Abnormal Skeletal Development Research Articles

VP13.05: Prenatal ultrasound diagnosis of fetal limb skeletal dysplasia

To explore the characteristics of fetal limb skeletal deformities by prenatal ultrasound. The prenatal ultrasound results of fetuses with skeletal dysplasia of extremities from October 2016 to January 2019 were retrospectively analysed. A total of 164 cases of abnormal limb skeletal development were detected from 12 weeks to 37 weeks of pregnancy. 15 cases presented with short and angled long bone, short ribs, small thorax, low ossification of skull, short nasal bone or abnormal spine, which were fatal skeletal malformations. There were four cases of fetal limb long bones less than -4SD and 51 cases of club foot. There were 12 cases of overlapping fingers, 4 of which were confirmed to be Trisomy 18. There were seven cases of middle phalangeal bone loss in both hands and 10 cases of dysplasia or deletion of radius with carpal abnormality. In addition, we found three cases of metacarpal and phalangeal abnormalities. Four cases of amniotic band syndrome were characterised by finger shortage, toe shortage, lower extremity abnormality, skull deformation, encephalocele, abdominal fissure, etc. A case of six fingers and six toes with bilateral fetal polycystic kidney and occipital encephalocele was confirmed as Meckel–Gruber syndrome after delivery. Two cases of cleft hand and foot were found. There were four cases of lower extremity rigidity, two cases of which were combined with femur less than -2SD, excessive amniotic fluid, mild neck edema and bilateral small lung, suspected of Pena Shokeir syndrome. One fetus was diagnosed as spinal stenosis, combined with extremely flat face and absent nasal bone. Postnatal x-ray showed that the terminal finger was short and gene chip examination showed no abnormality, which was a rare abnormality of macular achondroplasia. The common missed cases were polydactyly and syndactyly. Three-dimensional ultrasound is intuitive and can provide more information. Prenatal two-dimensional and three-dimensional ultrasound play an important role in the detection and classification of fetal limb skeletal malformations.

Magnetic resonance findings in a Cavalier King Charles spaniel with osteopetrosis, Chiari-like malformation and syringomyelia

Osteopetrosis (OP) is a rare genetic condition characterised by osteoclast impairment, deficient bone remodelling and increased bone density. Human patients with OP often present with fractures, osteomyelitis, anaemia, abnormal skeletal...

Genetic Disruption of Anoctamin 5 in Mice Replicates Human Gnathodiaphyseal Dysplasia (GDD).

Gnathodiaphyseal dysplasia (GDD; OMIM#166260) is a rare skeletal disorder which is mainly characterized by cemento-osseous lesions in mandibles, bone fragility, bowing and diaphyseal sclerosis of tubular bones. GDD is caused by point mutations in Anoctamin-5 (ANO5); however, the disease mechanisms remain unclear. Here we generated Ano5-knockout (KO) mice using a CRISPR/Cas 9 approach to study loss of function aspects of GDD mutations. Homozygous Ano5 knockout mice (Ano5-/-) replicate some typical traits of human GDD including massive jawbones, bowing tibia, sclerosis and cortical thickening of femoral and tibial diaphyses. Serum alkaline phosphatase (ALP) levels were elevated in Ano5-/- mice as in GDD patients. Calvaria-derived Ano5-/- osteoblast cultures show increased osteoblastogenesis, which is consistent with our previous in vitro observations. Bone matrix is hypermineralized, and the expression of bone formation-related factors is enhanced in Ano5-/- mice, suggesting that the osteogenic anomaly arises from a genetic disruption of Ano5. We believe this new mouse model will shed more light on the development of skeletal abnormalities in GDD on a cellular and molecular level.

IFT80 Improves Invasion Ability in Gastric Cancer Cell Line via ift80/p75NGFR/MMP9 Signaling.

