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.

Abnormal Thyroid Hormone Status Differentially Affects Bone Mass Accrual and Bone Strength in C3H/HeJ Mice: A Mouse Model of Type I Deiodinase Deficiency.

C3H/HeJ (C3H) mice are deficient of type I deiodinase (D1), an enzyme that activates thyroid hormone (TH), converting thyroxine (T4) to triiodothyronine (T3). Nevertheless, C3H mice present normal serum T3 and a gross euthyroid phenotype. To investigate if a global D1 deficiency interferes in the TH effects on bone, we compared bone growth, bone mass accrual and bone strength of C3H and C57BL/6J (B6) mice under abnormal TH status. Four-week-old female mice of both strains were grouped as Euthyroid, Hypothyroid (pharmacologically-induced), 1xT4 and 10xT4 (hypothyroid animals receiving 1- or 10-fold the physiological dose of T4 /day/16 weeks). Hypothyroidism and TH excess similarly impaired body weight (BW) gain and body growth in both mice strains. In contrast, whereas hypothyroidism only slightly impaired bone mineral density (BMD) accrual in B6 mice, it severely impaired BMD accrual in C3H mice. No differences were observed in serum and bone concentrations of T3 between hypothyroid animals of both strains. Interestingly, treatment with 10xT4 was less deleterious to BMD accrual in C3H than in B6 mice and resulted in less elevated T3 serum levels in B6 than in C3H mice, which is probably explained by the lower D1 activity in C3H mice. In addition, hypothyroidism decreased bone strength only in C3H but not in B6 mice, while TH excess decreased this parameter in both strains. These findings indicate that D1 deficiency contributes to the TH excess-induced differences in bone mass accrual in C3H vs. B6 mice and suggest that deiodinase-unrelated genetic factors might account for the different skeleton responses to hypothyroidism between strains.

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.

S-nitrosoglutathione reductase-dependent PPARγ denitrosylation participates in MSC-derived adipogenesis and osteogenesis.

Bone marrow-derived mesenchymal stem cells (MSCs) are a common precursor of both adipocytes and osteoblasts. While it is appreciated that PPARγ regulates the balance between adipogenesis and osteogenesis, the roles of additional regulators of this process remain controversial. Here, we show that MSCs isolated from mice lacking S-nitrosoglutathione reductase, a denitrosylase that regulates protein S-nitrosylation, exhibited decreased adipogenesis and increased osteoblastogenesis compared with WT MSCs. Consistent with this cellular phenotype, S-nitrosoglutathione reductase-deficient mice were smaller, with reduced fat mass and increased bone formation that was accompanied by elevated bone resorption. WT and S-nitrosoglutathione reductase-deficient MSCs exhibited equivalent PPARγ expression; however, S-nitrosylation of PPARγ was elevated in S-nitrosoglutathione reductase-deficient MSCs, diminishing binding to its downstream target fatty acid-binding protein 4 (FABP4). We further identified Cys 139 of PPARγ as an S-nitrosylation site and demonstrated that S-nitrosylation of PPARγ inhibits its transcriptional activity, suggesting a feedback regulation of PPARγ transcriptional activity by NO-mediated S-nitrosylation. Together, these results reveal that S-nitrosoglutathione reductase-dependent modification of PPARγ alters the balance between adipocyte and osteoblast differentiation and provides checkpoint regulation of the lineage bifurcation of these 2 lineages. Moreover, these findings provide pathophysiological and therapeutic insights regarding MSC participation in adipogenesis and osteogenesis.

Thyroid hormone interacts with the sympathetic nervous system to modulate bone mass and structure in young adult mice.

