Vitamin D receptor in chondrocytes promotes osteoclastogenesis and regulates FGF23 production in osteoblasts (original) (raw)
Loss of VDR in growth-plate chondrocytes does not affect their development. To evaluate the contribution of VDR in early endochondral-bone development, floxed VDR (VDRfl/fl) mice were intercrossed with transgenic mice in which Cre recombinase was driven by collagen 2a1 (Col2) promoter (Figure 1A). The efficiency of chondrocyte-specific VDR inactivation was assessed at the DNA, RNA, and functional levels. Correct excision of the floxed VDR exon was demonstrated by Southern blot analysis of tail-extracted DNA from 15-day-old Col2Cre+/–VDRfl/fl mice (further defined as mutant Cre+VDRfl/fl), which was not observed in WT Col2Cre–/–VDRfl/fl (Cre–VDRfl/fl) mice. At this age, the tail vertebrae still contain collagen 2–expressing cells (Figure 1B). Analysis of VDR mRNA expression by quantitative real-time PCR (qRT-PCR) revealed undetectable VDR levels in chondrocytes isolated from neonatal Cre+VDRfl/fl mice whereas VDR was abundantly expressed in Cre–VDRfl/fl chondrocytes (Figure 1C). This efficient VDR inactivation was confirmed by assessing 1,25(OH)2D3–induced gene transcription in cultured primary chondrocytes (Figure 1D). CYP24 mRNA expression was strongly increased by 1,25(OH)2D3 treatment (10–8 M) in Cre–VDRfl/fl chondrocytes, as anticipated, but not in Cre+VDRfl/fl chondrocytes. On the other hand, VDR activity and/or expression was not altered in cultured osteoblasts, kidney, or intestine (data not shown) of Cre+VDRfl/fl mice.
Inactivation of the VDR gene in chondrocytes. (A) Schematic representation of targeting construct pNT vector/VDR, the _VDR_WT allele, and the VDRfl 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 _Sac_I-digested genomic DNA from Cre–VDRfl/fl and Cre+VDRfl/fl mice using internal probe. (C) VDR gene expression in growth-plate chondrocytes of Cre–VDRfl/fl (Cre–) and Cre+VDRfl/fl (Cre+) mice (n = 6) was assessed by qRT-PCR analysis and calculated as a ratio to the HPRT mRNA copies. Cre–VDRfl/fl value was set at 100%. **P < 0.01 versus Cre–VDRfl/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.
Cre+VDRfl/fl mice were viable, showed normal growth curves during the examined period, and did not display any overt phenotype (data not shown). Longitudinal bone growth, assessed by measuring femur length, was similar between the 2 genotypes at all investigated ages, including E15.5, the neonatal period, 15 days (data not shown), and 8 weeks (Table 1). To determine whether VDR inactivation affects chondrocyte development, growth-plate morphology and gene expression were analyzed. No gross morphological changes, assessed on toluidine blue–stained sections of the tibiae, were noticed at any age in cartilage of Cre+VDRfl/fl mice (Figure 2, C and D; only histology of 15-day-old mice is shown.). The total length of the growth plate (data not shown) as well as the length of hypertrophic cartilage in tibiae of neonatal and 15-day-old mice were similar between the 2 genotypes (Figure 2E). Also, the amount of growth-plate mineralization in 8-week-old mice did not differ (Table 1). The mRNA level of Col2, a marker of proliferating chondrocytes, was assessed in neonatal, 15-day-old, and 8-week-old femora but was not significantly different between the 2 genotypes (Figure 2, F–H). In addition, its expression pattern in the growth plates of 15-day-old mice was indistinguishable between genotypes, as revealed by collagen 2 immunostaining (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI29463DS1). Compared with Cre–VDRfl/fl mice, collagen 10 (Col10) mRNA level, a specific marker of hypertrophic chondrocytes, was unaltered in neonatal Cre+VDRfl/fl mice (Figure 2F), decreased in 15-day-old mice (P < 0.05; Figure 2G), and at normal levels at the age of 8 weeks (Figure 2H). These data suggest that lack of VDR in chondrocytes does not manifestly impair chondrocyte development but suppresses temporarily the terminal differentiation of the chondrocytes.
Normal growth-plate development in chondrocyte-specific _VDR_-null mice. (A–D) Toluidine blue staining of the proximal tibiae from 15-day-old Cre–VDRfl/fl (A and B) and Cre+VDRfl/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–VDRfl/fl and Cre+VDRfl/fl mice, assessed by qRT-PCR and calculated as a ratio to HPRT mRNA copies. *P < 0.05 versus Cre–VDRfl/fl.