The assembly and maintenance of cilia depend on intraflagellar transport (IFT) proteins, which play an important role in development and homeostasis. IFT80 is a newly defined IFT protein and partial mutation of IFT80 in humans causes diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III, both characterized by abnormal skeletal development. However, the role and mechanism of IFT80 in the invasion of gastric cancer is unknown. We established SGC-7901 and MKN-45 gastric cancer cell lines that stably overexpressed IFT80, as verified by quantitative reverse transcription-PCR, Western blot, and immunofluorescence. Matrix metalloproteinase-9 (MMP9) plays an important role in tumor invasion, and its expression was assessed by quantitative reverse transcription-PCR, Western blotting, and immunofluorescence. The invasion ability of IFT80 on SGC-7901 and MKN-45 cells was examined by the Matrigel invasion assay. The relationship between p75NGFR, and the p75NGFR antagonists, PD90780 and IFT80, were detected by quantitative reverse transcription-PCR and Western blotting. We first detected an IFT80 expression pattern, and found that IFT80 was highly expressed in gastric cancer clinical samples. Overexpression of IFT80 in the gastric cancer cell lines, SGC-7901 and MKN-45, led to lengthening cilia. Additionally, overexpression of IFT80 significantly improved proliferation and invasion, but inhibited apoptosis, in gastric cancer cells. We further found that overexpression of IFT80 increased p75NGFR and MMP9 mRNA and protein expression. Treatment with the p75NGFR antagonist PD90780 inhibited the increased invasion ability resulting from overexpression of IFT80 in SGC-7901 and MKN-45 gastric cancer cells. Thus, these results suggest that IFT80 plays an important role in invasion of gastric cancer through regulating the ift80/p75NGFR/MMP9 signal pathways.

Genetic parameters of the upper-jaw abnormalities in Gilthead seabream Sparus aurata

The development of skeletal abnormalities is a frequent problem of quality in finfish hatcheries, resulting from unfavourable environmental conditions, often under the control of genetic background. In the present study, the heritability of jaw abnormalities in Gilthead seabream was estimated at the end of the larval rearing phase (39 dph, days post hatching). All larvae came from eggs obtained by mass spontaneous spawn of a common brood stock of a commercial hatchery. Skeletal abnormalities were examined after the double staining of the larvae for bone and cartilage. Macroscopically, three abnormality types were recorded in the studied larvae, pugheadness (Pug, 6.7%), lateral displacement of the upper jaw (LdUj, 2.3%) and crossbite (Crbt, 0.9%). The microscopic examination of the specimens revealed the frequent presence of internal abnormalities (e.g. fusions, size reductions, bending) which had not any clear effect on the external morphology of the fish (silent abnormalities). Each stained larva was genotyped and parentally assigned. Heritability estimates were insignificant for the three types of gross, externally-visible, abnormalities of the jaws (Pug, LdUj, Crbt). On the contrary, significant heritabilities were estimated for the internal abnormalities which are associated to LdUj (e.g. fusion of maxillaries with pre-maxillaries, 0.22 ± 0.06). Interestingly, the consideration of all the abnormalities of the upper jaw as a uniform trait resulted to the estimation of insignificant heritabilities, and therefore to the masking of the significant genetic component of the LdUj abnormality. Results are discussed in respect to their potential value for the selective breeding programs of Gilthead seabream.

Deletion of the Fanconi Anemia C Gene in Mice Leads to Skeletal Anomalies and Defective Bone Mineralization and Microarchitecture.

Fanconi anemia (FA) is a rare genetic disorder associated with a progressive decline in hematopoietic stem cells leading to bone marrow failure. FA is also characterized by a variety of developmental defects including short stature and skeletal malformations. More than half of children affected with FA have radial-ray abnormalities, and many patients have early onset osteopenia/osteoporosis. Although many Fanconi anemia genes have been identified and a molecular pathway defined, the underlying mechanism leading to bone defects remains elusive. To understand the role of FA genes in skeletal development and bone microarchitecture, we evaluated bone physiology during embryogenesis and in adult FancA- and FancC-deficient mice. We found that both FancA-/- and FancC-/- embryos have abnormal skeletal development shown by skeletal malformations, growth delay, and reduced bone mineralization. FancC-/- adult mice present altered bone morphology and microarchitecture with a significant decrease in cortical bone mineral density in a sex-specific manner. Mechanical testing revealed that male but not female FancC-/- mice show reduced bone strength compared with their wild-type littermates. Ex vivo cultures showed that FancA-/- and FancC-/- bone marrow-derived mesenchymal stem cells (BM MSC) have impaired differentiation capabilities together with altered gene expression profiles. Our results suggest that defective bone physiology in FA occurs in utero and possibly results from altered BM MSC function. These results provide valuable insights into the mechanism involved in FA skeletal defects. © 2018 American Society for Bone and Mineral Research.