To investigate whether thyroid hormone (TH) interacts with the sympathetic nervous system (SNS) to modulate bone mass and structure, we studied the effects of daily T3 treatment in a supraphysiological dose for 12 wk on the bone of young adult mice with chronic sympathetic hyperactivity owing to double-gene disruption of adrenoceptors that negatively regulate norepinephrine release, α(2A)-AR, and α(2C)-AR (α(2A/2C)-AR(-/-) mice). As expected, T3 treatment caused a generalized decrease in the areal bone mineral density (aBMD) of WT mice (determined by DEXA), followed by deleterious effects on the trabecular and cortical bone microstructural parameters (determined by µCT) of the femur and vertebra and on the biomechanical properties (maximum load, ultimate load, and stiffness) of the femur. Surprisingly, α(2A/2C)-AR(-/-) mice were resistant to most of these T3-induced negative effects. Interestingly, the mRNA expression of osteoprotegerin, a protein that limits osteoclast activity, was upregulated and downregulated by T3 in the bone of α(2A/2C)-AR(-/-) and WT mice, respectively. ß1-AR mRNA expression and IGF-I serum levels, which exert bone anabolic effects, were increased by T3 treatment only in α(2A/2C)-AR(-/-) mice. As expected, T3 inhibited the cell growth of calvaria-derived osteoblasts isolated from WT mice, but this effect was abolished or reverted in cells isolated from KO mice. Collectively, these findings support the hypothesis of a TH-SNS interaction to control bone mass and structure of young adult mice and suggests that this interaction may involve α2-AR signaling. Finally, the present findings offer new insights into the mechanisms through which TH regulates bone mass, structure, and physiology.

Inactivation of the adrenergic receptor ß2 disrupts glucose homeostasis in mice.

Three types of beta adrenergic receptors (ARß1-3) mediate the sympathetic activation of brown adipose tissue (BAT), the key thermogenic site for mice which is also present in adult humans. In this study, we evaluated adaptive thermogenesis and metabolic profile of a mouse with Arß2 knockout (ARß2KO). At room temperature, ARß2KO mice have normal core temperature and, upon acute cold exposure (4 °C for 4 h), ARß2KO mice accelerate energy expenditure normally and attempt to maintain body temperature. ARß2KO mice also exhibited normal interscapular BAT thermal profiles during a 30-min infusion of norepinephrine or dobutamine, possibly due to marked elevation of interscapular BAT (iBAT) and of Arß1, and Arß3 mRNA levels. In addition, ARß2KO mice exhibit similar body weight, adiposity, fasting plasma glucose, cholesterol, and triglycerides when compared with WT controls, but exhibit marked fasting hyperinsulinemia and elevation in hepatic Pepck (Pck1) mRNA levels. The animals were fed a high-fat diet (40% fat) for 6 weeks, ARß2KO mice doubled their caloric intake, accelerated energy expenditure, and induced Ucp1 expression in a manner similar to WT controls, exhibiting a similar body weight gain and increase in the size of white adipocytes to the WT controls. However, ARß2KO mice maintain fasting hyperglycemia as compared with WT controls despite very elevated insulin levels, but similar degrees of liver steatosis and hyperlipidemia. In conclusion, inactivation of the ARß2KO pathway preserves cold- and diet-induced adaptive thermogenesis but disrupts glucose homeostasis possibly by accelerating hepatic glucose production and insulin secretion. Feeding on a high-fat diet worsens the metabolic imbalance, with significant fasting hyperglycemia but similar liver structure and lipid profile to the WT controls.

ß(1) Adrenergic receptor is key to cold- and diet-induced thermogenesis in mice.

Brown adipose tissue (BAT) is predominantly regulated by the sympathetic nervous system (SNS) and the adrenergic receptor signaling pathway. Knowing that a mouse with triple ß-receptor knockout (KO) is cold intolerant and obese, we evaluated the independent role played by the ß(1) isoform in energy homeostasis. First, the 30  min i.v. infusion of norepinephrine (NE) or the ß(1) selective agonist dobutamine (DB) resulted in similar interscapular BAT (iBAT) thermal response in WT mice. Secondly, mice with targeted disruption of the ß(1) gene (KO of ß(1) adrenergic receptor (ß(1)KO)) developed hypothermia during cold exposure and exhibited decreased iBAT thermal response to NE or DB infusion. Thirdly, when placed on a high-fat diet (HFD; 40% fat) for 5 weeks, ß(1)KO mice were more susceptible to obesity than WT controls and failed to develop diet-induced thermogenesis as assessed by BAT Ucp1 mRNA levels and oxygen consumption. Furthermore, ß(1)KO mice exhibited fasting hyperglycemia and more intense glucose intolerance, hypercholesterolemia, and hypertriglyceridemia when placed on the HFD, developing marked non-alcoholic steatohepatitis. In conclusion, the ß(1) signaling pathway mediates most of the SNS stimulation of adaptive thermogenesis.

