Smoke carcinogens cause bone loss through the aryl hydrocarbon receptor and induction of Cyp1 enzymes

Abstract

Smoking is a major risk factor for osteoporosis and fracture, but the mechanism through which smoke causes bone loss remains unclear. Here, we show that the smoke toxins benzo(a)pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) interact with the aryl hydrocarbon receptor (Ahr) to induce osteoclastic bone resorption through the activation of cytochrome P450 1a/1b (Cyp1) enzymes. BaP and TCDD enhanced osteoclast formation in bone marrow cell cultures and gavage with BaP stimulated bone resorption and osteoclastogenesis in vivo. The osteoclastogenesis triggered by BaP or RANK-L was reduced in Ahr(-/-) cells, consistent with the high bone mass noted in Ahr(-/-) male mice. The receptor activator of NF-κB ligand (RANK-L) also failed to induce the expression of Cyp1 enzymes in Ahr(-/-) cells. Furthermore, the osteoclastogenesis induced by TCDD was lower in Cyp1a1/1a2(-/-) and Cyp1a1/1a2/1b1(-/-) cultures, indicating that Ahr was upstream of the Cyp enzymes. Likewise, the pharmacological inhibition of the Cyp1 enzymes with tetramethylsilane or proadifen reduced osteoclastogenesis. Finally, deletion of the Cyp1a1, Cyp1a2, and Cyp1b1 in triple knockout mice resulted in reduced bone resorption and recapitulated the high bone mass phenotype of Ahr(-/-) mice. Overall, the data identify the Ahr and Cyp1 enzymes not only in the pathophysiology of smoke-induced osteoporosis, but also as potential targets for selective modulation by new therapeutics.

Keywords: bone formation; osteoblast; skeletal remodeling; toxicology.

Fig. 1.

Ahr ligands BaP and TCDD increase osteoclastic resorption. (A, B, and D) TRAP-positive osteoclasts formed from RAW-C3 precursor cells or in bone marrow (BM) cell cultures treated with RANKL (concentrations as noted) and with benzo[a]pyrene (BaP) (A and B) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (D). (C) Effect of BaP gastric gavage (120 mg/kg, six doses) or corn oil (control) on bone resorption quantified as Resorbed S/B.Pm (TRAP-stained sections shown, ×10) vertebral bone or osteoclast formation in ex vivo BM cell cultures (C). ANOVA; comparing with zero-dose BaP or TCDD; *P ≤ 0.05; **P ≤ 0.01; n = 4 or 6 per group for ex vivo and in vivo studies, respectively.

Fig. 2.

Ahr is required for BaP- and RANK-L–induced osteoclast formation. TRAP-positive osteoclast number (A and B), TRAP production (C), and expression (measured by quantitative PCR) of trap, cathepsin K, and integrin β₃ (D) in bone marrow cell cultures from wild-type or Ahr^−/− mice treated with BaP (A) or RANK-L (B–D) (concentrations as shown). (E) Micro-CT images and structural parameters in femur metaphyseal bone, including volumetric bone mineral density (vBMD), bone volume/trabecular volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and connectivity density (Conn.D) from wild-type ∼4 mo-old male Ahr^+/+ and Ahr^−/− mice. (F) Cyp1a1 and Cyp1a2 expression in bone marrow cell cultures from Ahr^+/+ and Ahr^−/− mice treated with RANK-L (60 ng/mL). ANOVA; comparisons: quantitative PCR, triplicate, wild-type zero-dose vs. RANK-L, ^^P ≤ 0.01; wild type vs. Ahr^−/− both with RANK-L, **P ≤ 0.01; micro-CT, P values as shown, n = 5–7 mice per group; cell assays: 4 wells per group.

Fig. 3.

Cyp1 induction is necessary for TCDD- and RANK-L–induced osteoclastogenesis. (A and B) Induction of Cyp1a1, Cyp1a2, and Cyp1b1 mRNA (A) and protein (B) expression in bone marrow cell cultures isolated from mice treated with BaP (+, oral gavage, 120 mg/kg, 6 doses). (C and D) TRAP-positive osteoclast formation in bone marrow cell cultures derived from Cyp1a1/1a2^−/− double knockout, Cyp1a1/1a2/1b1^−/− triple knockout and Cyp1^+/+ wild-type mice either without (C) or after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (D). (E and F) Effect of RANK-L on the expression (quantitative PCR) of the osteoclast genes trap, cathepsin K and β₃ integrin (E) and the P450 genes Cyp1a1, Cyp1a2, and Cyp1b1 (F) in the three mouse genotypes. (G and H) Effect of the Cyp inhibitors proadifen and TMS (concentrations as shown) on RANK-L–induced TRAP-positive osteoclast formation (G), and expression of trap, β₃ integrin, and cathepsin K mRNA (H). RANK-L was used at 30 ng/mL; with 1 µM proadifen and 1 µg/mL TMS in H. ANOVA; comparisons either with wild-type littermates (C) or zero-dose controls (D–H); **P ≤ 0.01; comparisons between treatments as shown, ^P ≤ 0.05; ^^P ≤ 0.01; for cell cultures, four wells per group; quantitative PCR in triplicate.

Fig. 4.

Genetic deletion of Cyp1 enzymes reduces bone resorption and increases bone mass in vivo. (A) Bone resorption quantified as Resorbed S/B.Pm (TRAP-stained sections shown, ×10) in proximal tibial metaphyseal trabecular bone of ∼4 mo-old male Cyp1^+/+ wild-type (WT), and Cyp1a1/1a2^−/− and Cyp1a1/1a2/1b1^−/− knockout mice. (B) Structural parameters measured by micro-CT on vertebral bone, including volumetric bone mineral content (BMC), bone volume/trabecular volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.Sp), connectivity density (Conn.D), bone surface/bone volume (BS/BV), and bone surface/trabecular volume (BS/TV) from the three genotypes. ANOVA; comparisons between WT and respective knockout lines; *P ≤ 0.05; **P ≤ 0.01; n = 5–6 mice per group.