Characterization of Normal Murine Carpal Bone Development Prompts Re-Evaluation of Pathologic Osteolysis as the Cause of Human Carpal-Tarsal Osteolysis Disorders

Multicentric carpal-tarsal osteolysis; multicentric osteolysis, nodulosis, and arthropathy; and Winchester syndromes, skeletal dysplasias characterized by carpal/tarsal and epiphyseal abnormalities, are caused by mutations in v-maf musculoaponeurotic fibrosarcoma oncogene ortholog B (MAFB), matrix metalloproteinase (MMP) 2, and MMP14, respectively; however, the underlying pathophysiology is unclear. Osteoclast-mediated osteolysis has been regarded as the main mechanism, but does not explain the skeletal distribution. We hypothesized that MAFB, MMP-2, and MMP-14 have integral roles in carpal/tarsal and epiphyseal bone development. Normal neonatal mouse forepaws were imaged by micro-computed tomography and examined histologically. Murine forepaw ossification occurred sequentially. Subarticular regions of endochondral ossification showed morphologic and calcification patterns that were distinct from archetypical physeal endochondral ossification. This suggests that two different forms of endochondral ossification occur. The skeletal sites showing the greatest abnormality in the carpal-tarsal osteolysis syndromes are regions of subarticular ossification. Thus, abnormal bone formation in areas of subarticular ossification may explain the site-specific distribution of the carpal-tarsal osteolysis phenotype. MafB, Mmp-2, and Mmp-14 were expressed widely, and tartrate-resistant acid phosphatase staining notably was absent in the subarticular regions of the cartilage anlagen and entheses at a time point most relevant to the human osteolysis syndromes. Thus, abnormal peri-articular skeletal development and modeling, rather than excessive bone resorption, may be the underlying pathophysiology of these skeletal syndromes.

Thyroid-stimulating hormone stimulation downregulates autophagy and promotes apoptosis in chondrocytes

Subclinical hypothyroidism (SCH) patients have normal thyroid hormone levels but increased thyroid stimulating hormone (TSH) level in serum. It has been reported that high TSH is related to abnormal skeletal development in mice with hypothyroidism. However, the cellular mechanism is not fully understood. In the present study, we aim to investigate the direct effects of TSH stimulation on chondrocytes, and the putative role of autophagy in this process. By using EdU incorporation assay and flow cytometry for mitochondrial membrane potential assay, we demonstrated deceased proliferation and promoted apoptosis in TSH stimulated primary mouse chondrocytes. And the balance of Bcl-2 and BAX expression on protein level was broken. More interestingly, the expression of autophagic markers Beclin-1 and LC3II was reduced in TSH stimulated chondrocytes, accompanied by less autophagosomes and accumulated p62 protein, indicating an impaired autophagic flux. More interestingly, mTOR was upregulated and AMPK activity was decreased in TSH stimulated PMCs, suggesting that mTOR/AMPK pathway is get involved in the regulation of TSH on autophagy in PMCs. Collectively, we found an increased apoptosis and suppressed autophagy in TSH stimulated primary chondrocytes, which is meaningful in understanding the effects of increased TSH level on articular cartilage and the role of autophagy in this process, and thus provide a potential novel therapeutic target in related cartilage damages.

Altered mRNA Splicing, Chondrocyte Gene Expression and Abnormal Skeletal Development due to SF3B4 Mutations in Rodriguez Acrofacial Dysostosis.

The acrofacial dysostoses (AFD) are a genetically heterogeneous group of inherited disorders with craniofacial and limb abnormalities. Rodriguez syndrome is a severe, usually perinatal lethal AFD, characterized by severe retrognathia, oligodactyly and lower limb abnormalities. Rodriguez syndrome has been proposed to be a severe form of Nager syndrome, a non-lethal AFD that results from mutations in SF3B4, a component of the U2 small nuclear ribonucleoprotein particle (U2 snRNP). Furthermore, a case with a phenotype intermediate between Rodriguez and Nager syndromes has been shown to have an SF3B4 mutation. We identified heterozygosity for SF3B4 mutations in Rodriguez syndrome, confirming that the phenotype is a dominant disorder that is allelic with Nager syndrome. The mutations led to reduced SF3B4 synthesis and defects in mRNA splicing, primarily exon skipping. The mutations also led to reduced expression in growth plate chondrocytes of target genes, including the DLX5, DLX6, SOX9, and SOX6 transcription factor genes, which are known to be important for skeletal development. These data provide mechanistic insight toward understanding how SF3B4 mutations lead to the skeletal abnormalities observed in the acrofacial dysostoses.

