Thyroid diseases and bone health.

Thyroid hormones are essential for skeletal development and are important regulators of bone maintenance in adults. Childhood hypothyroidism causes delayed skeletal development, retarded linear growth and impaired bone mineral accrual. Epiphyseal dysgenesis is evidenced by classic features of stippled epiphyses on X-ray. In severe cases, post-natal growth arrest results in a complex skeletal dysplasia. Thyroid hormone replacement stimulates catch-up growth and bone maturation, but recovery may be incomplete dependent on the duration and severity of hypothyroidism prior to treatment. A severe phenotype characteristic of hypothyroidism occurs in children with resistance to thyroid hormone due to mutations affecting THRA encoding thyroid hormone receptor α (TRα). Discovery of this rare condition recapitulated animal studies demonstrating that TRα mediates thyroid hormone action in the skeleton. In adults, thyrotoxicosis is well known to cause severe osteoporosis and fracture, but cases are rare because of prompt diagnosis and treatment. Recent data, however, indicate that subclinical hyperthyroidism is associated with low bone mineral density (BMD) and an increased risk of fracture. Population studies have also shown that variation in thyroid status within the reference range in post-menopausal women is associated with altered BMD and fracture risk. Thus, thyroid status at the upper end of the euthyroid reference range is associated with low BMD and increased risk of osteoporotic fragility fracture. Overall, extensive data demonstrate that euthyroid status is required for normal post-natal growth and bone mineral accrual, and is fundamental for maintenance of adult bone structure and strength

Quantitative X-ray microradiography for high-throughput phenotyping of osteoarthritis in mice

Objective To investigate and validate digital X-ray microradiography as a novel, high-throughput and cost-effective screening approach to identify abnormal joint phenotypes in mice. Method Digital X-ray microradiography was used to quantify the subchondral bone mineral content (BMC) in the medial tibial plateau. Accuracy and reproducibility of the method were determined in 22 samples from C57BL/6(B6Brd;B6Dnk;B6N-Tyrc-Brd) wild-type mice. The method was then validated in wild-type mice that had undergone surgical destabilisation of medial meniscus (DMM) and in a genetically modified mouse strain with an established increase in trabecular bone mass. Results The measurement of subchondral BMC by digital X-ray microradiography had a coefficient of variation of 3.6%. Digital X-ray microradiography was able to demonstrate significantly increased subchondral BMC in the medial tibial plateau of male mice 4 and 8 weeks after DMM surgery and in female mice 8 weeks after surgery. Furthermore, digital X-ray microradiography also detected the increase in subchondral BMC in a genetically modified mouse strain with high trabecular bone mass. Conclusion Quantitation of subchondral BMC by digital X-ray microradiography is a rapid, sensitive and cost-effective method to identify abnormal joint phenotypes in mice of both genders at several ages

Automatic Quantification of Epidermis Curvature in H&E Stained Microscopic Skin Image of Mice

Changes in the curvature of the epidermis layer is often associated with many skin disorders, such as ichthyoses and generic effects of ageing. Therefore, methods to quantify changes in the curvature are of a scientific and clinical interest. Manual methods to determine curvature are both laborious and intractable to large scale investigations. This paper proposes an automatic algorithm to quantify curvature of microscope images of H&E-stained murine skin. The algorithm can be divided into three key stages. First, skin layers segmentation based on colour deconvolution to separate the original image into three channels of different representations to facilitate segmenting the image into multiple layers, namely epidermis, dermis and subcutaneous layers. The algorithm then further segments the epidermis layer into cornified and basal sub-layers. Secondly, it quantifies the curvature of the epidermis layer by measuring the difference between the epidermis edge and a straight line (theoretical reference line) connecting the two far sides of the epidermis edge. Finally, the curvature measurements extracted from a large number of images of mutant mice are used to identify a list of genes responsible for changes in the epidermis curvature. A dataset of 5714 H&E microscopic images of mutant and wild type mice were used to evaluate the effectiveness of the algorithm

Genetic and pharmacological targeting of transcriptional repression in resistance to thyroid hormone alpha

