Vitamin D receptor in chondrocytes promotes osteoclastogenesis and regulates FGF23 production in osteoblasts

Abstract

Genomic actions induced by 1alpha25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] are crucial for normal bone metabolism, mainly because they regulate active intestinal calcium transport. To evaluate whether the vitamin D receptor (VDR) has a specific role in growth-plate development and endochondral bone formation, we investigated mice with conditional inactivation of VDR in chondrocytes. Growth-plate chondrocyte development was not affected by the lack of VDR. Yet vascular invasion was impaired, and osteoclast number was reduced in juvenile mice, resulting in increased trabecular bone mass. In vitro experiments confirmed that VDR signaling in chondrocytes directly regulated osteoclastogenesis by inducing receptor activator of NF-kappaB ligand (RANKL) expression. Remarkably, mineral homeostasis was also affected in chondrocyte-specific VDR-null mice, as serum phosphate and 1,25(OH)(2)D levels were increased in young mice, in whom growth-plate activity is important. Both in vivo and in vitro analysis indicated that VDR inactivation in chondrocytes reduced the expression of FGF23 by osteoblasts and consequently led to increased renal expression of 1alpha-hydroxylase and of sodium phosphate cotransporter type IIa. Taken together, our findings provide evidence that VDR signaling in chondrocytes is required for timely osteoclast formation during bone development and for the endocrine action of bone in phosphate homeostasis.

Figure 1. Inactivation of the VDR gene in chondrocytes.

(A) Schematic representation of targeting construct pNT vector/VDR, the VDR^WT allele, and the VDR^fl allele after Cre excision of the floxed neo cassette and probe used for identifying correct Cre excision of floxed exon 2. Restriction sites are indicated. Ex, exon. (B) Southern blot of SacI-digested genomic DNA from Cre^–VDR^fl/fl and Cre⁺VDR^fl/fl mice using internal probe. (C) VDR gene expression in growth-plate chondrocytes of Cre^–VDR^fl/fl (Cre^–) and Cre⁺VDR^fl/fl (Cre⁺) mice (n = 6) was assessed by qRT-PCR analysis and calculated as a ratio to the HPRT mRNA copies. Cre^–VDR^fl/fl value was set at 100%. **P < 0.01 versus Cre^–VDR^fl/fl. (D) qRT-PCR analysis of CYP24 mRNA levels in primary chondrocyte culture stimulated with vehicle (veh) or 1,25(OH)₂D₃ (10^–10 M and 10^–8 M) for 48 hours and 72 hours. Values are corrected for HPRT mRNA copies and are shown as means ± SEM in log scale. **P < 0.01 versus vehicle.

Figure 2. Normal growth-plate development in chondrocyte-specific VDR-null mice.

(A–D) Toluidine blue staining of the proximal tibiae from 15-day-old Cre^–VDR^fl/fl (A and B) and Cre⁺VDR^fl/fl mice (C and D). Scale bar: 200 μm. (E) Quantification of the length of the hypertrophic cartilage zone in the proximal tibiae. (F–H) Gene expression of Col2 and Col10 in neonatal (F), 15-day-old (G), and 8-week-old (H) femora from Cre^–VDR^fl/fl and Cre⁺VDR^fl/fl mice, assessed by qRT-PCR and calculated as a ratio to HPRT mRNA copies. *P < 0.05 versus Cre^–VDR^fl/fl.

Figure 3. Chondrocyte-specific VDR inactivation results in increased metaphyseal bone volume in neonatal and 15-day-old mice.

(A) Quantification of BV/TV in the proximal tibiae metaphysis on von Kossa–stained sections shows a significant increase in BV/TV in Cre⁺VDR^fl/fl mice compared with Cre^–VDR^fl/fl mice. (B and C) von Kossa staining on tibiae sections of 15-day-old Cre^–VDR^fl/fl (B) and Cre⁺VDR^fl/fl mice (C). Scale bar: 400 μm. (D) Osteocalcin gene expression in femora (total bone or dissected diaphysis) of 15-day-old Cre^–VDR^fl/fl and Cre⁺VDR^fl/fl mice was assessed by qRT-PCR analysis and calculated as a ratio to the HPRT mRNA copies. Cre^–VDR^fl/fl value was set at 100%. *P < 0.05; **P < 0.01 versus Cre^–VDR^fl/fl.

Figure 4. Vascular invasion and osteoclast formation are impaired in E15.

Cre⁺VDR^fl/fl mice. (A–D, F–I) Sets of adjacent tibial sections were immunostained for CD31 (A–D) or TRAP (F–I) to visualize endothelial cells or osteoclasts in E15.5 Cre^–VDR^fl/fl (A, B, F, and G) and Cre⁺VDR^fl/fl (C, D, H, and I) mice, respectively. Scale bar: 200 μm. (E) Invasion of blood vessels from the perichondrium into the cartilage core was measured and expressed relative to the cartilage width in Cre⁺VDR^fl/fl and Cre^–VDR^fl/fl tibiae. (J) Quantification of the number of osteoclasts (Oc.N) in the primary ossification center. *P < 0.05 versus Cre^–VDR^fl/fl.