NANS-mediated synthesis of sialic acid is required for brain and skeletal development.

We identified biallelic mutations in NANS, the gene encoding the synthase for N-acetylneuraminic acid (NeuNAc; sialic acid), in nine individuals with infantile-onset severe developmental delay and skeletal dysplasia. Patient body fluids showed an elevation in N-acetyl-D-mannosamine levels, and patient-derived fibroblasts had reduced NANS activity and were unable to incorporate sialic acid precursors into sialylated glycoproteins. Knockdown of nansa in zebrafish embryos resulted in abnormal skeletal development, and exogenously added sialic acid partially rescued the skeletal phenotype. Thus, NANS-mediated synthesis of sialic acid is required for early brain development and skeletal growth. Normal sialylation of plasma proteins was observed in spite of NANS deficiency. Exploration of endogenous synthesis, nutritional absorption, and rescue pathways for sialic acid in different tissues and developmental phases is warranted to design therapeutic strategies to counteract NANS deficiency and to shed light on sialic acid metabolism and its implications for human nutrition.

A novel FGD1 mutation in a family with Aarskog-Scott syndrome and predominant features of congenital joint contractures.

Mutations in FGD1 cause Aarskog–Scott syndrome (AAS), an X-linked condition characterized by abnormal facial, skeletal, and genital development due to abnormal embryonic morphogenesis and skeletal formation. Here we report a novel FGD1 mutation in a family with atypical features of AAS, specifically bilateral upper and lower limb congenital joint contractures and cardiac abnormalities. The male proband and his affected maternal uncle are hemizygous for the novel FGD1 mutation p.Arg921X. This variant is the most carboxy-terminal FGD1 mutation identified in a family with AAS and is predicted to truncate the FGD1 protein at the second to last amino acid of the carboxy-terminal pleckstrin homology (PH) domain. Our study emphasizes the importance of the 3′ peptide sequence in the structure and/or function of the FGD1 protein and further demonstrates the need to screen patients with X-linked congenital joint contractures for FGD1 mutations.

High doses of caffeine reduce invivo osteogenic activity in prepubertal rats.

Caffeine adversely affects endochondral ossification during fetal skeletal growth, and results in increased incidence of delayed and abnormal fetal skeletal development. Chronic caffeine intake also decreases growth hormone secretion. Thus, it is conceivable that caffeine may disrupt bone growth during the peripubertal period. This study aimed to investigate the impact of high-caffeine consumption on bone growth throughout puberty. A total of 51 male rats (21days old) were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 and 180mgkg(-1) day(-1) for 4weeks. After death, the final length and weight of leg bones were measured, and the tibia processed for histomorphometric analysis. Caffeine caused a significant decrease in body mass gain. This was accompanied with proportional decreases in lean body mass and body fat. In addition, bone mass and osteogenic activity invivo were assessed using dual-energy X-ray absorptiometry and (18) F-NaF positron emission tomography. The results showed significant decreases of bone mass and invivo osteogenic activity in the caffeine-fed groups. Rats fed with caffeine showed a significantly shorter and lighter tibia and femur and the vertebral column compared with controls. In addition, caffeine does not increase the width of the growth plates (GPs), it slows the rate at which the GP closes due to a slower rate of growth. These results demonstrated that caffeine altered osteogenic activity, leading to delayed peripubertal longitudinal bone growth and maturation. Given that osteogenic cells undergo dynamic changes in metabolic activity and that the pubertal growth spurt is mainly stimulated by growth hormone/insulin-like growth factor-1 and sex steroids during pubertal development, caffeine could suppress ossification by interfering with both physiological changes in hormonal secretion and osteogenic activity during this critical period. Further study will be needed to investigate the cellular/molecular mechanism by which caffeine affects osteogenesis using invitro experimental models.

