21) wild‐type (WT) and EIIA‐Fgfr2 ^S252W/+ mice. TRAP‐positive purple spots indicate multinucleated osteoclasts (n = 3, scale bar: 500 μm). (b) Quantification of TRAP‐positive cells in the trabecular bones of WT and EIIA‐Fgfr2 ^S252W/+ mice. Graph shows the number of osteoclasts per bone surface (N. Oc./BS). (c–e) Representative images of MMP9 (c), MMP13 (d), and nuclear factor‐κB (NF‐κB) (p65). (e) IHC of tibia trabecular bones in WT and EIIA‐Fgfr2 ^S252W/+ mice (n = 3, scale bar: 200 μm)

Figure 4

Abnormally enhanced osteoclast formation and activity in EIIA‐Fgfr2 ^S252W/+  mice are attributed to abnormal osteoblast differentiation. (a) Osteoclast identification by tartrate‐resistant acid phosphatase (TRAP) staining. Bone marrow‐derived macrophages (BMMs) isolated from wild‐type (WT) and EIIA‐Fgfr2 ^S252W/+ mice were differentiated into osteoclasts in the presence of CSF1 (20 ng/ml) and receptor activator of nuclear factor‐κB ligand (RANKL) (80 ng/ml) for 5 days (scale bar: 100 μm). (b) Number of TRAP‐positive osteoclasts with more than three nuclei (n = 5 in each group). (c, d) Messenger RNA (mRNA) levels of Fgfr1 and Fgfr2 measured by quantitative real‐time PCR (qPCR) analysis of WT BMMs isolated from the spleen (c) and bone marrow (d). BMMs from the spleen were cultured in osteoclast differentiation medium for each indicated day and BMMs from bone marrow were cultured for 6 days. Relative mRNA expression was normalized to Gapdh expression. (e) ERK phosphorylation of WT and EIIA‐Fgfr2S252W/+ mice BMMs from bone marrow. BMMs were differentiated into osteoclasts in osteoclast differentiation medium for 5 days. (f, g) TRAP staining of BMMs from WT and EIIA‐Fgfr2 ^S252W/+ mice cocultured with primary calvaria osteoblasts (OBs) from WT and EIIA‐Fgfr2 ^S252W/+ mice, respectively, for 7 days with vitamin D3 (10 nM) and prostaglandin E2 (PGE2) (1 µM) in osteogenic medium for osteoclast differentiation (scale bar: 100 μm). (h) Number of TRAP‐positive osteoclasts. (i–l) Primary calvarial OBs of WT and EIIA‐Fgfr2 ^S252W/+ were cocultured with WT BMMs and mRNA levels of osteoclast marker genes were determined by reverse‐transcription (RT) quantitative PCR (qPCR). Data are expressed as the mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001. NS, not significant

Figure 5

Imbalanced Rankl/Opg  expression due to enhanced Rankl  expression in osteoblasts increases osteoclast formation in EIIA‐Fgfr2 ^S252W/+  mice. (a) Alkaline phosphatase (ALP) and alizarin red staining were performed in wild‐type (WT) and EIIA‐Fgfr2 ^S252W/+ primary calvaria osteoblasts (OBs) treated with osteogenic medium for 5 days and 2 weeks. (b–d) Relative mRNA expression of osteoclast differentiation factors in WT and EIIA‐Fgfr2 ^S252W/+ primary calvaria OBs treated with or without (0 day) osteogenic medium for each indicated day as determined by quantitative PCR (qPCR). (e) Relative Rankl/Opg ratio. (f) mRNA level of Runx2 in WT and EIIA‐Fgfr2 ^S252W/+ primary calvaria OBs cultured in osteogenic medium. Cells were collected and analyzed by reverse‐transcription (RT)‐qPCR after treatment for 1 or 3 days with or without osteogenic medium. (g–i) Expression levels of osteoclast differentiation‐regulating genes in MC3T3‐E1 cells as measured by RT‐qPCR after transfection of Fgfr2 WT or S252W plasmids. (j) Graph of the relative Rankl/Opg ratio

Figure 6

Increased osteoblast‐mediated osteoclast activation and reduced bone formation in tibia trabecular bones of Col1a1‐Fgfr2 ^S252W/+  mice. (a) Representative micro‐computed tomography (CT) images of the tibial growth plate at postnatal Day 21 (P21) in midsagittal and coronal views are shown for each genotype. (n ≥ 8, scale bar: 1 mm). (b–e) Histomorphometric analyses of three‐dimensional (3D) micro‐CT data. (f) Tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts as determined by TRAP staining of trabecular bone and secondary ossification center (SOC) of proximal tibiae in P21 wild‐type (WT) and Col1a1‐Fgfr2 ^S252W/+ mice (n = 3, scale bars: top, 200 μm; bottom, 100 μm). (g) Graph of the number of osteoclasts per bone surface. Data are expressed as the mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001. (h) Mechanisms of reduced long bone growth in Fgfr2 ^S252W/+ mice