Missense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder characterized by hypoplastic or absent clavicles, large fontanelles, dental anomalies and delayed skeletal development. The phenotype is suggestive of a generalized defect in ossification and is one of the most common skeletal dysplasias not associated with disproportionate stature. To date, no genetic determinants of ossification have been identified. CCD has been mapped to chromosome 6p21, where CBFA1, a gene encoding OSF2/CBFA1, a transcriptional activator of osteoblast differentiation, has been localized. Here, we describe two de novo missense mutations, Met175Arg and Ser191Asn, in the OSF2/CBFA1 gene in two patients with CCD. These two mutations result in substitution of highly conserved amino acids in the DNA-binding domain. DNA-binding studies with the mutant polypeptides show that these amino acid substitutions abolish the DNA-binding ability of OSF2/CBFA1 to its known target sequence. Concurrent studies show that heterozygous nonsense mutations in OSF2/CBFA1 also result in CCD, while mice homozygous for the osf2/cbfa1 mull allele exhibit a more severe lethal phenotype. Thus, these results together suggest that CCD is produced by haploinsufficiency of OSF2/CBFA1 and provide direct genetic evidence that the phenotype is secondary to an alteration of osteoblast differentiation.

[Treatment of the urethral sphincter insufficiency]. / Prise en charge de l'insuffisance sphinctérienne urétrale.

The intrinsic sphincter insufficiency is a cause of stress urinary incontinence. Its definition is clinical and based on urodynamics. It is mostly met with women, in context of the post-obstetrical period or older women in a multifactorial context. For men, it occurs mainly as complication of the surgery of the cancer of prostate or bladder. An initial, clinical and paraclinical assessment allows to confirm the diagnosis of intrinsic sphincter insufficiency, to estimate its severity, and to identify associated mechanisms of incontinence (urethral hypermobility, bladder overactivity) to choose the most adapted treatment. The perineal reeducation is the treatment of first intention in both sexes. At the menopausal woman, the local hormonotherapy is a useful additive. In case of failure or of incomplete efficiency, the treatment of the intrinsic sphincter insufficiency is surgical. Bulking agents, urethral slings, peri-urethral balloons and artificial sphincter are 4 therapeutic options to discuss according to history, the severity of the incontinence, the expectations of the patient.

[Treatment of detrusor-striated sphincter dyssynergia with permanent nitinol urethral stent: results after a minimum follow-up of 2 years]. / Résultats à moyen terme du traitement de la dyssynergie vésicosphinctérienne par sphinctérotomie prothétique permanente.

OBJECTIVES: The aim of this study was to assess the outcomes of nitinol permanent urethral stents used in detrusor-striated sphincter dyssynergia (DSD) treatment on male patients with a spinal cord injury. MATERIALS: We investigated retrospectively all patients treated from 2004 to 2012. A total of 22 patients were included, with an age ranging from 22 to 76 years old. The DSD syndrome was due to spinal cord injury (18) or various spinal cord diseases (four) and treated with a nitinol urethral stent (11 Ultraflex(®) and 11 Mémotherm(®)). Every patient had an urodynamical study. The follow-up reached at least 2 years. RESULTS: The mean follow-up was 56 months (± 14). Complementary procedures after stenting included: five stent prolongation or displacement (mean interval 7.6 months), six bladder neck incisions (12.2 months), three urethrotomy (42 months), ten obstruction treated by laser (47.3 months). Eight patients had a change of their urinary pattern: four underwent ileal conduit diversion, one had a continent urinary diversion, one chose self intermittent catheterization, two were under indwelling catheterization waiting for another treatment. Stent retrieval was either harmful or impossible for four of them. Three patients were free of complementary procedures. CONCLUSIONS: Nitinol urethral stent was an effective treatment initially. However, by the third year, urethral stenosis and hypertrophic growth of the urethral mucosa usually require iterative endoscopic procedures (0.31 per patient per year). Patients treated with permanent uretral stent deserve a yearly endoscopic follow-up. Safety and effectiveness of permanent uretral stent compared to surgical sphincterotomy to treat DSD are discussed.

A novel role for Twist-1 in pulp homeostasis.