Double disruption of α2A- and α2C-adrenoceptors results in sympathetic hyperactivity and high-bone-mass phenotype.

Evidence demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via ß(2)-adrenoceptor (ß2-AR) signaling. Here we show that female mice with chronic sympathetic hyperactivity owing to double knockout of adrenoceptors that negatively regulate norepinephrine release, α(2A)-AR and α(2C)-AR (α(2A) /α(2C)-ARKO), present an unexpected and generalized phenotype of high bone mass with decreased bone resorption and increased formation. In α(2A) /α(2C)-ARKO versus wild-type (WT) mice, micro-computed tomographic (µCT) analysis showed increased, better connected, and more plate-shaped trabeculae in the femur and vertebra and increased cortical thickness in the vertebra, whereas biomechanical analysis showed increased tibial and femoral strength. Tibial mRNA expression of tartrate-resistant acid phosphatase (TRACP) and receptor activator of NF-κB (RANK), which are osteoclast-related factors, was lower in knockout (KO) mice. Plasma leptin and brain mRNA levels of cocaine amphetamine-regulated transcript (CART), which are factors that centrally affect bone turnover, and serum levels of estradiol were similar between mice strains. Tibial ß(2)-AR mRNA expression also was similar in KO and WT littermates, whereas α(2A)-, α(2B)- and α(2C)-AR mRNAs were detected in the tibia of WT mice and in osteoblast-like MC3T3-E1 cells. By immunohistochemistry, we detected α(2A)-, α(2B)-, α(2C)- and ß(2)-ARs in osteoblasts, osteoclasts, and chondrocytes of 18.5-day-old mouse fetuses and 35-day-old mice. Finally, we showed that isolated osteoclasts in culture are responsive to the selective α(2)-AR agonist clonidine and to the nonspecific α-AR antagonist phentolamine. These findings suggest that ß(2)-AR is not the single adrenoceptor involved in bone turnover regulation and show that α(2)-AR signaling also may mediate the SNS actions in the skeleton.

The thyroid hormone receptor (TR) beta-selective agonist GC-1 inhibits proliferation but induces differentiation and TR beta mRNA expression in mouse and rat osteoblast-like cells.

Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) beta1 over TRalpha1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TRalpha1 than TRbeta1 mRNA in rat (approximately 20-90%) and mouse (approximately 90-98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TRalpha1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TRbeta1 mRNA expression increases (approximately 2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TRbeta1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TRbeta1 mRNA expression to a similar extent in both cell lineages (approximately 2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TRbeta selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts.

The monocarboxylate transporter 8 and L-type amino acid transporters 1 and 2 are expressed in mouse skeletons and in osteoblastic MC3T3-E1 cells.

