Lack of α2C-Adrenoceptor Results in Contrasting Phenotypes of Long Bones and Vertebra and Prevents the Thyrotoxicosis-Induced Osteopenia

Abstract

A series of studies have demonstrated that activation of the sympathetic nervous system (SNS) causes osteopenia via β2-adrenoceptor (β2-AR) signaling. However, in a recent study, we found an unexpected and generalized phenotype of high bone mass in female mice with chronic sympathetic hyperactivity, due to double gene inactivation of adrenoceptors that negatively regulate norepinephrine release, α2A-and α2C-AR (α2A/2C-AR-/-). These findings suggest that β2-AR is not the single adrenoceptor involved in bone turnover regulation and show that α2-AR signaling may also mediate the SNS actions in the skeleton. In addition, we found that α2A/2C-AR-/- animals are resistant to the thyrotoxicosis-induced osteopenia, suggesting that thyroid hormone (TH), when in supraphysiological levels, interacts with the SNS to control bone mass and structure, and that this interaction may also involve α2-AR signaling. In the present study, to further investigate these hypotheses and to discriminate the roles of α2-AR subtypes, we have evaluated the bone phenotype of mice with the single gene inactivation of α2C-AR subtype, which mRNA expression was previously shown to be down regulated by triiodothyronine (T3). A cohort of 30 day-old female α2CAR-/- mice and their wild-type (WT) controls were treated with a supraphysiological dose of T3 for 30 or 90 days, which induced a thyrotoxic state in both mouse lineages. The micro-computed tomographic (μCT) analysis showed that α2C-AR-/- mice present lower trabecular bone volume (BV/TV) and number (Tb.N), and increased trabecular separation (Tb.Sp) in the femur compared with WT mice; which was accompanied by decreased bone strength (determined by the three-point bending test) in the femur and tibia. The opposite was observed in the vertebra, where α2C-AR-/- mice show increased BV/TV, Tb.N and trabecular thickness (Tb.Th), and decreased Tb.Sp, compared with WT animals. In spite of the contrasting bone phenotypes of the femur and L5, thyrotoxicosis negatively regulated most of the micro architectural features of the trabecular bone in both skeletal sites of WT, but not of α2C-AR-/- mice. T3 treatment also decreased biomechanical properties (maximum load and ultimate load) in the femur and tibia of WT, but not of knockout mice. The mRNA expression of osteocalcin, a marker of mature osteoblasts, and tartrate-resistant acid phosphatase, which is expressed by osteoclasts and is involved in collagen degradation, was increased by T3 treatment only in WT, and not in α2C-AR-/- mice. Altogether, these findings suggest that α2C-AR subtype mediates the effects of the SNS in the bone in a skeletal site-dependent manner, and that thyrotoxicosis depends on α2C-AR signaling to promote bone loss, which sustains the hypothesis of a TH-SNS interaction to modulate bone remodeling and structure.

Fig 1. T3 and T4 serum levels in WT and α_2C-AR^-/- mice.

(A) Serum levels of T3. (B) Serum levels of T4. Animals were treated with saline or a supraphysiological dose of T3 (7 μg·100 g body wt^-1·day^-1). Significance between groups was determined by two-way ANOVA followed by Tukey’s test. Values are expressed as means ± SEM (n = 8-10/group). *P <0.05, versus the respective saline-treated animals (WT vs. WT+T3, KO vs. KO+T3).

Fig 2. Effect of T3-treatment on heart mass of WT and α_2C-AR^-/- mice.

30d and 90d, refer, respectively, to 30 and 90 days of treatment with saline or a supraphysiological dose of T3 (7 μg·100 g body wt^-1·day^-1). The dependent values were calculated dividing heart mass per body mass [(g of heart mass/g of body mass) x 100]. Significance between groups was determined by two-way ANOVA followed by Tukey’s test. Values are expressed as means ± SEM (n = 10–12 per group). ***P <0.001 versus the respective saline-treated animals (WT vs. WT+T3, KO vs. KO+T3).

Fig 3. Body mass of WT and α_2C-AR^-/- mice.