The molecular mechanisms that maintain the equilibrium of odontoblast progenitor cells in dental pulp are unknown. Here we tested whether homeostasis in dental pulp is modulated by Twist-1, a nuclear protein that partners with Runx2 during osteoblast differentiation. Our analysis of Twist-1(+/-) mice revealed phenotypic changes that involved an earlier onset of dentin matrix formation, increased alkaline phosphatase activity, and pulp stones within the pulp. RT-PCR analyses revealed Twist-1 expression in several adult organs, including pulp. Decreased levels of Twist-1 led to higher levels of type I collagen and Dspp gene expression in perivascular cells associated with the pulp stones. In mice heterozygous for both Twist-1 and Runx2 inactivation, the phenotype of pulp stones appeared completely rescued. These findings suggest that Twist-1 plays a key role in restraining odontoblast differentiation, thus maintaining homeostasis in dental pulp. Furthermore, Twist-1 functions in dental pulp are dependent on its interaction with Runx2.

Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers.

Bovine aortic smooth muscle cell (BASMC) cultures undergo mineralization on addition of the organic phosphate donor, beta-glycerophosphate (betaGP). Mineralization is characterized by apatite deposition on collagen fibrils and the presence of matrix vesicles, as has been described in calcified vascular lesions in vivo as well as in bone and teeth. In the present study, we used this model to investigate the molecular mechanisms driving vascular calcification. We found that BASMCs lost their lineage markers, SM22alpha and smooth muscle alpha-actin, within 10 days of being placed under calcifying conditions. Conversely, the cells gained an osteogenic phenotype as indicated by an increase in expression and DNA-binding activity of the transcription factor, core binding factor alpha1 (Cbfa1). Moreover, genes containing the Cbfa1 binding site, OSE2, including osteopontin, osteocalcin, and alkaline phosphatase were elevated. The relevance of these in vitro findings to vascular calcification in vivo was further studied in matrix GLA protein null (MGP(-/-)) mice whose arteries spontaneously calcify. We found that arterial calcification was associated with a similar loss in smooth muscle markers and a gain of osteopontin and Cbfa1 expression. These data demonstrate a novel association of vascular calcification with smooth muscle cell phenotypic transition, in which several osteogenic proteins including osteopontin, osteocalcin, and the bone determining factor Cbfa1 are gained. The findings suggest a positive role for SMCs in promoting vascular calcification.

Expression of core binding factor Osf2/Cbfa-1 and bone sialoprotein in tooth development.

The transcription factor Osf2/Cbfa1 is a key regulator of osteogenic differentiation while BSP, a major non-collagenous protein, is a marker of osteoblastic differentiation. To determine the relationship between Osf2/Cbfa1 and the formation of mineralized tissues in tooth development we have studied the temporal expression of Osf2/Cbfa1 and BSP mRNA using in situ hybridization. These studies show that Osf2/Cbfa1 is expressed early in mesenchymal and epithelial tissues destined to form the mineralized tissues of the tooth and periodontal tissues, whereas BSP provides a specific marker for the differentiated cells in each of these tissues. Expression of Osf2/Cbfa1, but not BSP, was observed in the periodontal ligament indicating that expression of Osf2/Cbfa1 is not restricted to mineralizing tissues.

DNA fragmentation during bone formation in neonatal rodents assessed by transferase-mediated end labeling.

To study the fate of bone cells, we used the transferase-mediated, biotin-dUTP nick end-labeling (TUNEL) assay to detect DNA fragmentation during the formation of intramembranous and endochondral bone in newly born hamsters, mice, and rats. In alveolar bone forming around the developing tooth crowns, DNA fragmentation was found in three cell types: TRAP-negative mononuclear cells at the bone surface, osteocytes, and some but not all nuclei of TRAP-positive osteoclasts. Osteoblasts did not undergo DNA fragmentation. A strong positive correlation was found between contacts of TUNEL-positive osteocytes and osteoclasts. Extracellular bone matrix also stained occasionally for the presence of DNA fragments. During endochondral bone formation, TUNEL staining was detected in late hypertrophic chondrocytes of the epiphyseal growth plate. During rapid longitudinal growth of long bones, TUNEL-positive hypertrophic chondrocytes were found coincident with or slightly after invasion of blood vessels from the diaphysis. However, during slow longitudinal growth and in secondary ossification centers, DNA fragmentation was seen in hypertrophic chondrocytes still located within their lacunae. We conclude that some of the osteocytes in deeper layers of bone die within their lacuna and disperse nuclear fragments over the extracellular matrix, that a majority of the osteocytes are phagocytosed and degraded by osteoclasts at sites of intense bone resorption, and that during endochondral ossification, substantial numbers of late hypertrophic chondrocyte cells undergo cell death.

Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin.

Osteocalcin, the gamma-carboxyglutamic acid-containing protein, which in most species is the predominant noncollagenous protein of bone and dentin, has been postulated to play roles in bone formation and remodeling. Recently, genetic studies showed that osteocalcin acts as an inhibitor of osteoblast function. Based on von Kossa staining and measurement of mineral apposition rates in tetracycline-labeled bones, osteocalcin knockout animals were reported to have no detectable alterations in bone mineralization. To test the hypothesis that, in addition to regulating osteoblastic activity, osteocalcin is involved in regulating mineral properties, a more sensitive assay of mineralization, Fourier transform infrared microspectroscopy (FT-IRM) was used to study thin sections of femora of 4-week-, 6-month- (intact and ovariectomized), and 9-month-old wild-type and osteocalcin-knockout mice. FT-IRM spectra provided spatially resolved measures of relative mineral and carbonate contents, and parameters indicative of apatite crystal size and perfection. No differences were detected in the mineral properties of the 4-week-old knockout and wild-type mice indicating that the mineralization process was not altered at this time point. Six-month-old wild-type animals had higher mineral contents (mineral:matrix ratios) in cortical as compared with trabecular bones; mineral contents in knockout and wild-type bones were not different. At each age studied, carbonate:phosphate ratios tended to be greater in the wild-type as compared with knockout animals. Detailed analysis of the phosphate nu1,nu3 vibrations in the spectra from 6-month-old wild-type animals indicated that the crystals were larger/more perfect in the cortical as opposed to the trabecular bones. In contrast, in the knockout animals' bones at 6 months, there were no differences between trabecular and cortical bone in terms of carbonate content or crystallite size and perfection. Spectral parameters of the cortical and trabecular bone of the knockout animals resembled those in the wild-type trabecular bone and differed from wild-type cortical bone. In ovariectomized 6-month-old animals, the mineral content (mineral:matrix ratio) in the wild-type cortices increased from periosteum to endosteum, whereas, in the knockout animals' bones, the mineral:matrix ratio was constant. Ovariectomized knockout cortices had lower carbonate:phosphate ratios than wild-type, and crystallite size and perfection resembled that in wild-type trabeculae, and did not increase from periosteum to endosteum. These spatially resolved data provide evidence that osteocalcin is required to stimulate bone mineral maturation.

Bone morphogenetic protein-7 (osteogenic protein-1, OP-1) and tooth development.

Bone morphogenetic proteins (BMPs) form a family of growth factors originally isolated from extracellular bone matrix that are capable of inducing bone formation ectopically. We studied the expression, tissue localization, and function of BMP-7 (OP-1) during tooth development in rodents. Patterns of BMP-7 gene expression and peptide distribution indicated that BMP-7 was present in dental epithelium during the dental lamina, bud, and cap stages. During the bell stage, BMP-7 mRNA expression and protein distribution shifted from dental epithelium toward the dental mesenchyme. With advancing differentiation of odontoblasts, BMP-7 protein staining in the dental papilla became restricted to the layer of fully functional odontoblasts in the process of depositing (pre)dentin. Secretory-stage ameloblasts exhibited weak immunostaining for BMP-7. A restricted pattern of staining in ameloblasts became apparent in post-secretory stages of amelogenesis. Also, cells of the forming periodontal ligament were immunopositive. Histological analysis of tooth development in neonatal BMP-7-deficient mice did not reveal obvious changes compared with wild-type mice. We conclude that, in developing dental tissues, BMP-7 has distribution and expression patterns similar to those of other BMP members but is not an essential growth factor for tooth development, possibly because of functional redundancy with other BMP members or related growth factors.

