Franz Varga

Thyroid hormones increase insulin-like growth factor mRNA levels in the clonal osteoblastic cell line MC3T3-E1

Thyroid hormones are known to affect skeletal growth and maturation by influencing both bone resorption and bone formation. Their exact mechanism of action, however, is still unknown. Local factors such as prostaglandins, TGF‐β or IGF‐I were suggested to mediate their effects. Thyroid hormones were reported to stimulate expression of IGF‐I mRNA in liver and kidney and to increase IGF‐I release from bone organ cultures and osteoblast‐like cells. Therefore we studied the effect of thyroid hormones on IGF‐I mRNA expression in MC3T3‐E1 cells. The cells were grown in culture for 5 to 7 days and treated with triiodothyronine (10−11 ‐ 10−6 M) and thyroxin (10−6 M) for 1–24 h. Cellular mRNA was isolated and subjected to Northern hybridization. The amount of IGF‐I mRNA, which is already expressed in this cell line under control conditions, was markedly enhanced by T3 and T4. This effect was found to be dose‐dependent with a maximum at 10−7M and could already be seen after 3 h increasing up to 24 h. Our findings indicate that IGF‐I expression in osteoblasts is directly regulated by thyroid hormones. We conclude that IGF‐I expression belongs to the phenotypic characteristics of mature osteoblasts, and that thyroid hormones play an important role in differentiation of MC3T3‐E1 cells along the osteoblastic lineage.

The expression of matrix metalloproteinase-13 and osteocalcin in mouse osteoblasts is related to osteoblastic differentiation and is modulated by 1,25-dihydroxyvitamin D3 and thyroid hormones

Matrix metalloproteinase‐13 (MMP‐13), is a key protein of bone matrix degradation, and is highly expressed by osteoblasts. We used the osteoblast‐like MC3T3‐E1 cell line and compared the stimulatory effects of the bone resorptive agents 1,25‐dihydroxyvitamin D3 (1,25‐(OH)2D3) 3,3′,5‐triido‐l‐thyronine (T3) on the expression of MMP‐13 mRNA. We showed that the stimulatory effects were time and dose dependent, and were also transduced to the protein level, with 1,25‐(OH)2D3being more potent.

The organization of adherens junctions in mouse osteoblast-like cells (MC3T3-E1) and their modulation by triiodothyronine and 1,25-dihydroxyvitamin D3

Abstract. Cadherin-mediated cell–cell adhesion is essential for the development and survival of multicellular tissues. Thus it is hypothesized that these molecules also play a fundamental role for the development and maintenance of bone by mediating cellular crosstalk between osteogenic cells and by providing targets for the sorting and migration of osteogenic precursors toward the bone surface. We describe the localization of cadherin-11 and N-cadherin along the cell margins of mouse osteoblast-like cells, the colocalization of pancadherin with α-catenin, β-catenin, p120, and vinculin, and the association of these complexes with the actin microfilaments. Furthermore, we measured the influence of cell confluency and the effects of the osteogenic hormones triiodothyronine (T3) and 1,25-dihydroxyvitamin D3 (D3) on these parameters. By mRNA studies we found the abundantly expressed cadherin-11 being unaffected during T3- and D3-induced osteoblastic differentiation. However, protein levels of N-cadherin and pancadherin were strongly suppressed by D3. We also observed a clear distinction in cadherin immunolocalization when comparing confluent control and confluent hormone-treated cultures. Immunoprecipitation experiments indicated that vinculin is part of the junctional complex, and that the association of pancadherin/β-catenin is strongly increased after treatment with T3 which might influence the functional competence of cell–cell contacts. Thus, this study demonstrates the molecular organization of adherens junctions in mouse osteoblastic MC3T3-E1 cells and their sensitivity to the osteogenic factors T3 and D3 in confluent cultures.