Background Thyroid hormones act in bone and cartilage via thyroid hormone receptor α (TRα). In the absence of T3, TRα interacts with co-repressors, including nuclear receptor co-repressor-1 (NCoR1), which recruit histone deacetylases (HDACs) and mediate transcriptional repression. Dominant-negative mutations of TRα cause resistance to thyroid hormone α (RTHα; OMIM 614450), characterized by excessive repression of T3 target genes leading to delayed skeletal development, growth retardation and bone dysplasia. Treatment with thyroxine has been of limited benefit even in mildly affected individuals and there is a need for new therapeutic strategies. We hypothesized that (i) the skeletal manifestations of RTHα are mediated by the persistent TRα/NCoR1/HDAC repressor complex containing mutant TRα, and (ii) treatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) would ameliorate these manifestations. Methods We determined the skeletal phenotypes of (i) Thra1PV/+ mice, a well characterized model of RTHα, (ii) Ncor1ΔID/ΔID mice, which express an NCoR1 mutant that fails to interact with TRα, and (iii) Thra1PV/+Ncor1ΔID/ΔID double mutant adult mice. Wild-type, Thra1PV/+, Ncor1ΔID/ΔID, and Thra1PV/+Ncor1ΔID/ΔID double mutant mice were also treated with SAHA to determine whether HDAC inhibition results in amelioration of skeletal abnormalities. Results Thra1PV/+ mice had a severe skeletal dysplasia characterized by short stature, abnormal bone morphology and increased bone mineral content. Despite normal bone length, Ncor1ΔID/ΔID mice displayed increased cortical bone mass, mineralization and strength. Thra1PV/+Ncor1ΔID/ΔID double mutant mice displayed only a small improvement of skeletal abnormalities compared to Thra1PV/+ mice. Treatment with SAHA to inhibit histone deacetylation had no beneficial or detrimental effects on bone structure, mineralization or strength in wild-type or mutant mice. Conclusions These studies indicate treatment with SAHA is unlikely to improve the skeletal manifestations of RTHα. Nevertheless, the findings (i) confirm that TRα1 has a critical role in the regulation of skeletal development and adult bone mass, (ii) suggest a physiological role for alternative co-repressors that interact with TR in skeletal cells, and (iii) demonstrate a novel role for NCoR1 in the regulation of adult bone mass and strength

The secreted protein augurin is a novel modulator of canonical Wnt signalling involved in osteoblast differentiation

Background ECRG4/C2ORF40 is a tumour suppressor gene downregulated in several cancer types, which encodes the secreted protein augurin. A wide number of functions in health and disease have been assigned to augurin, but the signalling pathways it regulates are still poorly characterized. Augurin expression is strongly upregulated during in vitro differentiation of neonatal mouse osteoblasts. Methods In vitro differentiation assays of calvarial osteoblasts isolated from Ecrg4 -/- and wild-type mice; transient transfection assays using reporters activated by Wnt signalling and other signal transduction pathways; Real-time quantitative polymerase chain reaction for measurement of gene expression; protein expression in Chinese hamster ovary cells and Escherichia coli; in situ binding assays of proteins expressed as fusions to alkaline phosphatase with cells expressing various membrane receptors. Results Osteoblasts from Ecrg4 -/- mice have an accelerated differentiation compared to wild-type and upregulation of Wnt markers. Augurin is a specific repressor of Wnt-stimulated transcriptional activity, both when coexpressed together with the reporter and when added to the culture medium as a soluble protein. We confirmed the previously described binding of augurin to LOX1, a scavenger receptor, but an inhibitor of this molecule did not impair augurin repression of Wnt-stimulated transcription specifically. Genome-wide association studies showed an association of ECRG4 genomic variation with body height and osteoarthritis. Conclusions Our study sheds new light on the wide spectrum of functions previously ascribed to augurin in brain function, stem cell biology, inflammation/immunity and cancer. Furthermore, our discovery paves the way to further characterization of the mechanisms involved in augurin repression of Wnt signalling and the development of agonists and antagonists for this protein, which have a wide array of potential applications in the clinic

