Mutations in distant regulatory elements can have a negative impact on human development and health, yet because of the difficulty of detecting these critical sequences, we predominantly focus on coding sequences for diagnostic purposes. We have undertaken a comparative sequence-based approach to characterize a large noncoding region deleted in patients affected by Van Buchem (VB) disease, a severe sclerosing bone dysplasia. Using BAC recombination and transgenesis, we characterized the expression of human sclerostin (SOST) from normal (SOST^wt) or Van Buchem (SOST^vbΔ) alleles. Only theSOST^wtallele faithfully expressed high levels of humanSOSTin the adult bone and had an impact on bone metabolism, consistent with the model that the VB noncoding deletion removes aSOST-specific regulatory element. By exploiting cross-species sequence comparisons with in vitro and in vivo enhancer assays, we were able to identify a candidate enhancer element that drives humanSOSTexpression in osteoblast-like cell lines in vitro and in the skeletal anlage of the embryonic day 14.5 (E14.5) mouse embryo, and discovered a novel function for sclerostin during limb development. Our approach represents a framework for characterizing distant regulatory elements associated with abnormal human phenotypes.

Expression of the osteocyte‐derived bone formation inhibitor sclerostin in adult bone requires a distant enhancer. We show that MEF2 transcription factors control this enhancer and mediate inhibition of sclerostin expression by PTH. Introduction: Sclerostin encoded by the SOST gene is a key regulator of bone formation. Lack of SOST expression is the cause for the progressive bone overgrowth disorders sclerosteosis and Van Buchem disease. We have previously identified a distant enhancer within the 52‐kb Van Buchem disease deletion downstream of the SOST gene that is essential for its expression in adult bone. Furthermore, we and others have reported that SOST expression is suppressed by PTH. The aim of this study was to identify transcription factors involved in SOST bone enhancer activity and mediating PTH responsiveness. Materials and Methods: Regulation of the SOST enhancer and promoter was studied by luciferase reporter gene assays. Transcription factor binding sites were mapped by footprint analysis and functional mutation analyses using transient transfections of osteoblast‐like UMR‐106 cells that exhibit endogenous SOST expression. Specific transcription factor binding was predicted by sequence analysis and shown by gel retardation assays and antibody‐induced supershifts. Expression of myocyte enhancer factors 2 (MEF2) was detected by in situ hybridization, quantitative RT‐PCR (qPCR), and immunohistochemistry. The role of MEF2s in SOST expression was assessed by reporter gene assays and siRNA‐mediated RNA knockdown. Results: PTH completely suppressed the transcriptional activity of the SOST bone enhancer but did not affect the SOST promoter. A MEF2 response element was identified in the bone enhancer. It was essential for transcriptional activation, bound MEF2 transcription factors, and mediated PTH responsiveness. Expression of MEF2s in bone was shown by qPCR, in situ hybridization, and immunohistochemistry. MEF2s and sclerostin co‐localized in osteocytes. Enhancer activity was stimulated by MEF2C overexpression and inhibited by co‐expression of a dominant negative MEF2C mutant. Finally, siRNA‐mediated knockdown of MEF2A, C, and D suppressed endogenous SOST expression in UMR‐106 osteoblast‐like cells. Conclusions: These data strongly suggest that SOST expression in osteocytes of adult bone and its inhibition by PTH is mediated by MEF2A, C, and D transcription factors controlling the SOST bone enhancer. Hence, MEF2s are implicated in the regulation of adult bone mass.

The Wnt antagonist Sost has emerged as a key regulator of bone homeostasis through the modulation of Lrp4/5/6 Wnt coreceptors. In humans, lack of Sclerostin causes sclerosteosis and van Buchem (VB) disease, two generalized skeletal hyperostosis disorders that result from hyperactive Wnt signaling. Unlike sclerosteosis, VB patients lack SOST coding mutations but carry a homozygous 52 kb noncoding deletion that is essential for the transcriptional activation of SOST in bone. We recently identified a putative bone enhancer, ECR5 , in the VB deletion region, and showed that the transcriptional activity of ECR5 is controlled by Mef2C transcription factor in vitro. Here we report that mice lacking ECR5 or Mef2C through Col1-Cre osteoblast/osteocyte-specific ablation result in high bone mass (HBM) due to elevated bone formation rates. We conclude that the absence of the Sost -specific long-range regulatory element ECR5 causes VB disease in rodents, and that Mef2C is the main transcription factor responsible for ECR5 -dependent Sost transcriptional activation in the adult skeleton.

