|Home | About | Journals | Submit | Contact Us | Français|
Childhood hypothyroidism causes delayed endochondral ossification, impaired bone mineralization and growth retardation. In contrast, thyrotoxicosis induces accelerated growth, advanced bone age and increased mineralization, but leads to premature closure of the epiphyseal growth plates and short stature. In severe cases accelerated intramembranous ossification results in early closure of the skull sutures and craniosynostosis. In adults, thyrotoxicosis causes increased bone turnover, accelerated bone loss and a greater than 3-fold increase in osteoporotic fracture. Thyroid hormone (T3) binds to nuclear T3 receptors (TRα and TRβ), which act as hormone-dependent transcription factors that regulate target gene expression in response to T3. Concentrations of circulating thyroid hormones are maintained in a narrow physiological range by the hypothalamic-pituitary-thyroid (HPT) axis, in which TRβ controls activity of a negative feedback loop. The TRα and TRβ receptors are expressed in temporo-spatial specific patterns during development and in different ratios in individual tissues.
To investigate the molecular mechanisms underlying abnormalities of skeletal development and adult bone turnover that result from alterations of thyroid status, we characterised the skeletal phenotypes of TR mutant mice harboring dominant negative mutations (TRα1PV/+, TRα1R384C/+, TRβPV/PV) or deletions (TRα0/0, TRβ-/-) of the genes encoding TRα and TRβ. Murine skeletal development was characterized by light microscopy using histological methods that included alizarin red and alcian blue staining of whole skeletal preparations, von Kossa staining of undecalcified histological sections of long bones and alcian blue and van Gieson staining of decalcified sections. Dynamic aspects of endochondral ossification were assessed by analysis of long bone and tail growth, and histomorphometric measurement of epiphyseal growth plate zones and mid-diaphyseal cortical bone. Intramembranous ossification was assessed by measurement of cranial indices and skull suture and fontanelle areas. Skeletal thyroid status was determined by in situ hybridization analysis of T3-target gene expression. In adult mice, 3D analysis of bone micro-architecture was determined in near mid-line longitudinal sections of macerated long bones by BSE SEM using an automated digital system (Zeiss DSM962). In further studies, long bones, tail vertebrae and incisors were embedded in poly-methyl-methacrylate and optically flat standard longitudinal section planes were obtained by diamond micro-milling. The quantitative distribution of micro-mineralization densities was determined in these specimens by comparison to halogenated dimethacrylate standards using qBSE SEM.
Endochondral ossification, linear growth and skeletal mineralization were retarded in TRα null mice (TRα0/0) and more severely delayed in TRα dominant-negative mutants (TRα1PV/+, TRα1R384C/+). In contrast, these parameters were all advanced in TRβ null mice (TRβ-/-), with a more severe phenotype evident in TRβ dominant-negative mutants (TRβPV/PV). TRα mutant mice displayed increased cortical bone width, and up to an 8 fold increase in trabecular bone volume with increased trabecular thickness, plate-like morphology and greater micro-architectural complexity (Figs. (Figs.11,,2).2). In contrast, analysis of all these parameters including quantitation of bone micro-mineralization density revealed that TRβ mutants were markedly osteoporotic with thinner trabeculae of rod-like morphology. Analysis of downstream signalling pathways involving the T3-target genes FGFR1, FGFR3, GHR and IGF-1R revealed a phenotype of skeletal hypothyroidism in all TRα mutant mice but skeletal thyrotoxicosis in TRβ mutants. We further demonstrated that TRα is expressed at 12-fold higher levels in bone than TRβ, whereas TRβ is known to be predominantly expressed in hypothalamus and pituitary. Accordingly, all TRα mutant mice were systemically euthyroid whereas TRβPV/PV and TRβ-/- displayed pituitary resistance to thyroid hormone with inappropriately elevated circulating thyroid hormone levels.
Our analysis of this large series of TR mutant mice with differing genetic backgrounds unequivocally demonstrates that TRα is the predominant TR isoform expressed in bone, and shows that the skeletal effects of disrupted TRβ signalling result from its effects on systemic thyroid status. Thus, TRα plays the major physiological role in skeletal development, linear growth and the maintenance of adult skeletal integrity in vivo.
These studies were supported by grants and fellowships obtained from The Medical Research Council, Wellcome Trust, EU FP6 Marie Curie Fellowship Fund and Arthritis Research Campaign. We wish to thank Mo Arora for technical assistance.