CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members, and its null mice display skeletal dysmorphisms. However, little is known concerning roles of the other CCN members in chondrocytes. Using both in vivo and in vitro approaches, we conducted a comparative analysis of CCN2-null and wildtype mice to study the roles of CCN2 and the other CCN proteins in cartilage development. Immunohistochemistry was used to evaluate the localization of CCN proteins and other chondrocyte-associated molecules in the two types of mice. Moreover, gene expression levels and the effects of exogenous CCN proteins on chondrocyte proliferation, differentiation, and the expression of chondrocyteassociated genes in their primary chondrocytes were evaluated. Ccn3 was dramatically upregulated in CCN2-null cartilage and chondrocytes. This upregulation was associated with diminished cell proliferation and delayed differentiation. Consistent with the in vivo findings, CCN2 deletion entirely retarded chondrocyte terminal differentiation and decreased the expression of several chondrocyte-associated genes in vitro, whereas Ccn3 expression drastically increased. In contrast, the addition of exogenous CCN2 promoted differentiation strongly and induced the expression of the associated genes, whereas decreasing the Ccn3 expression. These findings collectively indicate that CCN2 induces chondrocyte differentiation by regulating the expression of chondrocyte-associated genes but that these effects are counteracted by CCN3. The lack of CCN2 caused upregulation of CCN3 in CCN2-null mice, which resulted in the observed phenotypes, such as the resultant delay of terminal differentiation. The involvement of the PTHrP-Ihh loop in the regulation of CCN3 expression is also suggested.

The mammalian SWI/SNF chromatin-remodeling complex facilitates DNA access by transcription factors and the transcription machinery. The characteristic member of human SWI/SNF-A is BAF250/ARID1, of which there are two isoforms, BAF250a/ARID1a and BAF250b/ARID1b. Here we report that BAF250b complexes purified from mammalian cells contain elongin C (Elo C), a BC box binding component of an E3 ubiquitin ligase. BAF250b was found to have a BC box motif, associate with Elo C in a BC box-dependent manner, and, together with cullin 2 and Roc1, assemble into an E3 ubiquitin ligase. The BAF250b BC box mutant protein was unstable in vivo and was autoubiquitinated in a manner similar to that for the VHL BC box mutants. The discovery that BAF250 is part of an E3 ubiquitin ligase adds an enzymatic function to the chromatinremodeling complex SWI/SNF-A. The immunopurified BAF250b E3 ubiquitin ligase was found to target histone H2B at lysine 120 for monoubiquitination in vitro. To date, all H2B monoubiquitination was attributed to the human homolog of yeast Bre1 (RNF20/40). Mutation of Drosophila osa, the homolog of BAF250, or depletion of BAF250 by RNA interference (RNAi) in cultured human cells resulted in global decreases in monoubiquitinated H2B, implicating BAF250 in the cross talk of histone modifications.

Abstract. Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that promotes proliferation, motility, and morphogenesis in epithelial cells. Recently the HGF receptor, c-met protooncogene product, has been shown to be expressed in developing limb buds (Sonnenberg, E., D. Meyer, M. Weidner, and C. Birchmeiyer. 1993. J. Cell Biol. 123: 223-235), suggesting that some populations of mesenchymal cells in limb buds respond to HGF/SF. To test the possibility that HGF/SF is involved in regulation of cartilage development, we isolated chondrocytes from knee joints and costal cartilages of 23-d embryonic and 4-wk-old rabbits, and analyzed the effects of HGF/SF on migration and proliferation of these cells. We found that HGF/SF stimulated migration of cultured articular chondrocytes but did not scatter limb mesenchymal fibroblasts or synovial fibroblasts in culture. HGF/SF also stimulated proliferation of chondrocytes; a maximum three-fold stimulation in DNA synthesis was observed at the concentration of 3 ng/ml of HGF/SF. Moreover, HGF/SF had the ability to enhance proteoglycan synthesis in chondrocytes. The responsiveness of chondrocytes to HGF/SF was also supported by the observation that they expressed the HGF/SF receptor. Addition of the neutralizing antibody to rat HGF/ SF affected neither DNA synthesis nor proteoglycan synthesis in rat chondrocytes, suggesting a paracine mechanism of action of HGF/SF on these cells. In situ hybridization analysis showed that HGF/SF mRNA was restrictively expressed in the areas of future joint regions in developing limb buds and in the intercostal spaces of developing costal cartilages. These findings suggest that HGF/SF plays important roles in cartilage development through its multiple activities.

CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members, and Ccn2 null mutant mice display skeletal dysmorphisms. However, little is known concerning the roles of CCN2 during bone formation. We herein present a comparative analysis of wild-type and Ccn2 null mice to investigate the roles of CCN2 in bone development. Multiple histochemical methods were employed to analyze the effects of CCN2 deletion in vivo, and effects of CCN2 on the osteogenic response were evaluated with the isolated and cultured osteoblasts. As a result, we found a drastic reduction of the osteoblastic phenotype in Ccn2 null mutants. Importantly, addition of exogenous CCN2 promoted every step of osteoblast differentiation and rescued the attenuated activities of the Ccn2 null osteoblasts. These results suggest that CCN2 is required not only for the regulation of cartilage and subsequent events, but also for the normal intramembranous bone development.

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TBP-associated factor 4 (TAF4), an essential subunit of the TFIID complex acts as a coactivator for multiple transcriptional regulators, including Sp1 and CREB. However, little is known regarding the structural properties of the TAF4 subunit that lead to the coactivator function. Here, we report the crystal structure at 2.0-Å resolution of the human TAF4-TAFH domain, a conserved domain among all metazoan TAF4, TAF4b, and ETO family members. The hTAF4-TAFH structure adopts a completely helical fold with a large hydrophobic groove that forms a binding surface for TAF4 interacting factors. Using peptide phage display, we have characterized the binding preference of the hTAF4-TAFH domain for a hydrophobic motif, D⌿⌿ ⌿⌽, that is present in a number of nuclear factors, including several important transcriptional regulators with roles in activating, repressing, and modulating posttranslational modifications. A comparison of the hTAF4-TAFH structure with the homologous ETO-TAFH domain reveals several critical residues important for hTAF4-TAFH target specificity and suggests that TAF4 has evolved in response to the increased transcriptional complexity of metazoans.TFIID ͉ transcription ͉ x-ray crystallography ͉ TAFH domain T he general transcription factor TFIID, composed of the TATAbinding protein (TBP) and at least 14 additional TBPassociated factors (TAFs) (1), plays an important role in the regulation of gene transcription by RNA polymerase II. It contributes to a large number of activities necessary for regulated transcription, such as core promoter recognition and chromatin modification and recognition (2, 3). Individual TAFs are important for mediating distinct activator-specific transcriptional regulation in vivo (4). Studies of yeast strains having temperature-sensitive mutations indicate that Ϸ84% of yeast genes depend on one or more TAFs (5), indicating the importance of this factor in RNA Pol II transcription.The human TAF4 (hTAF II 130/dTAF II 110) subunit of the TFIID complex was the first of the TAF subunits demonstrated to possess coactivator activity for the glutamine-rich activators Sp1 (6), CREB (7), and nuclear receptors RAR and TR (8). More recently, TAF4 has been demonstrated to play a critical role in maintaining the stability of the TFIID complex (9). The hTAF4 sequence contains four glutamine-rich tracts mediating interactions with the activators Sp1 and CREB (7) and two highly conserved domains CI and CII [supporting information (SI) Fig. 5A]. The misregulation of Sp1 activation mediated by hTAF4 has been implicated in Huntington's disease and a number of other neurodegenerative diseases that result from polyglutamine expansions (10). A region (residues 870-911) of the CII domain has been shown to interact with the histone-fold motif of hTAF12 and form a novel histone-like pair (11). The CI region of hTAF4 is highly conserved among all metazoan TAF4, TAF4b, and ETO family members and is also known as the TAF homology (TAFH) domain (6, 12) (SI Fig. 5B). The ETO-TAFH domain has been demonstrated t...

An assessment of bone quality in the anterior alveolar ridge may well reflect age-related systemic pathological conditions and should be used in dental implant treatment planning to avoid associated risk factors.

After a Le Fort I osteotomy, nasal and labial changes are sometimes undesirable. The aim of this study was to perform a three-dimensional evaluation of the morphologic changes of the nose and lips after a Le Fort I osteotomy with a three-dimensional laser scanner.Twelve female patients who underwent a Le Fort I osteotomy with bilateral sagittal split ramus osteotomy (mean age, 24.2 y) were selected. Three-dimensional facial morphology was measured immediately after surgery and 6 months after surgery with a three-dimensional laser scanner. Analysis of the three-dimensional data was performed with three-dimensional image analyzing software. The evaluation was performed by (1) linear and angular analyses of landmarks, (2) three-dimensional curvature, and (3) visual qualitative analysis using superimposing mapping images. The results were compared control with data taken from volunteers (mean age, 24.5 y).Lateral expansion of nasal alae was commonly observed after surgery. The labial changes were mainly due to movements of the jawbone, and obvious flattening was not detected in this study. There was no relationship between the nasal changes and maxillary advancement.The nasal morphologic changes after a Le Fort I osteotomy consist mainly of a widening of the nasal alae caused by the release of the muscle insertion and their retraction. This change was not influenced by the direction of maxillary movements.