Bone parameters in 8-week-old mice
BV/TV is increased in juvenile Cre+VDRfl/fl mice. During endochondral ossification, cartilage becomes progressively replaced by mineralized bone, which is a highly coordinated process, depending partially on factors produced by chondrocytes. To investigate the effect of VDR inactivation in chondrocytes on this process, trabecular bone parameters were quantified at several ages. At 8 weeks, static and dynamic bone parameters were normal, as evidenced by similar values between the 2 genotypes for trabecular and cortical bone mineral density (BMD) analyzed by peripheral quantitative computed tomography of the femur, BV/TV quantified on von Kossa–stained sections, and bone formation ratio (BFR/BS) assessed on unstained sections of the tibiae (Table 1).
In contrast, BV/TV was significantly increased, by 50%, in neonatal and 15-day-old Cre+VDRfl/fl compared with Cre–VDRfl/fl mice (Figure 3A). In addition, von Kossa staining revealed that trabecular bone extended more deeply into the metaphyseal area of the proximal tibiae in 15-day-old Cre+VDRfl/fl mice (Figure 3, B and C).
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+VDRfl/fl mice compared with Cre–VDRfl/fl mice. (B and C) von Kossa staining on tibiae sections of 15-day-old Cre–VDRfl/fl (B) and Cre+VDRfl/fl mice (C). Scale bar: 400 μm. (D) Osteocalcin gene expression in femora (total bone or dissected diaphysis) of 15-day-old Cre–VDRfl/fl and Cre+VDRfl/fl mice was assessed by qRT-PCR analysis and calculated as a ratio to the HPRT mRNA copies. Cre–VDRfl/fl value was set at 100%. *P < 0.05; **P < 0.01 versus Cre–VDRfl/fl.
Vascularization and osteoclast invasion are delayed when chondrocytes lack VDR. The observed changes in bone mass in 15-day-old Cre+VDRfl/fl mice may have resulted from increased bone formation and/or decreased bone resorption. Osteoblast function was not manifestly changed by VDR inactivation in chondrocytes, as suggested by the normal mRNA expression of the osteoblastic marker osteocalcin (Figure 3D) and runt-related transcription factor 2 (Runx2) (data not shown) in 15-day-old femora. In addition, in vitro osteogenic differentiation of bone marrow stromal cells (CFU-osteoblast) isolated from 15-day-old mice did not differ between genotypes, as comparable number and size of colonies staining positive for alkaline phosphatase or alizarin red were obtained (data not shown).
On the other hand, bone resorption may be impaired; this is often associated at these stages with changes in vascularization. These processes were investigated at E15.5 and in neonatal and 15-day-old mice. Initial blood vessel invasion in Cre–VDRfl/fl tibiae, assessed by CD31 staining, was observed at E15.5, showing endothelial cells located at the bone collar/perichondrium but also invading the cartilage core (Figure 4, A and B). In contrast, in Cre+VDRfl/fl mice, endothelial cells were solely detected along the bone collar/perichondrium (Figure 4, C and D). At the same time, tartrate-resistant acid phosphatase–positive (TRAP-positive) multinuclear osteoclasts accompanying endothelial cells had just started to invade the tibial cartilage, forming the primary ossification center in Cre–VDRfl/fl tibiae (Figure 4, F and G) whereas these cells were hardly observed in Cre+VDRfl/fl tibiae (Figure 4, H and I). Quantification revealed that blood vessel invasion in Cre+VDRfl/fl tibiae was only 57% of the Cre–VDRfl/fl level (P < 0.05; Figure 4E) and that the number of TRAP-positive cells even decreased, with 70% in Cre+VDRfl/fl tibiae compared with Cre–VDRfl/fl (P < 0.05; Figure 4J).
Vascular invasion and osteoclast formation are impaired in E15. Cre+VDRfl/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–VDRfl/fl (A, B, F, and G) and Cre+VDRfl/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+VDRfl/fl and Cre–VDRfl/fl tibiae. (J) Quantification of the number of osteoclasts (Oc.N) in the primary ossification center. *P < 0.05 versus Cre–VDRfl/fl.