BACKGROUND: Several plasma membrane transporters have been shown to mediate the cellular influx and/or efflux of iodothyronines, including the sodium-independent organic anion co-transporting polypeptide 1 (OATP1), the sodium taurocholate co-transporting polypeptide (NTCP), the L-type amino acid transporter 1 (LAT1) and 2 (LAT2), and the monocarboxylate transporter 8 (MCT8). The aim of this study was to investigate if the mRNAs of these transporters were expressed and regulated by thyroid hormone (TH) in mouse calvaria-derived osteoblastic MC3T3-E1 cells and in the fetal and postnatal bones of mice. METHODS: The mRNA expression of the iodothyronine transporters was investigated with real-time polymerase chain reaction analysis in euthyroid and hypothyroid fetuses and litters of mice and in MC3T3-E1 cells treated with increasing doses of triiodothyronine (T(3); 10(-10) to 10(-6) M) or with 10(-8) M T(3) for 1-9 days. RESULTS: MCT8, LAT1, and LAT2 mRNAs were detected in fetal and postnatal femurs and in MC3T3-E1 cells, while OATP1 and NTCP mRNAs were not. LAT1 and LAT2 mRNAs were not affected by TH status in vivo or in vitro or by the stage of bone development or osteoblast maturation (analyzed by the expression of osteocalcin and alkaline phosphatase, which are key markers of osteoblastic differentiation). In contrast, the femoral mRNA expression of MCT8 decreased significantly during post-natal development, whereas MCT8 mRNA expression increased as MC3T3-E1 cells differentiated. We also showed that MCT8 mRNA was up-regulated in the femur of hypothyroid animals, and that it was down-regulated by treatment with T(3) in MC3T3-E1 cells. CONCLUSIONS: This is the first study to demonstrate the mRNA expression of LAT1, LAT2, and MCT8 in the bone tissue of mice and in osteoblast-like cells. In addition, the pattern of MCT8 expression observed in vivo and in vitro suggests that MCT8 may be important to modulate TH effects on osteoblast differentiation and on bone development and metabolism.

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton.

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To study this, pregnant mice were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved up to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocytes, in addition to a slight delay in endochondral and intramembranous ossification. This suggests that up to E16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly ( approximately 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast, D2 mRNA expression increased significantly by E18.5 and markedly ( approximately 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development.

Thyroid hormone receptor beta-specific agonist GC-1 increases energy expenditure and prevents fat-mass accumulation in rats.

It is well known that thyroid hormone affects body composition; however, the effect of the thyroid hormone receptor beta (TRbeta)-selective thyromimetic GC-1 on this biological feature had not been demonstrated. In the current study, we compared the effects of a 6-week treatment with triiodothyronine (T3; daily injections of 3 or 6 microg/100 g body weight) or GC-1 (equimolar doses) on different metabolic parameters in adult female rats. Whereas all animals gained weight (17-25 g) in a way not basically affected by T3 or GC-1 treatment, only T3 treatment selectively increased food intake (50-70%). Oxygen consumption was significantly and equally increased (50-70%) by T3 and GC-1. Analysis of body composition by dual-energy X-ray absorptiometry (DEXA) revealed that, whereas control animals gained about 80% of fat mass, T3- or GC-1-treated animals lost 70-90 and approximately 20% respectively. Direct analysis of the carcass showed that T3 treatment promoted a 14-74% decrease in fat content but GC-1 treatment promoted only a 15-23% reduction. The gain in lean mass by DEXA and the carcass protein content were not affected by T3 or GC-1 treatment. However, the mass of individual skeletal muscles was negatively affected by T3 but only barely by GC-1. These findings highlight the potential use of GC-1 for the treatment of obesity and the metabolic syndrome.

Marked disturbance of calcium homeostasis in mice with targeted disruption of the Trpv6 calcium channel gene.

