Global Disruption of α2A Adrenoceptor Barely Affects Bone Tissue but Minimizes the Detrimental Effects of Thyrotoxicosis on Cortical Bone

Abstract

Evidence shows that sympathetic nervous system (SNS) activation inhibits bone formation and activates bone resorption leading to bone loss. Because thyroid hormone (TH) interacts with the SNS to control several physiological processes, we raised the hypothesis that this interaction also controls bone remodeling. We have previously shown that mice with double-gene inactivation of α2A- and -adrenoceptors (α2A/2C-AR^-/-) present high bone mass (HBM) phenotype and resistance to thyrotoxicosis-induced osteopenia, which supports a TH-SNS interaction to control bone mass and suggests that it involves α2-AR signaling. Accordingly, we detected expression of α2A-AR, α2B-AR and α2C-AR in the skeleton, and that triiodothyronine (T3) modulates α2C-AR mRNA expression in the bone. Later, we found that mice with single-gene inactivation of α2C-AR (α2C-AR^-/-) present low bone mass in the femur and HBM in the vertebra, but that both skeletal sites are resistant to TH-induce osteopenia, showing that the SNS actions occur in a skeletal site-dependent manner, and that thyrotoxicosis depends on α2C-AR signaling to promote bone loss. To further dissect the specific roles of α2-AR subtypes, in this study, we evaluated the skeletal phenotype of mice with single-gene inactivation of α2A-AR (α2A-AR^-/-), and the effect of daily treatment with a supraphysiological dose of T3, for 4 or 12 weeks, on bone microarchitecture and bone resistance to fracture. Micro-computed tomographic (μCT) analysis revealed normal trabecular and cortical bone structure in the femur and vertebra of euthyroid α_2A-AR^-/- mice. Thyrotoxicosis was more detrimental to femoral trabecular bone in α2A-AR^-/- than in WT mice, whereas this bone compartment had been previously shown to present resistance to thyrotoxicosis in α2C-AR^-/- mice. Altogether these findings reveal that TH excess depends on α2C-AR signaling to negatively affect femoral trabecular bone. In contrast, thyrotoxicosis was more deleterious to femoral and vertebral cortical bone in WT than in α2A-AR^-/- mice, suggesting that α2A-AR signaling contributes to TH actions on cortical bone. These findings further support a TH-SNS interaction to control bone physiology, and suggest that α2A-AR and α2C-AR signaling pathways have key roles in the mechanisms through which thyrotoxicosis promotes its detrimental effects on bone remodeling, structure and resistance to fracture.

Keywords: bone remodeling; cortical bone; sympathetic nervous system; thyroid hormone; thyrotoxicosis; trabecular bone; α2A-adrenoceptor.

Figure 1

Serum levels of T3 and T4 in α_2A-AR^−/− and WT mice. Animals were treated with a supraphysiological dose of T3 (7 μg/100 g BW/day) or saline (-) for 4 or 12 weeks, by daily i.p. injections. Values are expressed as mean ± SEM (n = 7 per group). ^*P < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001 vs. the respective saline-treated mice, by Two-way ANOVA followed by Tukey's test.

Figure 2

Effect of thyrotoxicosis on body weight of α_2A-AR^−/− and WT mice. Animals were treated with a supraphysiological dose of T3 (7 μg/100 g BW/day) or saline for 12 weeks, by daily i.p. injections. Body weight was measured every week. Values are expressed as mean ± SEM (n = 7 per group). ⁺⁺P < 0.01, and ⁺⁺⁺p < 0.001 vs. WT; and ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 vs. the respective saline-treated mice, by Two-way ANOVA followed by Tukey's test.

Figure 3

Effect of thyrotoxicosis on fat mass, muscle mass and heart mass of α_2A-AR^−/− and WT mice. Animals were treated with a supraphysiological dose of T3 (7 μg/100 g body mass/day) or saline (-) for 4 or 12 weeks, by daily i.p. injections. Values are expressed as mean ± SEM (n = 7 per group). ^**P < 0.01 and ^***p < 0.001 vs. the respective saline-treated mice, by Two-way ANOVA followed by Tukey's test. P-values above the bars indicate differences between WT and KO mice.