Figure 5. Decreased vascularization and osteoclast number in neonatal and 15-day-old Cre⁺VDR^fl/fl mice.

(A–D) Endothelial cells and osteoclasts were visualized by CD31 (A and B) and TRAP (C and D) staining, respectively, in neonatal Cre^–VDR^fl/fl (A and C) and Cre⁺VDR^fl/fl (B and D) tibiae. Scale bar: 200 μm. (E) Intercapillary distance, measured at 0, 75, and 150 μm from the growth-plate border in neonatal and 15-day-old tibiae was larger in Cre⁺VDR^fl/fl mice compared with Cre^–VDR^fl/fl mice. (F) The number of osteoclasts at the terminal row of hypertrophic chondrocytes in neonatal tibiae was significantly lower in Cre⁺VDR^fl/fl mice. (G) Osteoclast surface (Oc.S) was significantly decreased in 15-day-old Cre⁺VDR^fl/fl tibiae. (H) Calcitonin receptor, RANKL, and VEGF mRNA levels in neonatal and 15-day-old Cre⁺VDR^fl/fl femora was determined by qRT-PCR, corrected for HPRT mRNA copies, and expressed relative to Cre^–VDR^fl/fl mice set at 100%. *P < 0.05 versus Cre^–VDR^fl/fl.

Figure 6. Signaling of 1,25(OH)₂D₃ in chondrocytes promotes osteoclast differentiation by the RANKL pathway.

(A–F) Microscopic observation of TRAP-positive multinuclear cells formed after 1 week of coculturing osteoblasts or chondrocytes with Cre^–VDR^fl/fl splenocytes. Osteoclast formation was similar whether osteoblasts from Cre^–VDR^fl/fl (A) or Cre⁺VDR^fl/fl mice (B) were used and treated with 1,25(OH)₂D₃ (10^–8 M). Chondrocytes from Cre^–VDR^fl/fl mice induced TRAP-positive cells when stimulated with 1,25(OH)₂D₃ (C) or 10^–6 M PGE₂ (E) whereas chondrocytes from Cre⁺VDR^fl/fl mice induced osteoclasts only when treated with PGE₂ (F) and not with 1,25(OH)₂D₃ (D). Scale bar: 100 μm. (G) qRT-PCR analysis of RANKL mRNA expression in primary osteoblast or chondrocyte cultures from Cre^–VDR^fl/fl and Cre⁺VDR^fl/fl mice stimulated by 10^–8 M 1,25(OH)₂D₃ or 10^–6 M PGE₂ for 2 days. Values are calculated as ratio to HPRT mRNA copies and expressed relative to vehicle set as 100%. *P < 0.05; **P < 0.01 versus vehicle.

Figure 7. Chondrocyte-specific VDR inactivation affects phosphate–vitamin D homeostasis by decreasing FGF23 expression in bone.

(A and B) Immunohistochemical staining of renal NPT2a in 15-day-old Cre^–VDR^fl/fl (A) and Cre⁺VDR^fl/fl (B) mice. Scale bar: 50 μm. (C–G) qRT-PCR analysis of renal NPT2a (C), CYP27B1 (D), CYP24 (E), and VDR (F) mRNA expression in 15-day-old mice and of FGF23 (G) mRNA expression in 1-week-old and 15-day-old femora, corrected for HPRT mRNA levels. Cre⁺VDR^fl/fl values are expressed relative to Cre^–VDR^fl/fl set at 100%. (H) Serum FGF23 level was significantly decreased in 15-day-old Cre⁺VDR^fl/fl mice. *P < 0.05; **P < 0.01 versus Cre^–VDR^fl/fl.

Figure 8. Signaling of 1,25(OH)₂D₃ in chondrocytes supports FGF23 expression in osteoblasts.

(A–D) qRT-PCR analysis of FGF23 mRNA expression in primary osteoblast cultures (A), in E16.5 metatarsal cultures (B), in cocultures of chondrocytes and osteoblasts (C), and in primary osteoblasts cultured in the Transwell system with chondrocytes cultured on the membrane (D), corrected for HPRT mRNA copies. Cultures derived from Cre^–VDR^fl/fl or Cre⁺VDR^fl/fl mice were treated with 1,25(OH)₂D₃ (10^–8 M) for 48 hours or vehicle. FGF23 mRNA expression in 1,25(OH)₂D₃-treated metatarsals is depicted as an increase relative to its vehicle-treated contralateral (B). (E) FGF23 protein level was measured in the culture media of the Transwell system after 1,25(OH)₂D₃ treatment. In the cocultures, only Cre^–VDR^fl/fl osteoblasts were used whereas the genotype of the chondrocytes varied as indicated. *P < 0.05; **P < 0.005 versus Cre^–VDR^fl/fl.