PYY is a negative regulator of bone mass and strength

Objective Bone loss in anorexia nervosa and following bariatric surgery is associated with an elevated circulating concentration of the gastrointestinal, anorexigenic hormone, peptide YY (PYY). Selective deletion of the PYY receptor Y1R in osteoblasts or Y2R in the hypothalamus results in high bone mass, but deletion of PYY in mice has resulted in conflicting skeletal phenotypes leading to uncertainty regarding its role in the regulation of bone mass. As PYY analogs are under development for treatment of obesity, we aimed to clarify the relationship between PYY and bone mass. Methods The skeletal phenotype of Pyy knockout (KO) mice was investigated during growth (postnatal day P14) and adulthood (P70 and P186) using X-ray microradiography, micro-CT, back-scattered electron scanning electron microscopy (BSE-SEM), histomorphometry and biomechanical testing. Results Bones from juvenile and Pyy KO mice were longer (P < 0.001), with decreased bone mineral content (P < 0.001). Whereas, bones from adult Pyy KO mice had increased bone mineral content (P < 0.05) with increased mineralisation of both cortical (P < 0.001) and trabecular (P < 0.001) compartments. Long bones from adult Pyy KO mice were stronger (maximum load P < 0.001), with increased stiffness (P < 0.01) and toughness (P < 0.05) compared to wild-type (WT) control mice despite increased cortical vascularity and porosity (P < 0.001). The increased bone mass and strength in Pyy KO mice resulted from increases in trabecular (P < 0.01) and cortical bone formation (P < 0.05). Conclusions These findings demonstrate that PYY acts as a negative regulator of osteoblastic bone formation, implicating increased PYY levels in the pathogenesis of bone loss during anorexia or following bariatric surgery

Hypochondroplasia gain-of-function mutation in FGFR3 causes defective bone mineralization in mice

Hypochondroplasia (HCH) is a mild dwarfism caused by missense mutations in fibroblast growth factor receptor 3 (FGFR3), with the majority of cases resulting from a heterozygous p.Asn540Lys gain-of-function mutation. Here, we report the generation and characterization of the first mouse model (Fgfr3Asn534Lys/+) of HCH to our knowledge. Fgfr3Asn534Lys/+ mice exhibited progressive dwarfism and impairment of the synchondroses of the cranial base, resulting in defective formation of the foramen magnum. The appendicular and axial skeletons were both severely affected and we demonstrated an important role of FGFR3 in regulation of cortical and trabecular bone structure. Trabecular bone mineral density (BMD) of long bones and vertebral bodies was decreased, but cortical BMD increased with age in both tibiae and femurs. These results demonstrate that bones in Fgfr3Asn534Lys/+ mice, due to FGFR3 activation, exhibit some characteristics of osteoporosis. The present findings emphasize the detrimental effect of gain-of-function mutations in the Fgfr3 gene on long bone modeling during both developmental and aging processes, with potential implications for the management of elderly patients with hypochondroplasia and osteoporosis

Impact of severity, duration, and etiology of hyperthyroidism on bone turnover markers and bone mineral density in men