(A) WT versus α_2C-AR^-/- mice. (B) WT versus WT+T3. (C) α_2C-AR^-/-versus α_2C-AR^-/-+T3. Animals were treated with saline or a supraphysiological dose of T3 (7 μg·100 g body wt^-1·day^-1). The significance between all groups, presented in figures A, B and C, was determined in a single test by two-way ANOVA, followed by Tukey’s test. The groups were separated in figures A, B and C to more clearly show the differences between groups. Values represent the mean ± SEM (n = 10–12 per group). *P< 0.05, **P< 0.01, versus the respective saline-treated animals (WT vs. WT+T3, KO vs. KO+T3), ⁺P < 0.05 (WT vs. KO).

Fig 4. Effect of T3-treatment on the structural parameters of the trabecular and cortical bone of the distal metaphysis of the femur in WT and α_2C-AR^-/- mice assessed by μCT.

(A–D) Effect of 30-day treatment on trabecular parameters. (E-H) Effect of 90-day treatment on trabecular parameters. (I-L) Effect of 30-day treatment on cortical parameters. (M-P) Effect of 90-day treatment on cortical parameters. Animals were treated with saline or a supraphysiological dose of T3 (7 μg·100 g body wt^-1·day^-1). Significance between groups was determined by two-way ANOVA followed by Tukey’s test. Values are expressed as means ± SEM (n = 10–12 per group). *P<0.05 and **P< 0.01 vs. the respective saline-treated animals (WT vs. WT+T3, KO vs. KO+T3). ⁺P< 0.05, ⁺⁺P< 0.01 and ⁺⁺⁺P< 0.001 for differences between WT and KO mice, as indicated. BV/TV, trabecular bone volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular speculation; T.Ar, total area; B.Ar, bone area; Ma.Ar, medullary area; Ec.Pm, endocortical perimeter.

Fig 5. Effect of T3-treatment on the structural parameters of the trabecular bone of the vertebral body of L5 in WT and α_2C-AR^-/- mice assessed by μCT.

(A–D) Effect of 30-day treatment on trabecular parameters. (E-H) Effect of 90-day treatment on trabecular parameters. (I-L) Effect of 30-day treatment on cortical parameters. (M-P) Effect of 90-day treatment on cortical parameters. Animals were treated with saline or a supraphysiological dose of T3 (7 μg·100 g body wt^-1·day^-1). Significance between groups was determined by two-way ANOVA followed by Tukey’s test. Values are expressed as means ± SEM (n = 10–12 per group). *P <0.05 and **P< 0.01 vs. the respective saline-treated animals (WT vs. WT+T3, KO vs. KO+T3). ⁺P< 0.05, ⁺⁺P< 0.01 and ⁺⁺⁺P< 0.001 for differences between WT and KO mice, as indicated. BV/TV, trabecular bone volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular speculation.

Fig 6. Effect of T3-treatment on the biomechanical parameters of the femur and tibia in WT mice and α_2C-AR^-/- mice.

Data were assessed by means of the 3-point bending test. Animals were treated with saline or a supraphysiological dose of T3 (7 μg·100 g body wt^-1·day^-1). Values are expressed as means ± SEM (n = 10–12 per group). Significance between groups was determined by two-way ANOVA followed by Tukey’s test. *P <0.05 and **P< 0.01 vs. the respective saline-treated animals (WT vs. WT+T3, KO vs. KO+T3). ⁺P< 0.05, ⁺⁺P < 0.01 and ⁺⁺⁺P < 0.001 for differences between WT and KO mice, as indicated.

Fig 7. Effect of T3-treatment on the relative mRNA expression of bone metabolism-related genes.

(A-L) Genes expressed in the femur. (M-X) Genes expressed in the vertebra. (A-F and M-R) Effect of 30-day treatment. (G-L and S-X) Effect of 90-day treatment. Receptor activator of nuclear factor-ҡB (RANK), RANK ligand (RANKL), osteoprotegerin (OPG), osteocalcin (OC) and tartrate-resistant acid phosphatase (TRAP). mRNA expression was determined by real-time PCR analysis. Animals were treated with saline or a supraphysiological dose of T3 (7 μg·100 g body wt^-1·day^-1) for 30 or 90 days. Values are expressed as means ± EPM (n = 4 to 5/group). Significance between groups was determined by two-way ANOVA followed by Tukey’s test. *P < 0.05 and **P< 0.01, ***P<0.001 vs. the respective saline-treated animals (WT vs. WT+T3, KO vs. KO+T3), ⁺P< 0,05, ⁺⁺P< 0,01 and ⁺⁺⁺P< 0,001 for differences between WT and KO mice, as indicated.