Novel PHEX Mutation Associated with Hypophosphatemic Rickets

Background: X-linked hypophosphatemia (XLH) is the most prevalent heritable form of rickets. It is a dominantly inherited disorder, characterized by renal phosphate wasting, abnormal vitamin D and PTH metabolism, and defective bone mineralization. Inactivating mutations in the gene encoding PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) have been found to be associated with XLH. Methods: We report about a 54-year-old male patient who exhibited the typical features of XLH, and in whom mutational analysis using PCR and sequencing was performed. Additionally, extensive laboratory and radiological investigations were carried out. Results: A 1-bp deletion in exon 2 of the PHEX gene was detected (177delC), which, to the best of our knowledge, has not been reported yet. This deletion results in a premature stop codon (C59X), suggesting a truncation of the PHEX protein. Furthermore, elevated FGF23 and PTH levels as well as an increased axial bone mineral density score were measured. Conclusions: We present a male patient with XLH, who harbors a novel mutation in the PHEX gene, which might be the cause for his disease. Our data support previous findings and therefore contribute to the decipherment of the pathogenetic pathways of XLH.

Persistent torticollis, facial asymmetry, grooved tongue, and dolicho-odontoid process in connection with atlas malformation complex in three family subjects

Congenital clefts and other malformations of the atlas are incidental findings identified while investigating the cervical spine following trauma. A persistent bifid anterior and posterior arch of the atlas beyond the age of 3–4xa0years is observed in skeletal dysplasias, Goldenhar syndrome, Conradi syndrome, and Down’s syndrome. There is a high incidence of both anterior and posterior spina bifida of the atlas in patients with metabolic disorders, such as Morquio’s syndrome [Baraitser and Winter in London dysmorphology database, Oxford University Press, 2005; Torriani, Lourenco in Rev Hosp Clin Fac Med Sao Paulo 53: 73–76, 2002]. We report two siblings and their mother, with congenital, persistent torticollis, plagiocephaly, facial asymmetry, grooved tongues, and asymptomatic “dolicho-odontoid process”. All are of normal intelligence. No associated Neurological dysfunction, paresis, apnoea, or failures to thrive were encountered. Radiographs of the cervical spine were non-contributory, but 3D CT scanning of this area allowed further visualisation of the cervico–cranial malformation complex in this family and might possibly explain the sudden early juvenile mortality. Agenesis of the posterior arch of the atlas and bifidity/clefting of anterior arch of the atlas associated with asymptomatic “dolicho-odontoid process” were the hallmark in the proband and his female sibling. Some of the features were present in the mother. All the family subjects were investigated. To the best of our knowledge the constellation of malformation complex in this family has not been previously reported.

The imbalance between osteoprotegerin and cathepsin K in the serum of patients with longstanding rheumatoid arthritis

Osteoprotegerin (OPG) and soluble receptor activator of NF-kappa B ligand (sRANKL) together regulate the bone metabolism among other cytokines, whereby cathepsin K has a potent collagen-degrading activity. An imbalance of this system may be partly responsible for the skeletal complications of RA. Expanding on a previous study, we investigated the relationship between OPG, sRANKL and cathepsin K levels in the serum of patients with longstanding RA. We measured serum levels of OPG, sRANKL and cathepsin K of 100 patients with active, longstanding RA. We detected elevated serum levels of cathepsin K (median 54.8xa0pmol/l) and OPG (median 4.8xa0pmol/l), but normal sRANKL levels (median 0.2xa0pmol/l). Cathepsin K did not show a correlation with the overexpressed OPG (Pxa0=xa00.64) and sRANKL (Pxa0=xa00.81). The radiological destruction correlates significantly with cathepsin K (Pxa0=xa00.004) and OPG (Pxa0=xa00.007). We speculate that the increased levels of OPG are effective in compensating the action of sRANKL, but do not directly prevent bone degradation, as reflected by the elevated serum levels of cathepsin K.

1,25-Dihydroxy Vitamin D3 and Tri-iodothyronine Stimulate the Expression of a Protein Immunologically Related to Osteocalcin