In neonatal tibiae, the number of blood vessels invading the terminal row of the hypertrophic chondrocytes of the growth plate was reduced in Cre+VDRfl/fl mice (Figure 5B) as compared with Cre–VDRfl/fl mice (Figure 5A). Accordingly, intercapillary distance was significantly larger in neonatal Cre+VDRfl/fl tibiae (P < 0.05; Figure 5E). Also, in 15-day-old Cre+VDRfl/fl mice, the intercapillary distance was significantly increased both at the terminal row of the growth plate and in the metaphysis, 150 μm distal from the hypertrophic chondrocytes (Figure 5E). The changes in vascularization were accompanied by alterations in osteoclast formation, as shown by the reduced TRAP positivity in Cre+VDRfl/fl mice (Figure 5D) compared with Cre–VDRfl/fl mice (Figure 5C). The number of TRAP-positive cells at the border of the growth plate was decreased by 50% in neonatal Cre+VDRfl/fl tibiae (Figure 5F). This was reflected in a significant reduction of the calcitonin receptor mRNA level, a marker of differentiated osteoclasts, in Cre+VDRfl/fl mice (Figure 5H). Also, at 15 days, osteoclast surface in trabecular bone was decreased in Cre+VDRfl/fl tibiae (Figure 5G). These 2 processes, osteoclast formation and angiogenesis, are regulated by the secreted factors RANKL and VEGF, respectively. In agreement with the histological findings, mRNA expression of both factors was significantly reduced in neonatal Cre+VDRfl/fl femora compared with Cre–VDRfl/fl mice (P < 0.05; Figure 5H). These data indicate that vascular invasion at the growth plate and resorption of trabecular bone is decreased in Cre+VDRfl/fl mice, which can explain the observed increased bone volume.
Decreased vascularization and osteoclast number in neonatal and 15-day-old Cre+VDRfl/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–VDRfl/fl (A and C) and Cre+VDRfl/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+VDRfl/fl mice compared with Cre–VDRfl/fl mice. (F) The number of osteoclasts at the terminal row of hypertrophic chondrocytes in neonatal tibiae was significantly lower in Cre+VDRfl/fl mice. (G) Osteoclast surface (Oc.S) was significantly decreased in 15-day-old Cre+VDRfl/fl tibiae. (H) Calcitonin receptor, RANKL, and VEGF mRNA levels in neonatal and 15-day-old Cre+VDRfl/fl femora was determined by qRT-PCR, corrected for HPRT mRNA copies, and expressed relative to Cre–VDRfl/fl mice set at 100%. *P < 0.05 versus Cre–VDRfl/fl.
Chondrocytes promote osteoclastogenesis by 1,25(OH)2D3–induced RANKL expression. Since Cre+VDRfl/fl mice displayed retarded osteoclast invasion into cartilage and reduced RANKL gene expression in neonatal femora, we investigated whether 1,25(OH)2D3 genomic signaling in chondrocytes regulates osteoclastogenesis, using cocultures with spleen cells. TRAP-positive multinuclear cells were formed in 1,25(OH)2D3-treated (10–8 M) cocultures of calvarial-derived osteoblasts and splenocytes irrespective of the genotype (Figure 6, A and B; only results of Cre–VDRfl/fl splenocytes are shown). Under similar experimental conditions, chondrocytes isolated from Cre–VDRfl/fl mice induced TRAP-positive multinuclear osteoclasts in cocultures with splenocytes from either genotype (Figure 6C). However, no osteoclasts were formed when Cre+VDRfl/fl chondrocytes were used (Figure 6D). This effect was specific for the 1,25(OH)2D3/VDR signaling pathway, as prostaglandin E2 (PGE2) (10–6 M) induced osteoclast formation irrespective of the chondrocyte genotype (Figure 6, E and F).
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–VDRfl/fl splenocytes. Osteoclast formation was similar whether osteoblasts from Cre–VDRfl/fl (A) or Cre+VDRfl/fl mice (B) were used and treated with 1,25(OH)2D3 (10–8 M). Chondrocytes from Cre–VDRfl/fl mice induced TRAP-positive cells when stimulated with 1,25(OH)2D3 (C) or 10–6 M PGE2 (E) whereas chondrocytes from Cre+VDRfl/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–VDRfl/fl and Cre+VDRfl/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.
