Hyperkyphosis is not dependent on bone mass and quality in the mouse model of Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant disease affecting cardiovascular, ocular and skeletal systems. It is caused by mutations in the fibrillin-1 (FBN1) gene, leading to structural defects of connective tissue and increased activation of TGF-ß. Angiotensin II (ang-II) is involved in TGF-ß activity and in bone mass regulation. Inhibition of TGF-ß signaling by blockage of the ang-II receptor 1 (AT1R) via losartan administration leads to improvement of cardiovascular and pulmonary phenotypes, but has no effect on skeletal phenotype in the haploinsufficient mouse model of MFS mgR, suggesting a distinct mechanism of pathogenesis in the skeletal system. Here we characterized the skeletal phenotypes of the dominant-negative model for MFS mgΔlpn and tested the effect of inhibition of ang-II signaling in improving those phenotypes. As previously shown, heterozygous mice present hyperkyphosis, however we now show that only males also present osteopenia. Inhibition of ang-II production by ramipril minimized the kyphotic deformity, but had no effect on bone microstructure in male mutant animals. Histological analysis revealed increased thickness of the anterior longitudinal ligament (ALL) of the spine in mutant animals (25.8 ± 6.3 vs. 29.7 ± 7.7 µm), coupled with a reduction in type I (164.1 ± 8.7 vs. 139.0 ± 4.4) and increase in type III (86.5 ± 10.2 vs. 140.4 ± 5.6) collagen in the extracellular matrix of this ligament. In addition, we identified in the MFS mice alterations in the erector spinae muscles which presented thinner muscle fibers (1035.0 ± 420.6 vs. 655.6 ± 239.5 µm2) surrounded by increased area of connective tissue (58.17 ± 6.52 vs. 105.0 ± 44.54 µm2). Interestingly, these phenotypes were ameliorated by ramipril treatment. Our results reveal a sex-dependency of bone phenotype in MFS, where females do not present alterations in bone microstructure. More importantly, they indicate that hyperkyphosis is not a result of osteopenia in the MFS mouse model, and suggest that incompetent spine ligaments and muscles are responsible for the development of that phenotype.

White, brown, and bone marrow adipose tissue behavior in DHEA-induced PCOS mice.

METHODS: Thirty-one female C57BL/6J mice were divided into four groups: two were treated with subcutaneous dehydroepiandrosterone (DHEA) implants and divided into normal and hypercaloric diet (HFD). Two were control and divided into normal and HFD. Presence of insulin resistance, growth, and adipocyte markers expression of white and brown adipose tissues and growth and inflammatory cytokines expression of bone marrow adipose tissue were evaluated. RESULTS: Hypercaloric diet groups presented higher total weight gain and huge growth in all fat sites, except bone marrow. They also demonstrated greater expression of adipocyte markers in sites of white adipose tissue. DHEA + HFD group showed more insulin intolerance than all other groups. DHEA shows to abrogate AdipoQ expression in all fatty tissues. CONCLUSIONS: DHEA alone does not influence adipose tissue growth, but contributes to increased insulin resistance and influences the expression of adipokines. Proximal MAT showed different behavior from the other fat depot.

Thyrotoxicosis Involves ß2-Adrenoceptor Signaling to Negatively Affect Microarchitecture and Biomechanical Properties of the Femur.

Background: Thyrotoxicosis increases bone turnover, resulting in net bone loss. Sympathetic nervous system (SNS) activation, via ß2-adrenoceptor (ß2-AR) signaling, also has osteopenic effects. Because thyroid hormones (TH) interact with the SNS to regulate several physiological processes, we hypothesized that this interaction also occurs to regulate bone mass. Previous studies support this hypothesis, as α2-AR knockout (KO) mice are less susceptible to thyrotoxicosis-induced osteopenia. Here, we evaluated whether TH-SNS interactions in bone involve ß2-AR signaling. Methods: Thyrotoxicosis was induced in 120-day-old female and male mice with ß2-AR gene inactivation (ß2-AR-/-) by daily treatment with supraphysiological doses of triiodothyronine (T3) for 12 weeks. The impact of thyrotoxicosis on femoral bone microarchitecture, remodeling, fracture risk, and gene expression of the receptor activator of nuclear factor-kappa-B (RANK)-RANK ligand (RANKL)-osteoprotegerin (OPG) pathway was evaluated. In addition, the effect of the ß2-AR-specific agonist clenbuterol (CL) on cAMP accumulation was determined in osteoblastic (MC3T3-E1) cells treated with T3 and/or 17ß-estradiol (E2). Results: Thyrotoxicosis negatively affected trabecular bone microarchitecture in wild-type (WT) females, but this effect was milder or nonexistent in ß2-AR-/- animals, whereas the opposite was seen in males. T3 treatment increased the femoral RANKL/OPG mRNA ratio and the endosteal perimeter and medullary area of the diaphysis in WT females and males, but not in ß2-AR-/- mice, suggesting that T3 promotes endosteal resorption in cortical bone, in a mechanism that involves ß2-AR signaling. T3 treatment increased endocortical mineral apposition rate only in WT females but not in ß2-AR-/- mice, suggesting that TH also induce bone formation in a ß2-AR signaling-dependent mechanism. T3 treatment decreased femoral resistance to fracture only in WT females, but not in KO mice. E2 and CL similarly increased cAMP accumulation in MC3T3-E1 cells; whereas T3 alone had no effect, but it completely blocked E2-stimulated cAMP accumulation, suggesting that some T3 effects on bone may involve E2/cAMP signaling in osteoblasts. Conclusions: These findings sustain the hypothesis that T3 interacts with the SNS to regulate bone morphophysiology in a ß2-AR signaling-dependent mechanism. The data also reveal sex as an important modifier of skeletal manifestations of thyrotoxicosis, as well as a modifier of the TH-SNS interactions to control bone microarchitecture, remodeling, and resistance to fracture.

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

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 α2A-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.

Thyroid Hormone and Skeletal Development.

Thyroid hormone (TH) is essential for skeletal development from the late fetal life to the onset of puberty. During this large window of actions, TH has key roles in endochondral and intramembranous ossifications and in the longitudinal bone growth. There is evidence that TH acts directly in skeletal cells but also indirectly, specially via the growth hormone/insulin-like growth factor-1 axis, to control the linear skeletal growth and maturation. The presence of receptors, plasma membrane transporters, and activating and inactivating enzymes of TH in skeletal cells suggests that direct actions of TH in these cells are crucial for skeletal development, which has been confirmed by several in vitro and in vivo studies, including mouse genetic studies, and clinical studies in patients with resistance to thyroid hormone due to dominant-negative mutations in TH receptors. This review examines progress made on understanding the mechanisms by which TH regulates the skeletal development.