Molecular determinants of arterial calcification.

Calcification of extracellular matrix (ECM) can be either physiological or pathological. Physiological calcification (or mineralization) of ECM is restricted to bones, teeth and, to a lesser extent, growth plate cartilages. Pathological calcification appears often in the ECM of arteries where it is a frequent complication of atherosclerosis. However, calcification of the ECM of arteries is not restricted to atherosclerosis. Indeed, human diseases have been described that are characterized by calcification of the aortic media in the absence of any atherosclerotic lesions. The existence of these rare diseases, along with several mouse models recently generated and discussed below, indicates that the formation of atherosclerotic lesions and the calcification of the artery ECM are controlled by different genetic pathways. This emerging knowledge has implications for our understanding of ECM calcification beyond atherosclerosis.

Functional hierarchy between two OSE2 elements in the control of osteocalcin gene expression in vivo.

Osteocalcin gene expression is initiated perinatally and is restricted to mature osteoblasts and odontoblasts. Because their pattern of expression is highly restricted, the osteocalcin genes are excellent tools to study osteoblast-specific gene expression. To define the mechanisms of osteocalcin cell-specific gene expression in vivo, we generated transgenic mice harboring deletion mutants of the promoter region of OG2, one of the mouse osteocalcin genes. We show here that only 647 base pairs of this promoter are sufficient to confer cell-specific and time-specific expression to a reporter gene in vivo. This promoter fragment contains two copies of OSE2. This osteoblast-specific cis-acting element binds Osf2, a recently characterized osteoblast-specific transcription factor (Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. (1997) Cell 89, 747-754). We also demonstrate that the proximal OSE2 element is critical to confer an osteoblast-specific, developmentally regulated pattern of expression to a reporter gene. The other OSE2 element, located more upstream and presenting a lower affinity for Osf2, affects only weakly OG2 promoter activity. These data demonstrate the crucial role of Osf2 in controlling osteocalcin gene expression. Since osteocalcin synthesis is a hallmark of the differentiated osteoblast phenotype, these results suggest that, beyond its developmental function, Osf2 is also required for the maintenance of the osteoblast phenotype postnatally.

Studies of osteocalcin function in dentin formation in rodent teeth.

Osteocalcin (OC) is a major non-collagenous protein synthesized by osteoblasts, odontoblasts and cementoblasts. We examined the function of OC in dentinogenesis by exposing rat and hamster tooth organ cultures to 1,25(OH)2vit D3 or to bovine OC added to the culture medium. We furthermore examined dentinogenesis in tooth explants cultured in the presence of warfarin (an inhibitor of gamma-carboxylation of OC). Finally, we analyzed dentin from osteocalcin null mutant mice. Exposure to 1,25(OH)2vit D3 increased OC synthesis by odontoblasts in vitro at the transcriptional and protein levels but had no apparent effect on matrix formation or 45Ca uptake. High levels of bovine OC temporarily suppressed the initial formation of dentin and enamel and uptake of 45Ca. This effect was not seen when tooth explants were exposed to thermally decarboxylated OC. Exposure of tooth explants to warfarin had no significant effect on dentinogenesis. Dentin obtained from two-month-old OC null mutants looked structurally normal and did not show marked differences in dentin matrix thickness and mineral content compared to wild type. We concluded that, although OC at supraphysiological levels has the potential to affect dentin mineralization probably through its Gla-residues, the locally produced levels of OC are not sufficient to markedly influence dentinogenesis.

Cbfa1 is required for epithelial-mesenchymal interactions regulating tooth development in mice.