As osteoclastogenesis is dependent on the ratio of RANKL to osteoprotegerin (OPG), the mRNA level of these factors was quantified in 1,25(OH)2D3- or PGE2-treated primary cultures and compared with vehicle treatment. RANKL gene expression was significantly induced in osteoblasts (P < 0.001) regardless of the genotype and stimulus (Figure 6G). In addition, 1,25(OH)2D3 (10–8 M) increased RANKL expression 30-fold in Cre–VDRfl/fl chondrocytes (P < 0.001) whereas no induction was noticed in Cre+VDRfl/fl chondrocytes (Figure 6G). On the other hand, PGE2 significantly induced RANKL expression in chondrocytes from either genotype (Figure 6G). OPG mRNA levels did not differ in any condition. Thus, these findings clearly indicate that chondrocytes can promote osteoclast differentiation by expressing RANKL, which is strongly induced by 1,25(OH)2D3 genomic signaling.
1,25(OH)2D3 genomic signaling in chondrocytes modulates phosphaturic factor expression. In systemic _VDR_-null mice, mineral homeostasis becomes manifestly disturbed after weaning, concurrent with the development of skeletal changes. In the present study, we investigated serum biochemistry at the age of 15 days, when endochondral ossification was clearly affected in Cre+VDRfl/fl mice. Chondrocyte-specific VDR inactivation did not affect calcium and parathyroid hormone (PTH) levels (Table 2). On the other hand, phosphate and 1,25(OH)2D serum concentrations were significantly increased in Cre+VDRfl/fl mice compared with Cre–VDRfl/fl mice (P < 0.05; Table 2). These changes were no longer observed in 8-week-old mice, as both serum phosphate (13.5 ± 0.6 mg/dl in Cre–VDRfl/fl versus 12.9 ± 0.8 mg/dl in Cre+VDRfl/fl) and 1,25(OH)2D (128 ± 13 pg/ml in Cre–VDRfl/fl versus 121 ± 1 pg/ml in Cre+VDRfl/fl) showed normal values.
Serum biochemistry in 15-day-old mice
Since phosphate homeostasis is mainly regulated by renal phosphate reabsorption, we assessed the expression of NPT2a, the major player in this process, expressed at the apical membrane of proximal tubular cells. Immunohistochemical staining showed more abundant NPT2a expression in 15-day-old Cre+VDRfl/fl kidneys (Figure 7B) compared with Cre–VDRfl/fl kidneys (Figure 7A), which is in agreement with the increased serum phosphate level. Quantification of renal NPT2a mRNA level by qRT-PCR revealed a 4-fold increase in Cre+VDRfl/fl mice (P < 0.05; Figure 7C), confirming the immunohistochemical analysis. Concerning 1,25(OH)2D serum levels, these are mainly regulated by renal CYP27B1 expression, which hydroxylates 25-hydroxyvitamin D to its active form. In accordance with the increased 1,25(OH)2D serum levels, CYP27B1 mRNA level was significantly increased in Cre+VDRfl/fl kidney (P < 0.01; Figure 7D) while gene expression of the catabolic enzyme CYP24 was not changed (Figure 7E). It is noteworthy that no difference in renal VDR mRNA levels was observed between the 2 genotypes (Figure 7F).
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–VDRfl/fl (A) and Cre+VDRfl/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+VDRfl/fl values are expressed relative to Cre–VDRfl/fl set at 100%. (H) Serum FGF23 level was significantly decreased in 15-day-old Cre+VDRfl/fl mice. *P < 0.05; **P < 0.01 versus Cre–VDRfl/fl.
These data suggest a link between the altered renal expression of these 2 genes and VDR inactivation in chondrocytes. A plausible factor that may affect 1,25(OH)2D as well as phosphate serum levels is FGF23, as FGF23 suppresses the expression of the renal phosphate transporter NPT2a and of CYP27B1 (16). In addition, bone has been identified as the largest source and regulatory site of FGF23, and FGF23 expression is induced by 1,25(OH)2D3 (17). Accordingly, FGF23 mRNA level was significantly decreased in Cre+VDRfl/fl femora of 1-week-old and 15-day-old mice, as revealed by qRT-PCR (P < 0.01; Figure 7G). In addition, FGF23 serum levels were 43% lower in 15-day-old Cre+VDRfl/fl mice compared with Cre–VDRfl/fl mice (P < 0.01; Figure 7H). As with the age-related changes of serum phosphate and 1,25(OH)2D levels, no difference in FGF23 serum levels was observed between the 2 genotypes in 8-week-old mice (71.4 ± 6.3 pg/ml in Cre–VDRfl/fl versus 76.9 ± 12.2 pg/ml in Cre+VDRfl/fl mice). These data indicate that the increased phosphate and 1,25(OH)2D serum concentrations before weaning are likely caused by decreased FGF23 production in bone, leading to increased renal NPT2a and CYP27B1 mRNA expression.