Figure 4

Effect of thyrotoxicosis on μCT parameters of trabecular and cortical bone of the femur in α_2A-AR^−/− and WT mice. Animals were treated with a supraphysiological dose of T3 (7 μg/100 g body mass/day) or saline (-) for 4 or 12 weeks, by daily i.p. injections. Values are expressed as mean ± SEM (n = 7 per group). ^*P < 0.05, ^**p < 0.01, and ^***p < 0.001 vs. the respective saline-treated mice, by Two-way ANOVA followed by Tukey's test. BV/TV, trabecular bone volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; SMI, structure model index; Tb.Pf, trabecular pattern function; Tb.Po, trabecular porosity; BMD, bone mineral density; Tt.Ar, total tissue area; Ct.Ar, cortical area; Ma.Ar, medullary area; Ct.Th, cortical thickness; Ps.Pm, periosteal perimeter; Ec.Pm, endocortical perimeter; and Ct.Po, cortical porosity. (A–H) Trabecular bone microarchitecture. (I–P) Cortical bone microarchitecture. (Q) μCT images of the distal methaphysis of the femur.

Figure 5

Effect of thyrotoxicosis on μCT parameters of trabecular and cortical bone of the vertebral body of L5 in α_2A-AR^−/− and WT mice. Animals were treated with a supraphysiological dose of T3 (7 μg/100 g body mass/day) or saline (-) for 4 or 12 weeks, by daily i.p. injections. Values are expressed as mean ± SEM (n = 7 per group). ^*P < 0.05, ^**p < 0.01, and ^***p < 0.001 vs. the respective saline-treated mice, by Two-way ANOVA followed by Tukey's test. BV/TV, trabecular bone volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; SMI, structure model index; Tb.Pf, trabecular pattern function; Tb.Po, trabecular porosity; BMD, bone mineral density; Tt.Ar, total tissue area; Ct.Ar, cortical area; Ma.Ar, medullary area; Ct.Th, cortical thickness; Ps.Pm, periosteal perimeter; Ec.Pm, endocortical perimeter; and Ct.Po, cortical porosity. (A–H) Trabecular bone microarchitecture. (I–P) Cortical bone microarchitecture.

Figure 6

Effect of thyrotoxicosis on biomechanical parameters of the femur of α_2A-AR^−/− and WT mice. Animals were treated with a supraphysiological dose of T3 (7 μg/100 g body mass/day) or saline (-) for 4 or 12 weeks, by daily i.p. injections. Values are expressed as mean ± SEM (n = 7 per group). ^*P < 0.05 and ^**p < 0.01 vs. the respective saline-treated mice, by Two-way ANOVA followed by Tukey's test.

Figure 7

Effect of thyrotoxicosis on the relative mRNA expression of α_2A-, α_2B-, α_2C-, β1- and β2-adrenoceptors in the femur. Animals were treated with a supraphysiological dose of T3 (7 μg/100 g body mass/day) or saline (-) for 12 weeks, by daily i.p. injections. mRNA expression in the whole femur was determined by real-time PCR analysis. Values are expressed as mean ± SEM (n = 7 per group). ^*P < 0.05 vs. the respective saline-treated mice, by unpaired Student t-test or by Two-way ANOVA followed by Tukey's test.

Figure 8

Schematic representation of a possible balance between α₂-AR and β2-AR signaling to control femoral trabecular bone in the distal metaphysis of the femur in α_2A/2C-AR^−/−, α_2C-AR^−/− and α_2A-AR^−/− and WT mice. All KO mouse models present increased norepinephrine (NE) release and, therefore, increased sympathetic activation. (A) In α_2A/2C-AR^−/− mice, the lack of α2A-AR predominantly results in anabolic actions that overcomes the catabolic effects of β2-AR activation and the lack of α_2C-AR anabolic actions, resulting in increased trabecular bone volume (BV/TV). (B) In α2C-AR^−/− mice, the osteopenic actions of α2A-AR and β2-AR and the lack of α2C-AR-mediated anabolism results in lower BV/TV. (C) In α2A-AR^−/− mice, a balance between α2C-AR-mediated anabolism and β2-AR-mediated catabolism could result in normal BV/TV. (D) In WT mice, since NE release is normal, α2-AR and β2-AR are functioning at basal levels, and in equilibrium, to maintain bone mass. (C,D) T3 inhibits α2C-AR signaling, which suppresses the α2C-AR-mediated trabecular anabolism, leading to decreased BV/TV. ↑, ↓ and = vs. WT. This is a simplified scheme that does not includes other adrenoceptors that may mediate some of the effects observed in this study.