Osteoblasts and odontoblasts, cells that are responsible for the formation of bone and dentin matrices respectively, share several molecular characteristics. Recently, Cbfa1 was shown to be a critical transcriptional regulator of osteoblast differentiation. Mutations in this gene cause cleidocranial dysplasia (CCD), an autosomal dominant disorder in humans and mice characterized by defective bone formation. CCD also results in dental defects that include supernumerary teeth and delayed eruption of permanent dentition. The dental abnormalities in CCD suggest an important role for this molecule in the formation of dentition. Here we describe results of studies aimed at understanding the functions of Cbfa1 in tooth formation. RT-PCR and in situ hybridization analyses show that Cbfa1 has a unique expression pattern in dental mesenchyme from the bud to early bell stages during active epithelial morphogenesis. Unlike that observed in osteoblast differentiation, Cbfa1 is downregulated in fully differentiated odontoblasts and is surprisingly expressed in ectodermally derived ameloblasts during the maturation phase of enamel formation. The role of Cbfa1 in tooth morphogenesis is further illustrated by the misshapen and severely hypoplastic tooth organs in Cbfa1-/- mice. These tooth organs lacked overt odontoblast and ameloblast differentiation and normal dentin and enamel matrices. Epithelial-mesenchymal recombinants demonstrate that dental epithelium regulates mesenchymal Cbfa1 expression during the bud and cap stages and that these effects are mimicked by the FGFs but not by the BMPs as shown by our bead implantation assays. We propose that Cbfa1 regulates the expression of molecules in mesenchyme that act reciprocally on dental epithelium to control its growth and differentiation. Taken together, our data indicate a non-redundant role for Cbfa1 in tooth development that may be distinct from that in bone formation. In odontogenesis, Cbfa1 is not involved in the early signaling networks regulating tooth initiation and early morphogenesis but regulates key epithelial-mesenchymal interactions that control advancing morphogenesis and histodifferentiation of the epithelial enamel organ.

Nuclear DNA fragmentation during postnatal tooth development of mouse and hamster and during dentin repair in the rat.

The TUNEL (transferase-mediated, dUTP-biotin nick end labeling) method for in situ labeling of DNA strands was utilized to localize DNA fragmentation in cells involved in tooth formation in the neonatal mouse and hamster. Positive reactions for the presence of DNA fragments were obtained in some epithelial cells of the cervical loop region of incisors, late secretory, transitional and early maturation stage ameloblasts, stratum intermedium cells and in shortened ameloblasts just before eruption. Also, cells of the periodontal ligament of the continuously erupting incisors stained positive shortly before eruption. Odontoblasts were negative but became strongly positive during the formation of physiological osteodentin at the tip of developing incisors. Osteodentin matrix and the surfaces of unerupted enamel and cementum just prior to eruption stained for DNA fragments as well. DNA fragmentation could be elicited in odontoblasts and underlying pulpal tissues of mature erupted molars after mechanical injury to the odontoblast processes during cavity preparation. We conclude that, in rodents, DNA fragmentation and cell death are biological processes which take place in a variety of cells involved in formation of teeth. The TUNEL staining technique is a simple but powerful tool to examine the fate of cells and tissues undergoing either programmed cell death (apoptosis) or fragmentation of nuclear DNA induced by external factors leading to pathological changes.

CBFA1 mutation analysis and functional correlation with phenotypic variability in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a dominantly inherited skeletal dysplasia caused by mutations in the osteoblast-specific transcription factor CBFA1. To correlate CBFA1 mutations in different functional domains with the CCD clinical spectrum, we studied 26 independent cases of CCD and a total of 16 new mutations were identified in 17 families. The majority of mutations were de novo missense mutations that affected conserved residues in the runt domain and completely abolished both DNA binding and transactivation of a reporter gene. These, and mutations which result in premature termination in the runt domain, produced a classic CCD phenotype by abolishing transactivation of the mutant protein with consequent haploinsufficiency. We further identified three putative hypomorphic mutations (R391X, T200A and 90insC) which result in a clinical spectrum including classic and mild CCD, as well as an isolated dental phenotype characterized by delayed eruption of permanent teeth. Functional studies show that two of the three mutations were hypomorphic in nature and two were associated with significant intrafamilial variable expressivity, including isolated dental anomalies without the skeletal features of CCD. Together these data show that variable loss of function due to alterations in the runt and PST domains of CBFA1 may give rise to clinical variability, including classic CCD, mild CCD and isolated primary dental anomalies.