Detailed information about stage-specific changes in gene expression is crucial for understanding the gene regulatory networks underlying development and the various signal transduction pathways contributing to morphogenesis. Here we describe the global gene expression dynamics during early murine limb development, when cartilage, tendons, muscle, joints, vasculature and nerves are specified and the musculoskeletal system of limbs is established. We used whole-genome microarrays to identify genes with differential expression at 5 stages of limb development (E9.5 to 13.5), during fore- and hind-limb patterning. We found that the onset of limb formation is characterized by an up-regulation of transcription factors, which is followed by a massive activation of genes during E10.5 and E11.5 which levels off at later time points. Among the 3520 genes identified as significantly up-regulated in the limb, we find ∼30% to be novel, dramatically expanding the repertoire of candidate genes likely to function in the limb. Hierarchical and stage-specific clustering identified expression profiles that are likely to correlate with functional programs during limb development and further characterization of these transcripts will provide new insights into specific tissue patterning processes. Here, we provide for the first time a comprehensive analysis of developmentally regulated genes during murine limb development, and provide some novel insights into the expression dynamics governing limb morphogenesis.

The goal of this study was to assess the long-term biocompatibility of single-wall carbon nanotubes (SWNTs) for tissue engineering of articular cartilage. We hypothesized that SWNT nanocomposite scaffolds in cartilage tissue engineering can provide an improved molecular-sized substrate for stimulation of chondrocyte growth, as well as structural reinforcement of the scaffold's mechanical properties. The effect of SWNT surface functionalization (-COOH or -PEG) on chondrocyte viability and biochemical matrix deposition was examined in two-dimensional cultures, in three-dimensional (3D) pellet cultures, and in a 3D nanocomposite scaffold consisting of hydrogels+SWNTs. Outcome measures included cell viability, histological and SEM evaluation, GAG biochemical content, compressive and tensile biomechanical properties, and gene expression quantification, including extracellular matrix (ECM) markers aggrecan ( Agc ), collagen-1 ( Col1a1 ), collagen-2 ( Col2a1 ), collagen-10 ( Col10a1 ), surface adhesion proteins fibronectin (Fn) , CD44 antigen (CD44), and tumor marker ( Tp53 ). Our findings indicate that chondrocytes tolerate functionalized SWNTs well, with minimal toxicity of cells in 3D culture systems (pellet and nanocomposite constructs). Both SWNT-PEG and SWNT-COOH groups increased the GAG content in nanocomposites relative to control. The compressive biomechanical properties of cell-laden SWNT-COOH nanocomposites were significantly elevated relative to control. Increases in the tensile modulus and ultimate stress were observed, indicative of a tensile reinforcement of the nanocomposite scaffolds. Surface coating of SWNTs with -COOH also resulted in increased Col2a1 and Fn gene expression throughout the culture in nanocomposite constructs, indicative of increased chondrocyte metabolic activity. In contrast, surface coating of SWNTs with a neutral -PEG moiety had no significant effect on Col2a1 or Fn gene expression, suggesting that the charged nature of the -COOH surface functionalization may promote ECM expression in this culture system. The results of this study indicate that SWNTs exhibit a unique potential for cartilage tissue engineering, where functionalization with bioactive molecules may provide an improved substrate for stimulation of cellular growth and repair.

Increased osteoblast activity in sclerostin‐knockout (Sost^−/−) mice results in generalized hyperostosis and bones with small bone marrow cavities resulting from hyperactive mineralizing osteoblast populations. Hematopoietic cell fate decisions are dependent on their local microenvironment, which contains osteoblast and stromal cell populations that support both hematopoietic stem cell quiescence and facilitate B‐cell development. In this study, we investigated whether high bone mass environments affect B‐cell development via the utilization of Sost^−/− mice, a model of sclerosteosis. We found the bone marrow of Sost^−/− mice to be specifically depleted of B cells because of elevated apoptosis at all B‐cell developmental stages. In contrast, B‐cell function in the spleen was normal. Sost expression analysis confirmed that Sost is primarily expressed in osteocytes and is not expressed in any hematopoietic lineage, which indicated that the B‐cell defects in Sost^−/− mice are non‐cell autonomous, and this was confirmed by transplantation of wild‐type (WT) bone marrow into lethally irradiated Sost^−/− recipients. WT→Sost^−/− chimeras displayed a reduction in B cells, whereas reciprocal Sost^−/−→WT chimeras did not, supporting the idea that the Sost^−/− bone environment cannot fully support normal B‐cell development. Expression of the pre‐B‐cell growth stimulating factor, Cxcl12, was significantly lower in bone marrow stromal cells of Sost^−/− mice, whereas the Wnt target genes Lef‐1 and Ccnd1 remained unchanged in B cells. Taken together, these results demonstrate a novel role for Sost in the regulation of bone marrow environments that support B cells. © 2012 American Society for Bone and Mineral Research.