REGULATION OF BMP-6 IN EARLY OSTEOBLAST DIFFERENTIATION

描述(改编自申请人的摘要)：复杂的过程 成骨细胞分化涉及许多系统、旁分泌和自分泌 各种因素。其中最关键的是骨形态发生蛋白 (BMPS)。BMP是一个局部活跃的生长和分化家族 因子，大多数成员都能诱导和调节骨细胞 差异化。申请者对糖皮质激素(GC)的研究 早期成骨细胞分化的增强导致了这一假说 BMP-6具有独特的重要作用。本提案的重点是 了解BMP-6的作用和调节机制 在成骨细胞分化早期的表达。这是假设的 认为：(1)BMP-6的表达是成骨细胞启动的必要条件 (2)GC对BMP-6表达的调节 主要通过翻译前机制；以及(3) BMP-6基因的GC调控需要BMP-6启动子来实现 表情。骨形态发生蛋白-6启动成骨细胞的必要条件 将通过证明BMP-6反义来检验其差异性 寡核苷酸阻断GC诱导的分化；建议 外源性BMP-6的加入应能恢复正常分化 用BMP-6反义寡核苷酸处理培养物。翻译前 BMP-6表达的调节将通过测量GC诱导的 BMP-6mRNA合成及稳定性的变化。最后，该计划的推动者 将确定BMP-6基因和所需的特定区域 测定GC对BMP-6转录的促进作用。这一地区将成为 与BMP-6启动子区域相比，该区域是 BMP-6在其他情况下的表达增加，包括自发的 成骨细胞的分化和其他骨形态发生蛋白的诱导。临床 应用BMP诱导骨形成可提高成骨成功率 在具有挑战性的整形外科应用中的骨愈合，例如脊柱 融合、骨折不愈合和节段性长骨缺损。尽管 骨形态发生蛋白在低等脊椎动物模型、骨骼中的令人鼓舞的结果 在非人类灵长类动物中形成需要更大剂量的BMP，使某些 临床应用非常昂贵。更好地理解 监管特定的BMP，并制定战略，以提高 最关键的成骨诱导因子的体内表达或活性， 会有很大的临床价值。增加对BMP-6的了解 启动子可能导致涉及寡核苷酸治疗或 病毒介导的局部增强BMP-6表达的因子， 或者在特定的细胞群体中。

DESCRIPTION (Adapted from the Applicant's Abstract): The complex process of osteoblast differentiation involves many systemic, paracrine and autocrine factors. Among the most critical are the bone morphogenetic proteins (BMPs). BMPs are a family of locally active growth and differentiation factors, with most members capable of inducing and modulating bone cell differentiation. The applicants' study of the glucocorticoid (GC) enhancement of early osteoblast differentiation has led to the hypothesis that BMP-6 has a uniquely important role. The present proposal focuses on understanding mechanisms involved in the role and regulation of BMP-6 expression during early osteoblast differentiation. It is hypothesized that: (1) BMP-6 expression is required for initiation of osteoblast differentiation; (2) the GC regulation of the BMP-6 expression occurs primarily via pretranslational mechanisms; and (3) a specific region of the BMP-6 promoter is required to convey the GC regulation of BMP-6 gene expression. The requirement for BMP-6 to initiate osteoblast differentiation will be tested by demonstrating that BMP-6 antisense oligonucleotides block GC-induced differentiation; it is suggested that addition of exogenous BMP-6 should restore normal differentiation of cultures treated with BMP-6 antisense oligonucleotides. Pretranslational regulation of BMP-6 expression will be examined by measuring GC-induced changes in BMP-6 mRNA synthesis and stability. Finally, the promoter of the BMP-6 gene will be identified and the specific region that is required for GC-enhancement of BMP-6 transcription determined. This region will be compared with the region of the BMP-6 promoter which is required for increased BMP-6 expression in other contexts, including spontaneous osteoblast differentiation and induction by other BMPs. Clinical application of the BMP-induced bone formation could increase successful healing of bone in challenging orthopedic applications, such as spine fusions, fracture nonunions and segmental long bone defects. Despite encouraging results with BMPs in lower vertebrate animal models, bone formation in nonhuman primates has required larger BMP doses, making certain clinical applications very expensive. An improved understanding of the regulation of specific BMPs, and development of strategies to enhance the in vivo expression or activity of the most critical osteoinductive factors, would have great clinical value. An increased understanding of the BMP-6 promoter may lead to strategies involving oligonucleotide therapy or viral-mediated delivery of factors to enhance expression of BMP-6 locally, or in a targeted population of cells.