RECQL4 Regulates p53 Function In Vivo During Skeletogenesis

RECQ DNA helicases play critical roles in maintaining genomic stability, but their role in development has been less well studied. Rothmund-Thomson syndrome, RAPADILINO, and Baller-Gerold syndrome are rare genetic disorders caused by mutations in the RECQL4 gene. These patients have significant skeletal developmental abnormalities including radial ray, limb and craniofacial defects. To investigate the role of Recql4 in the developing skeletal system, we generated Recql4 conditional knockout mice targeting the skeletal lineage. Inactivation of Recql4 using the Prx1-Cre transgene led to limb abnormalities and craniosynostosis mimicking the major bone findings in human RECQL4 patients. These Prx1-Cre(+) ;Recql4(fl/fl) mice as well as Col2a1-Cre(+) ;Recql4(fl/fl) mice exhibited growth plate defects and an increased p53 response in affected tissues. Inactivation of Trp53 in these Recql4 mutants resulted in genetic rescue of the skeletal phenotypes, indicating an in vivo interaction between Recql4 and Trp53, and p53 activation as an underlying mechanism for the developmental bone abnormalities in RECQL4 disorders. Our findings show that RECQL4 is critical for skeletal development by modulating p53 activity in vivo.

A Transgenic Mouse Model of OI Type V Supports a Neomorphic Mechanism of the IFITM5 Mutation

Osteogenesis imperfecta (OI) type V is characterized by increased bone fragility, long bone deformities, hyperplastic callus formation, and calcification of interosseous membranes. It is caused by a recurrent mutation in the 5' UTR of the IFITM5 gene (c.-14C > T). This mutation introduces an alternative start codon, adding 5 amino acid residues to the N-terminus of the protein. The mechanism whereby this novel IFITM5 protein causes OI type V is yet to be defined. To address this, we created transgenic mice expressing either the wild-type or the OI type V mutant IFITM5 under the control of an osteoblast-specific Col1a1 2.3-kb promoter. These mutant IFITM5 transgenic mice exhibited perinatal lethality, whereas wild-type IFITM5 transgenic mice showed normal growth and development. Skeletal preparations and radiographs performed on E15.5 and E18.5 OI type V transgenic embryos revealed delayed/abnormal mineralization and skeletal defects, including abnormal rib cage formation, long bone deformities, and fractures. Primary osteoblast cultures, derived from mutant mice calvaria at E18.5, showed decreased mineralization by Alizarin red staining, and RNA isolated from calvaria showed reduced expression of osteoblast differentiation markers such as Osteocalcin, compared with nontransgenic littermates and wild-type mice calvaria, consistent with the in vivo phenotype. Importantly, overexpression of wild-type Ifitm5 did not manifest a significant bone phenotype. Collectively, our results suggest that expression of mutant IFITM5 causes abnormal skeletal development, low bone mass, and abnormal osteoblast differentiation. Given that neither overexpression of the wild-type Ifitm5, as shown in our model, nor knock-out of Ifitm5, as previously published, showed significant bone abnormalities, we conclude that the IFITM5 mutation in OI type V acts in a neomorphic fashion.

Responses of different body compartments to acute dietary phosphorus deficiency in juvenile triploid rainbow trout (Oncorhynchus mykiss , Walbaum)