UNLABELLED: We report the phenotype of mice with targeted disruption of the Trpv6 (Trpv6 KO) epithelial calcium channel. The mice exhibit disordered Ca(2+) homeostasis, including defective intestinal Ca(2+) absorption, increased urinary Ca(2+) excretion, decreased BMD, deficient weight gain, and reduced fertility. Although our Trpv6 KO affects the closely adjacent EphB6 gene, the phenotype reported here is not related to EphB6 dysfunction. INTRODUCTION: The mechanisms underlying intestinal Ca(2+) absorption are crucial for overall Ca(2+) homeostasis, because diet is the only source of all new Ca(2+) in the body. Trpv6 encodes a Ca(2+)-permeable cation channel responsible for vitamin D-dependent intestinal Ca(2+) absorption. Trpv6 is expressed in the intestine and also in the skin, placenta, kidney, and exocrine organs. MATERIALS AND METHODS: To determine the in vivo function of TRPV6, we generated mice with targeted disruption of the Trpv6 (Trpv6 KO) gene. RESULTS: Trpv6 KO mice are viable but exhibit disordered Ca(2+) homeostasis, including a 60% decrease in intestinal Ca(2+) absorption, deficient weight gain, decreased BMD, and reduced fertility. When kept on a regular (1% Ca(2+)) diet, Trpv6 KO mice have deficient intestinal Ca(2+) absorption, despite elevated levels of serum PTH (3.8-fold) and 1,25-dihydroxyvitamin D (2.4-fold). They also have decreased urinary osmolality and increased Ca(2+) excretion. Their serum Ca(2+) is normal, but when challenged with a low (0.25%) Ca(2+) diet, Trpv6 KO mice fail to further increase serum PTH and vitamin D, ultimately developing hypocalcemia. Trpv6 KO mice have normal urinary deoxypyridinoline excretion, although exhibiting a 9.3% reduction in femoral mineral density at 2 months of age, which is not restored by treatment for 1 month with a high (2%) Ca(2+) "rescue" diet. In addition to their deranged Ca(2+) homeostasis, the skin of Trpv6 KO mice has fewer and thinner layers of stratum corneum, decreased total Ca(2+) content, and loss of the normal Ca(2+) gradient. Twenty percent of all Trpv6 KO animals develop alopecia and dermatitis. CONCLUSIONS: Trpv6 KO mice exhibit an array of abnormalities in multiple tissues/organs. At least some of these are caused by tissue-specific mechanisms. In addition, the kidneys and bones of Trpv6 KO mice do not respond to their elevated levels of PTH and 1,25-dihydroxyvitamin D. These data indicate that the TRPV6 channel plays an important role in Ca(2+) homeostasis and in other tissues not directly involved in this process.

Thyroid hormone receptor-beta-selective agonist GC-24 spares skeletal muscle type I to II fiber shift.

Triiodothyronine (T3) is known to play a key role in the function of several tissues/organs via the thyroid hormone receptor isoforms alpha (TRalpha) and beta (TRbeta). We have investigated the effects of GC-24, a novel synthetic TRbeta-selective compound, on skeletal muscle fiber-type determination, cross-sectional area, and gene expression in rat skeletal muscles. For fiber typing, cross sections of soleus and extensor digitorum longus (EDL) muscles were stained for myosin ATPase activity at various pHs. Serum T3, T4, and cholesterol levels were also determined. Analysis of highly T3-responsive genes, viz., myosin heavy chain IIa (MHCIIa) and sarcoendoplasmic reticulum adenosine triphosphatase (SERCA1), was performed by quantitative real-time polymerase chain reaction. Equimolar doses of T3 and GC-24 had a similar cholesterol-lowering effect. T3, but not GC-24, decreased fiber type I and increased fiber type II abundance in soleus and EDL muscles. Conversely, in EDL, both T3 and GC-24 decreased the mean cross-sectional area of type I fibers. MHCIIa gene expression was reduced (approximately 50%) by T3 and unchanged by GC-24. SERCA1 gene expression was strongly induced by T3 (approximately 20-fold) and mildly induced by GC-24 (approximately two-fold). These results show that GC-24 does not significantly alter the composition of skeletal muscle fiber type and further strengthens the putative use of GC compounds as therapeutic agents.

The thyroid hormone receptor beta-specific agonist GC-1 selectively affects the bone development of hypothyroid rats.