<p>Abstract</p> <p>Background</p> <p>Hyperthyroidism is accompanied by osteoporosis with higher incidence of fracture rates. The present work aimed to study bone status in hyperthyroidism and to elucidate the impact of severity, duration, and etiology of hyperthyroidism on biochemical markers of bone turnover and bone mineral density (BMD).</p> <p>Methods</p> <p>Fifty-two male patients with hyperthyroidism, 31 with Graves' disease (GD) and 21 with toxic multinodular goiter (TNG), with an age ranging from 23 to 65 years were included, together with 25 healthy euthyroid men with matched age as a control group. In addition to full clinical examination, patients and controls were subjected to measurement of BMD using dual-energy X-ray absorptiometery scanning of the lower half of the left radius. Also, some biochemical markers of bone turnover were done for all patients and controls.</p> <p>Results</p> <p>Biochemical markers of bone turnover: included serum bone specific alkaline phosphatase, osteocalcin, carboxy terminal telopeptide of type l collagen also, urinary deoxypyridinoline cross-links (DXP), urinary DXP/urinary creatinine ratio and urinary calcium/urinary creatinine ratio were significantly higher in patients with GD and TNG compared to controls (P < 0.01). However, there was non-significant difference in these parameters between GD and TNG patients (P > 0.05). BMD was significantly lower in GD and TNG compared to controls, but the Z-score of BMD at the lower half of the left radius in patients with GD (-1.7 ± 0.5 SD) was not significantly different from those with TNG (-1.6 ± 0.6 SD) (>0.05). There was significant positive correlation between free T3 and free T4 with biochemical markers of bone turnover, but negative correlation between TSH and those biochemical markers of bone turnover. The duration of the thyrotoxic state positively correlated with the assessed bone turnover markers, but it is negatively correlated with the Z-score of BMD in the studied hyperthyroid patients (r = -0.68, P < 0.0001).</p> <p>Conclusion</p> <p>Men with hyperthyroidism have significant bone loss with higher biochemical markers of bone turnover. The severity and the duration of the thyrotoxic state are directly related to the derangement of biochemical markers of bone turnover and bone loss.</p

Sequence analysis and transcript expression of the MEN1 gene in sporadic pituitary tumours

The majority of pituitary tumours are monoclonal in origin and arise sporadically or occasionally as part of multiple endocrine neoplasia type 1 (MEN1). Whilst a multi-step aetiology involving both oncogenes and tumour suppressor genes has been proposed for their development, the target(s) of these changes are less clearly defined. Both familial and sporadic pituitary tumours have been shown to harbour allelic deletion on 11q13, which is the location of the recently cloned MEN1 gene. We investigated 23 sporadic pituitary tumours previously shown to harbour allelic deletion on 11q13 with the marker PYGM centromeric and within 50 kb of the MEN1 locus. In addition, the use of intragenic polymorphisms in exon 9 and at D11S4946, and of telomeric loci at D11S4940 and D11S4936, revealed that five of 20 tumours had loss of heterozygosity (LOH) telomeric to the menin gene. However, the overall pattern of loss in informative cases was indicative of non-contiguous deletion that brackets the menin gene. Sequence analysis of all MEN1 coding exons and flanking intronic sequence, in tumours and matched patient leucocyte DNA, did not reveal mutation(s) in any of the 23 tumours studied. A benign polymorphism in exon 9 was encountered at the expected frequency, and in seven patients heterozygous for the polymorphism the tumour showed retention of both copies of the menin gene. Reverse transcription polymerase chain reaction analysis of ten evaluable tumours and four normal pituitaries revealed the presence of the menin transcript. Whilst these findings suggest that gene silencing is unlikely to be mechanistic in sporadic pituitary tumorigenesis, they do not exclude changes in the level or stability of the transcript or translation to mature protein. Our study would support and extend very recent reports of a limited role for mutations in the MEN1 gene in sporadic pituitary tumours. Alternatively, these findings may point to an, as yet, unidentified tumour suppressor gene in this region

A Component of Retinal Light Adaptation Mediated by the Thyroid Hormone Cascade

Analysis with DNA-microrrays and real time PCR show that several genes involved in the thyroid hormone cascade, such as deiodinase 2 and 3 (Dio2 and Dio3) are differentially regulated by the circadian clock and by changes of the ambient light. The expression level of Dio2 in adult rats (2–3 months of age) kept continuously in darkness is modulated by the circadian clock and is up-regulated by 2 fold at midday. When the diurnal ambient light was on, the expression level of Dio2 increased by 4–8 fold and a consequent increase of the related protein was detected around the nuclei of retinal photoreceptors and of neurons in inner and outer nuclear layers. The expression level of Dio3 had a different temporal pattern and was down-regulated by diurnal light. Our results suggest that DIO2 and DIO3 have a role not only in the developing retina but also in the adult retina and are powerfully regulated by light. As the thyroid hormone is a ligand-inducible transcription factor controlling the expression of several target genes, the transcriptional activation of Dio2 could be a novel genomic component of light adaptation