Rainbow trout (Oncorhynchus mykiss) is an important production species as well as one of the most studied fish models, particularly regarding nutritional physiology. Due to negative environmental impacts linked with rainbow trout farm effluents, significant restrictions have been established in numerous regions to reduce dietary phosphorus (P) outputs. However, questions have arisen regarding the link between abnormal skeletal development and mineralization and insufficient dietary P availability during rapid fish growth (juvenile fish). Despite significant work to understand the dynamics of P-deficiency and the resulting impact on tissue mineralization, the extent of the early responses in rainbow trout fed low-P diets is not well described. The aim of this study was to explore the early-responses of scales, vertebrae and carcass P and ash in rainbow trout fed low-P vs sufficient-P controls. Two practical diets (sufficient: 0.45% available P and deficient: 0.29% available P) were fed over a 5-week period to triploid rainbow trout (initial mass 60.8 ± 1.6 g). Ash and P contents were used to assess mineral status of the different tissues. The relative loss of mineral and coefficient of variation were also calculated to compare the relative response and the inter-individual variability. After 4 weeks of P deprivation, no detectable effects were observed on growth performance, mortality or feed intake. However, as early as the second week onward, ash and P levels in scales and carcasses were significantly lower (p < 0.05) in fish fed a P-deficient diet compared to those fed a P-sufficient diet. At week 4, the reduction in mineralization reached 19.3 and 18.4% for P and ash contents in scales, respectively. By the same week, P and ash contents in carcasses reached 15.1 and 12.8%, respectively. Interestingly, the absence of significant mineral loss in vertebrae did not prevent the emergence of a high incidence of vertebral deformities in P-deficient fish (45.6 ± 11.0%) when compared to P-sufficient fish (1.5 ± 2.1%). The main vertebral deformities observed in P-deficient trout were specific to impede bone matrix mineralization such as pronounced biconcave (35.5%) and undersized and widely spaced vertebral bodies (9.3%). For the scales compartment (ash and P status) and mineral parameter, the coefficients of variation showed a higher inter-individual variability in P-deficient vs P-sufficient fish, while no such effect was observed in carcass or vertebrae compartment. This study provides a useful comparison of various commonly-used indicators of bone mineral status as well as an approach to assess variability of fish response under low-dietary P level.

ZBTB42 mutation defines a novel lethal congenital contracture syndrome (LCCS6).

Lethal congenital contracture syndrome (LCCS) is a lethal autosomal recessive form of arthrogryposis multiplex congenita (AMC). LCCS is genetically heterogeneous with mutations in five genes identified to date, all with a role in the innervation or contractile apparatus of skeletal muscles. In a consanguineous Saudi family with multiple stillbirths presenting with LCCS, we excluded linkage to all known LCCS loci and combined autozygome analysis and whole-exome sequencing to identify a novel homozygous variant in ZBTB42, which had been shown to be enriched in skeletal muscles, especially at the neuromuscular junction. Knockdown experiments of zbtb42 in zebrafish consistently resulted in grossly abnormal skeletal muscle development and myofibrillar disorganization at the microscopic level. This severe muscular phenotype is successfully rescued with overexpression of the human wild-type ZBTB42 gene, but not with the mutant form of ZBTB42 that models the human missense change. Our data assign a novel muscular developmental phenotype to ZBTB42 in vertebrates and establish a new LCCS6 type caused by ZBTB42 mutation.

Vitamin D and the Goldilocks Principle: Too Little, Too Much, or Just Right?

Vitamin D and the Goldilocks Principle: Too Little, Too Much, or Just Right?

Is Vitamin D a Key Factor in Muscle Health?