UNLABELLED: We investigated the effects of GC-1, a TRbeta-selective thyromimetic, on bone development of hypothyroid rats. Whereas T3 reverted the IGF-I deficiency and the skeletal defects caused by hypothyroidism, GC-1 had no effect on serum IGF-I or on IGF-I protein expression in the epiphyseal growth plate of the femur, but induced selective effects on bone development. Our findings indicate that T3 exerts some essential effects on bone development that are mediated by TRbeta1. INTRODUCTION: We investigated the role of the thyroid hormone receptor beta1 (TRbeta1) on skeletal development of rats using the TRbeta-selective agonist GC-1. MATERIALS AND METHODS: Twenty-one-day-old female rats (n = 6/group) were rendered hypothyroid (Hypo) and treated for 5 weeks with 0.3 ug/100 g BW/day of T3 (1xT3), 5xT3, or equimolar doses of GC-1 (1xGC-1 and 5xGC-1). Serum triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH), and insulin-like growth factor (IGF)-I concentrations were determined by radioimmunoassay (RIA). BMD and longitudinal bone growth were determined by DXA. Trabecular bone histomorphometry and epiphyseal growth plate (EGP) morphometry were performed in the distal femur. Expressions of IGF-I protein and of collagen II and X mRNA were evaluated by immunohistochemistry and in situ hybridization, respectively. To determine hormonal effects on ossification, skeletal preparations of hypothyroid-, 5xGC-1-, and 5xT3-treated neonatal rats were compared. RESULTS: Hypothyroidism impaired longitudinal body growth and BMD gain, delayed ossification, reduced the number of hypertrophic chondrocytes (HCs; 72% versus Euthyroid [Eut] rats; p < 0.001), and resulted in disorganized columns of EGP chondrocytes. Serum IGF-I was 67% reduced versus Eut rats (p < 0.001), and the expression of IGF-I protein and collagen II and X mRNA were undetectable in the EGP of Hypo rats. T3 completely or partially normalized all these parameters. In contrast, GC-1 did not influence serum concentrations or EGP expression of IGF-I, failed to reverse the disorganization of proliferating chondrocyte columns, and barely affected longitudinal growth. Nevertheless, GC-1 induced ossification, HC differentiation, and collagen II and X mRNA expression and increased EGP thickness to Eut values. GC-1-treated rats had higher BMD gain in the total tibia, total femur, and in the femoral diaphysis than Hypo animals (p < 0.05). These changes were associated with increased trabecular volume (48%, p < 0.01), mineralization apposition rate (2.3-fold, p < 0.05), mineralizing surface (4.3-fold, p < 0.01), and bone formation rate (10-fold, p < 0.01). CONCLUSIONS: Treatment of hypothyroid rats with the TRbeta-specific agonist GC-1 partially reverts the skeletal development and maturation defects resultant of hypothyroidism. This finding suggests that TRbeta1 has an important role in bone development.

Type 2 iodothyronine selenodeiodinase is expressed throughout the mouse skeleton and in the MC3T3-E1 mouse osteoblastic cell line during differentiation.

Thyroid hormone affects multiple aspects of bone metabolism, but little is known about thyroid hormone deiodination in bone cells except that cultures of skeletal cells and bone organ express types 1 and 2 iodothyronine deiodinases (D1 and D2) mRNAs. In the present study, outer ring deiodination (ORD) activity was detected in bone extracts of multiple sites of the mouse skeleton, bone marrow, and the MC3T3-E1 osteoblastic cell line. In all tissues, ORD was detected using 125I-rT3 or 125I-T4 as substrates and was found to be 6-n-propylthiouracil insensitive, display a Michaelis constant (T4) of approximately 1 nM, increase about 3-fold in hypo- and virtually disappear in thyrotoxicosis. Extracts of calvaria had the lowest ORD activity, whereas tibial and femoral extracts had roughly three times as much. The absence of ORD activity in bone extracts from mice with targeted disruption of the Dio2 gene confirms the principal role of D2 in this tissue. In the MC3T3-E1 osteoblasts, D2 activity increased in a time-dependent manner after plating, and with the content of selenium in the media, reaching a maximum 5-7 d later as cells attained more than 90% confluence. In these cells D2 half-life is about 30-40 min, which is further accelerated by exposure to substrate and stabilized by the proteasome inhibitor, MG132. Treatment with vitamin D [1,25(OH)2VD]-induced D2 activity by 2- to 3-fold as early as 24 h, regardless of the level of cell confluence, but estradiol, PTH, forskolin, leptin, TNFalpha, TGFbeta, and dexamethasone did not affect D2. Given the role of D2 in other cell types and processes, it is likely that bone ORD not only plays a role in bone development and adult bone T3 homeostasis but also contributes to extrathyroidal T3 production and maintenance of serum T3.