Osteocalcin (OC), a bone-specific protein, is a marker of late osteoblastic differentiation. Its expression is influenced by various growth factors and hormones. We investigated the effect of 1,25-dihydroxy vitamin D3 (D3) and tri-iodothyronine (T3) on OC expression in osteoblast-like MC3T3-E1 cells. A heterologous OC green fluorescence protein (GFP) fusion vector was established and expressed to study possible effects on protein transport. Immunostaining of endogenous OC revealed a significant increase in the percentage of positive cells after D3 and T3 treatment. This was consistent for MC3T3-E1 cells as well as nonosteogenic NIH-3T3 and mammary carcinoma cells, but not for neuroblastoma cells. The perinuclear immunostaining corresponded to the NBD C6 ceramide Golgi staining. Conversely, we found a strong induction of OC in MC3T3-E1 cells at the mRNA and protein levels only with T3 and not with D3. OC mRNA and protein expression was not detected in NIH fibroblasts. OC GFP transfection experiments indicate rapid transport and secretion of OC, because OC GFP was not found to be accumulated at intracellular compartments after hormone treatment. We conclude that the strong perinuclear immunostaining does not represent OC but a protein immunologically related to OC, as indicated by preabsorption experiments. The expression of this OC epitope-sharing protein is regulated by both D3 and T3 in the osteoblastic MC3T3-E1 and in nonosteogenic cells.

Craniovertebral malformation complex in a child with Weismann-Netter-Stuhl syndrome.

OBJECTIVEnBowing of the legs is usually thrown into the basket of vitamin D deficiency rickets; therefore, a significant number of affected children can be misdiagnosed and improperly managed. This case illustrates how the careful clinical and radiological assessment of such a case can lead to the adequate understanding of its etiology.nnnDESCRIPTIONnWe report a sporadic case of a 2-year-old male child who presented with radiological features that were compatible with Weismann-Netter-Stuhl syndrome. In addition, we observed craniovertebral malformation complex. He was of normal intelligence. To our knowledge, the combination of Weismann-Netter-Stuhl syndrome and presence of a hypoplastic occipitalized atlas and further C2-C3 fusion has not been reported before. The diagnosis of Weismann-Netter-Stuhl is discussed. Classically, Weismann-Netter-Stuhl syndrome is characterized by short stature, mental retardation (in some individuals), dural calcification, and anterior bowing of the tibiae. However, we believe that careful clinical and radiological examinations can reveal more striking data which might positively reflect on the whole process of management.nnnCOMMENTSnWe postulate that the congenital limitations in neck movements in our patient developed because of the marked fusion of the hypoplastic and occipitalized atlas and simultaneous C2-C3 fusion. Therefore, if this form of malformation is disregarded, there may be involvement of the atlantoaxial structure, and this can possibly lead to serious neurological and even life-threatening complications. The use of CT scanning for the detection of such abnormalities can be remarkably important.

Complexo de malformação craniovertebral em uma criança com síndrome de Weismann-Netter-Stuhl

OBJECTIVE: Bowing of the legs is usually thrown into the basket of vitamin D deficiency rickets; therefore, a significant number of affected children can be misdiagnosed and improperly managed. This case illustrates how the careful clinical and radiological assessment of such a case can lead to the adequate understanding of its etiology. DESCRIPTION: We report a sporadic case of a 2-year-old male child who presented with radiological features that were compatible with Weismann-Netter-Stuhl syndrome. In addition, we observed craniovertebral malformation complex. He was of normal intelligence. To our knowledge, the combination of Weismann-Netter-Stuhl syndrome and presence of a hypoplastic occipitalized atlas and further C2-C3 fusion has not been reported before. The diagnosis of Weismann-Netter-Stuhl is discussed. Classically, Weismann-Netter-Stuhl syndrome is characterized by short stature, mental retardation (in some individuals), dural calcification, and anterior bowing of the tibiae. However, we believe that careful clinical and radiological examinations can reveal more striking data which might positively reflect on the whole process of management. COMMENTS: We postulate that the congenital limitations in neck movements in our patient developed because of the marked fusion of the hypoplastic and occipitalized atlas and simultaneous C2-C3 fusion. Therefore, if this form of malformation is disregarded, there may be involvement of the atlantoaxial structure, and this can possibly lead to serious neurological and even life-threatening complications. The use of CT scanning for the detection of such abnormalities can be remarkably important.

Vertebral hyperostosis, ankylosed vertebral fracture and atlantoaxial rotatory subluxation in an elderly patient with a history of infantile idiopathic scoliosis; a case report

This is a case report of a 48-year-old-woman with scoliosis since early childhood. Recent radiographic spinal assessment revealed the presence of distinctive spinal abnormalities. To the best of our knowledge this is the first clinical report describing a constellation of unusual changes in an elderly woman with a history of infantile idiopathic scoliosis.