Abstract
Increased vitamin A (retinol) intake has been suggested to increase bone fragility. In the present study, we investigated effects of retinoids on bone resorption in cultured neonatal mouse calvarial bones and their interaction with glucocorticoids (GC). All-trans-retinoic acid (ATRA), retinol, retinalaldehyde, and 9-cis-retinoic acid stimulated release of (45)Ca from calvarial bones. The resorptive effect of ATRA was characterized by mRNA expression of genes associated with osteoclast differentiation, enhanced osteoclast number, and bone matrix degradation. In addition, the RANKL/OPG ratio was increased by ATRA, release of (45)Ca stimulated by ATRA was blocked by exogenous OPG, and mRNA expression of genes associated with bone formation was decreased by ATRA. All retinoid acid receptors (RARα/β/γ) were expressed in calvarial bones. Agonists with affinity to all receptor subtypes or specifically to RARα enhanced the release of (45)Ca and mRNA expression of Rankl, whereas agonists with affinity to RARβ/γ or RARγ had no effects. Stimulation of Rankl mRNA by ATRA was competitively inhibited by the RARα antagonist GR110. Exposure of calvarial bones to GC inhibited the stimulatory effects of ATRA on (45)Ca release and Rankl mRNA and protein expression. This inhibitory effect was reversed by the glucocorticoid receptor (GR) antagonist RU 486. Increased Rankl mRNA stimulated by ATRA was also blocked by GC in calvarial bones from mice with a GR mutation that blocks dimerization (GR(dim) mice). The data suggest that ATRA enhances periosteal bone resorption by increasing the RANKL/OPG ratio via RARα receptors, a response that can be inhibited by monomeric GR.
Highlights
Supplementation of the diet with vitamin A is a common occurrence in developed countries
Stimulation of Bone Resorption and RANKL in Mouse Calvarial Bones Is Mediated by retinoic acid receptors (RARs)␣—We showed in an earlier study that RAR␣ mediated the inhibition by All-trans-retinoic acid (ATRA) of osteoclastogenesis in mouse bone marrow cells stimulated by RANKL [24]
Assessment of gene expression showed that 10Ϫ7 M concentrations of ATRA, TTNPB, and GR104, but not A7980 or GR103, enhanced Rankl mRNA in calvarial bones at 24 h (Fig. 6D)
Summary
Materials—Mouse OPG fused to human IgG1 Fc (OPG/Fc chimera), Escherichia coli-derived mouse RANKL Lys158– Asp316 (catalog no. 462-TEC), the ELISA kits for mouse RANKL and mouse OPG, and recombinant human interleukin-1 receptor antagonistic protein were purchased from R&D Systems; ATRA, 9-cis-RA, TTNPB (Ro 13-7410) (4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid), A7980 (AC-41848) (1-[2,4dichlorophenyl)methyl-6,7,8,9-tetrahydro-3-phenyl-5H-imidazol[1,2-a]azepinium bromide hydrate), dexamethasone, hydrocortisone, RU 486 (17-hydroxy-11-(4-dimethylaminophenyl)17-(1-propynyl)-estra-4,9-dien-3-one), retinol, retinalaldehyde, and essentially fatty acid-free bovine serum albumin were from Sigma; GR110 (Ro-41–5253) (4-[(E)-2-(7-heptoxy4,4-dimethyl-1,1-dioxo-2,3-dihydrothiochromen-6-yl)prop-1enyl]benzoic acid), GR103 (N-(4-hydroxyphenyl)retinamide), and GR104 (AM-580) (4-[(5,6,7,8-tetrahydro-5,6,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid were from Biomol; bovine parathyroid hormone 1–34 (PTH) was from Bachem; ␣-modification of minimum essential medium and fetal calf serum (FCS) were from Invitrogen; CrossLabs for Culture ELISA (CTX) was from Immunodiagnostics a/s; L-[53H]proline was from PerkinElmer Life Sciences; [45Ca]CaCl2 was from Amersham Biosciences; oligonucleotide primers were from Invitrogen or Applied Biosystems; the HotStar Taq polymerase kit was from Qiagen Ltd.; the KapaTM Probe Fast quantitative PCR kit and Kapa2GTM Robust HotStart Ready Mix were from Kapa; the PCR core kit was from Roche Applied Science; fluorescent labeled probes (reporter fluorescent dye VIC at the 5Ј-end and quencher fluorescent dye TAMRA at the 3Ј-end), high capacity cDNA reverse transcription kit, Power SYBR Green PCR Master Mix, and the kits for quantitative real-time PCR were from Applied Biosystems; the RNAqueous-4PCR kit was from Ambion Inc. (Austin, TX); culture dishes, multiwell plates, and glass chamber slides were from Nunc Inc.; and suspension culture dishes were from Corning Glass. 2–3-day-old mice were injected with 1.5 Ci of 45Ca, and the amounts of radioactivity in bone and culture medium were analyzed by liquid scintillation at the end of the culture period. Matrix degradation was assessed by analyzing the amount of type I collagen degradation fragments in culture media released from calvarial halves cultured as described above by using a commercially available ELISA. Semiquantitative RT-PCR analysis of the mRNA expression of Calcr (calcitonin receptor gene), Acp (acid phosphatase gene), Ctsk (cathepsin K gene), Rankl, Rank, Opg, and M-csf was performed using either a HotStar Taq polymerase kit, a PCR core kit, or the Kapa2GTM Probe Fast quantitative PCR kit and compared at the logarithmic phase of the PCR. All experiments were performed at least twice with comparable results, and all data are presented as means Ϯ S.E
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