[The molecular and structural effects of thyroid hormone in bones]. / O efeito molecular e estrutural do hormônio tiroideano no esqueleto.

During development, thyroid hormone deficiency results in delayed skeletal maturation and epiphyseal dysgenesis, resulting in reduced growth and skeletal abnormalities. Thyroid hormone also has effects on bones of adults. Thyrotoxicosis is frequently associated with increased bone turnover and decreased bone mass. However, the mechanisms that mediate its effects on bone tissue are poorly understood. Thyroid hormone acts indirectly in the skeleton, by increasing the secretion of growth hormone and insulin-like growth factor-1; or directly, by modulating target genes via specific nuclear receptors. In vitro findings, such as the presence of thyroid receptors (TRs) and the induction of genes and proteins in skeletal cells by thyroid hormone, emphasize the importance of direct actions. The aim of this review is to summarize the in vivo and in vitro findings related to the effects of thyroid hormone on the skeleton.

O efeito molecular e estrutural do hormônio tiroideano no esqueleto / The molecular and structural effects of thyroid hormone in the skeleton

O hormônio tiroideano é essencial para o desenvolvimento, maturação e metabolismo ósseos normais. Durante o desenvolvimento, a deficiência do hormônio tiroideano resulta em atraso na maturação do esqueleto e disgênese das epífises, resultando em redução do crescimento e anormalidades esqueléticas. O hormônio tiroideano também tem efeito no osso do adulto. A tirotoxicose é freqüentemente associada ao aumento do metabolismo ósseo e diminuição da massa óssea. Embora a importância do hormônio tiroideano no desenvolvimento e metabolismo ósseos seja clara, os mecanismos que medeiam os efeitos desse hormônio no tecido ósseo apenas começam a ser desvendados. O hormônio tiroideano pode atuar indiretamente no esqueleto, aumetando a secreção de hormônio do crescimento (GH) e insulin-like growth factor-1 (IGF-1); ou diretamente, modulando genes alvo via receptores nucleares específicos. Não se sabe, entretanto, se os principais efeitos do hormônio tiroideano no osso são resultado de ações diretas ou indiretas. Achados in vitro, tais como a presença de receptores de hormônio tiroideano (TR) e a indução de genes e proteínas em células esqueléticas pelo hormônio tiroideano, evidenciam a importância de ações diretas. Esta revisão tem como meta sumarizar os achados in vivo e in vitro relacionados aos efeitos do hormônio tiroideano no esqueleto.

Spared bone mass in rats treated with thyroid hormone receptor TR beta-selective compound GC-1.

Thyrotoxicosis is frequently associated with increased bone turnover and decreased bone mass. To investigate the role of thyroid hormone receptor-beta (TR beta) in mediating the osteopenic effects of triiodothyronine (T3), female adult rats were treated daily (64 days) with GC-1 (1.5 microg/100 g body wt), a TR beta-selective thyromimetic compound. Bone mass was studied by dual-energy X-ray absorptiometry of several skeletal sites and histomorphometry of distal femur, and the results were compared with T3-treated (3 microg/100 g body wt) or control animals. As expected, treatment with T3 significantly reduced bone mineral density (BMD) in the lumbar vertebrae (L2-L5), femur, and tibia by 10-15%. In contrast, GC-1 treatment did not affect the BMD in any of the skeletal sites studied. The efficacy of GC-1 treatment was verified by a reduction in serum TSH (-52% vs. control, P < 0.05) and cholesterol (-21% vs. control, P < 0.05). The histomorphometric analysis of the distal femur indicated that T3 but not GC-1 treatment reduced the trabecular volume, thickness, and number. We conclude that chronic, selective activation of the TR beta isoform does not result in bone loss typical of T3-induced thyrotoxicosis, suggesting that the TR beta isoform is not critical in this process. In addition, our findings suggest that the development of TR-selective T3 analogs that spare bone mass represents a significant improvement toward long-term TSH-suppressive therapy.