Historically, vitamin D is viewed as fundamental to bone mineralization. However, it is well established that muscle strength and muscle mass are important determinants of bone density, bone geometry, and fracture risk. Vitamin D therefore plays a key role in bone metabolism not only through its direct effects on osteoblasts, calcium absorption by the intestines, but also through its effects on muscle fiber size and muscle function (1–3). Vitamin D may influence muscle physical function in 2 ways: indirectly through calcium-related effects and directly via its role in muscle cell regulation. On a cellular level, a variety of mechanisms by which vitamin D impacts upon the function of skeletal muscle have been elucidated. These can be broadly divided into genomic effects that arise from the binding of the nuclear receptor (NR) (vitamin D receptor [VDR]), and nongenomic effects. In its biologically active form 1,25(OH)2 D binds to VDR, which is a ligand-dependent nuclear transcription factor (4). VDR is heterodimerized with retinoid X receptor (RXR) and the complex activates positive vitamin D response element genes transcription. This increases synthesis of proteins related to several pathways in muscle functions, including Ca -binding proteins, such as calmodulin and calbindin D-9K. It also results in an increase in synthesis of other muscle cytoskeletal proteins important for muscle function and trophicity, namely IGF-binding protein-3, thereby inducing muscle hypertrophy. However, in some tissues, RXR levels are greater than VDR levels (5). Therefore, changes in VDR levels are rate limiting for VDR/RXR-mediated target gene expression. Coimmunoprecipitation analyses have also demonstrated direct binding of VDR with c-src under the influence of 1,25(OH)2 D, which mediates the stimulation of muscle growth and differentiation by its subsequent effects on MAPK-signaling pathways (6). Cellular 1,25(OH)2 D also elicits fast-acting responses that cannot be explained by activation of the slow nuclear VDR genomic pathway that requires hours for protein synthesis. This response is believed to be mediated by the activation of a plasma membrane-associated VDR that stimulates several interacting second-messenger pathways that transmit the signal to the cytoplasm (7). However, a cross talk between 1,25(OH)2 D activation of the plasma-membrane-bound VDR and the nuclear VDR action can exist. Both types affect Ca handling and muscle cell proliferation and differentiation. The presence of VDR in muscle has been reported on the basis of immunohistochemistry and detection of VDR mRNA by RT-PCR (7), but these results are subject to challenge due to the experimental conditions (8). Differences in the expression of VDR through the various stages of muscle differentiation may have accounted for these discrepancies. Deletion of VDR in mice (VDR knockout [KO] mouse model) has provided valuable insights into the biologic function of the vitamin D endocrine system. VDRKO mice present with abnormal skeletal muscle development and deregulatedexpressionofmyoregulatory transcription factors (9). These mice display atrophy of type I and II muscle fibers. These changes were observed in VDRKO mice on high-calcium diet, suggesting that the absence of VDR was the predominant cause rather than systemic biochemical changes. A positive association between vitamin D status and muscle function has been observed in rodent models or in humans. Vitamin D deficiency impairs excitation-contraction coupling in skeletal muscle leading to impairment in muscleforce generation. In a rat model, a decrease in sarcoplasmic

Excess TSH causes abnormal skeletal development in young mice with hypothyroidism via suppressive effects on the growth plate

Hypothyroidism in the young leads to irreversible growth failure. hyt/hyt Mice have a nonfunctional TSH receptor (TSHR) and are severely hypothyroid, but growth retardation was not observed in adult mice. We found that epiphysial cartilage as well as cultured chondrocytes expressed functional TSHR at levels comparable to that seen in the thyroid, and that addition of TSH to cultured chondrocytes suppressed expression of chondrocyte differentiation marker genes such as Sox-9 and type IIa collagen. Next, we compared the long bone phenotypes of two distinct mouse models of hypothyroidism: thyroidectomized (THYx) mice and hyt/hyt mice. Although both THYx and hyt/hyt mice were severely hypothyroid and had similar serum Ca(2+) and growth hormone levels, the tibia was shorter and the proliferating and hypertrophic zones in the growth plate was significantly narrower in THYx mice than in hyt/hyt mice. Supplementation of hyt/hyt mice thyroid hormone resulted in a wider growth plate compared with that of wild-type mice. Expressions of chondrocyte differentiation marker genes Sox-9 and type IIa collagen in growth plate from THYx mice were 52 and 60% lower than those of hyt/hyt mice, respectively. High serum TSH causes abnormal skeletal development in young mice with hypothyroidism via suppressive effects on the growth plate.

NF-κB signaling and bone resorption

The transcription factor NF-κB is a family of proteins involved in signaling pathways essential for normal cellular functions and development. Deletion of various components of this pathway resulted with abnormal skeletal development. Research in the last decade has established that NF-κB signaling mediates RANK ligand-induced osteoclastogenesis. Consistently, it was shown that inhibition of NF-κB was an effective approach to inhibit osteoclast formation and bone resorptive activity. Identification of the molecular machinery underlying NF-κB activation permitted osteoclast-specific deletion of the major components of this pathway. As a result, it was clear that deletion of members of the proximal IKK kinase complex and the distal NF-κB subunits and downstream regulators affected skeletal development. These studies provided several targets of therapeutic intervention in osteolytic diseases. NF-κB activity has been also described as the centerpiece of inflammatory responses and is considered a potent mediator of inflammatory osteolysis. Indeed, inflammatory insults exacerbate physiologic RANKL-induced NF-κB signals leading to exaggerated responses and to inflammatory osteolysis. These superimposed NF-κB activities appear to underlie several bone pathologies. This review will describe the individual roles of NF-κB molecules in bone resorption and inflammatory osteolysis.