Genomic action of 1,25(OH)2D3 in chondrocytes regulates FGF23 expression in osteoblasts in vitro. In bone, the cells identified as producing FGF23 are osteoblasts and osteocytes. To elucidate whether chondrocytes express FGF23 and whether this expression is regulated by 1,25(OH)2D3, in vitro experiments were performed. Treatment with 1,25(OH)2D3 (10–8 M, 48 hours) induced FGF23 mRNA expression 100-fold in primary osteoblasts derived from either Cre+VDRfl/fl or Cre–VDRfl/fl mice (Figure 8A). On the other hand, the FGF23 message was undetectable in cultured primary chondrocytes, both in basal conditions and after 1,25(OH)2D3 treatment (data not shown). These data indicate that FGF23 expression is restricted to osteoblasts and suggest that the reduced FGF23 expression in Cre+VDRfl/fl mice most likely results from altered expression of a chondrocyte-derived factor due to lack of VDR action. Several in vitro experiments were performed to investigate this hypothesis. First, metatarsals at E16.5, a stage when interaction between osteoblasts and chondrocytes is abundantly present, were cultured for 24 hours, after which 1 metatarsal was treated with 1,25(OH)2D3 (10–8 M) and the contralateral with vehicle. The relative induction of FGF23 mRNA by 1,25(OH)2D3 treatment was significantly higher in Cre–VDRfl/fl than in Cre+VDRfl/fl metatarsals (P < 0.005; Figure 8B). However, the number of osteoblasts in the metatarsals may be different between the 2 genotypes, as vascular invasion and formation of the primary ossification center is delayed in Cre+VDRfl/fl metatarsals (Figure 4). We therefore introduced a coculture system of primary osteoblasts and chondrocytes to characterize more precisely their interaction on FGF23 expression. Primary chondrocytes were isolated from both genotypes and cultured during 4 days, after which osteoblasts isolated from Cre–VDRfl/fl calvaria were added to the culture. Treatment with 1,25(OH)2D3 induced FGF23 mRNA expression in cocultures regardless of the presence of VDR in chondrocytes. However, the increase in FGF23 mRNA expression by 1,25(OH)2D3 treatment was significantly impaired when Cre+VDRfl/fl chondrocytes were used in cocultures, as compared with Cre–VDRfl/fl chondrocytes (P < 0.001; Figure 8C), indicating that a 1,25(OH)2D3-responsive chondrocyte-derived factor contributes to FGF23 expression in osteoblasts.
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–VDRfl/fl or Cre+VDRfl/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–VDRfl/fl osteoblasts were used whereas the genotype of the chondrocytes varied as indicated. *P < 0.05; **P < 0.005 versus Cre–VDRfl/fl.
Finally, to elucidate whether FGF23 mRNA expression in osteoblasts was modulated by a secreted factor produced by chondrocytes, a Transwell system was used with primary chondrocytes from either genotype cultured on the insert and primary osteoblasts derived from Cre–VDRfl/fl calvaria on the bottom plate. No manifest differences in the growth and differentiation of osteoblasts and chondrocytes were observed between the different conditions, as osteocalcin expression by the osteoblasts and Col10 expression by the chondrocytes were comparable (data not shown). The induction of FGF23 mRNA level by 1,25(OH)2D3 was significantly higher in osteoblasts cultured with Cre–VDRfl/fl chondrocytes than in cultures with Cre+VDRfl/fl chondrocytes (P < 0.001; Figure 8D). In addition, 1,25(OH)2D3-treated osteoblasts showed a more pronounced induction of FGF23 mRNA levels when cultured with Cre–VDRfl/fl chondrocytes in the Transwell system than when cultured alone (compare FGF23 mRNA level in Figure 8D with level in Figure 8A). In agreement with the results of mRNA expression, FGF23 protein level was more increased in conditioned media from cocultures with Cre–VDRfl/fl chondrocytes than from cultures with Cre+VDRfl/fl chondrocytes (P < 0.01; Figure 8E). These results indicate that vitamin D genomic signaling in chondrocytes contributes to FGF23 expression in osteoblasts by altering the expression of a secreted factor.