BONE FACTORS AND ENDOCHONDRAL OSSIFICATION

骨因素和软骨内骨化

• 批准号: 6214263
• 负责人: Barbara D. Boyan
• 金额: $ 6.22万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-03-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6214263
• 关键词: 1,25 dihydroxycholecalciferol; 25 hydroxycholecalciferol; SDS polyacrylamide gel electrophoresis; autoradiography; bone development; cartilage development; cholecalciferol; chondrocytes; gene expression; high performance liquid chromatography; hormone regulation /control mechanism; immunologic assay /test; immunoprecipitation; laboratory rat; mitogen activated protein kinase; normal ossification; polymerase chain reaction; protein biosynthesis; protein kinase C; radiotracer; receptor binding; tissue /cell culture; transforming growth factors; vitamin D; vitamin D receptors

DESCRIPTION (Adapted from the Applicant's Abstract): This research will examine the basic mechanisms underlying the role of transforming growth factor beta-1 (TGFb) in endochondral bone formation by focusing on the interrelationship between TGFb and 1,25(OH)2D3 (1,25D) and 24,25(OH)D3 (24,25D), using a well-characterized cell culture model that allows comparison of cells at two distinct stages of maturation: resting zone (RC) and growth zone (GC) chondrocytes. The following hypotheses will be addressed: TGFb production and activation are regulated by vitamin D metabolites via genomic and non-genomic mechanisms; matrix vesicle enzymes activate TGFb in the extracellular matrix; TGFb regulates production of 1,25D and 24,25D in a cell maturation-dependent manner and some of TGFb's effects are due to TGFb-dependent production of these hormones; TGFb-dependent regulation of chondrocyte maturation is mediated by protein kinase C (PKC); and, TGFb regulates endochondral development via signaling pathways that are distinct from those used by 1,25D and 24,25D. To test these hypotheses, the following Specific Aims will be pursued: (1) to characterize the regulation of active TGFb production by 1,25D and 24,25D. The investigators will determine if 1, 25D and 24,25D regulate TGFb production by modulating LTGFb gene expression, protein synthesis, or activation, or by changing LTGFb binding protein (LTBP) mRNA levels, protein synthesis, or incorporation into the extracellular matrix. Whether vitamin D metabolites act via traditional vitamin D receptors (VDR) or rapid membrane-mediated effects will be assessed using specific analogues of vitamin D with low affinity for the VDR. The mechanisms by which matrix vesicles activate LTGFb will be assessed by determining which enzymes are responsible, and whether they are regulated by vitamin D metabolites through genomic or non-genomic pathways. Once the LTGFb activator is identified, they will initiate experiments to purify and sequence the protein and clone its gene; (2) to characterize the extrarenal regulation of vitamin D metabolite production by TGFb. The applicants will examine the direct regulation of 25D hydroxylation by measuring both 1alpha- and 24- hydroxylase mRNA levels and activity in the TGFb-treated RC chondrocytes. Whether TGFb exerts its effects via regulation of the hydroxylase co-factor, cytochrome P450, will be determined using specific inhibitors. The investigators will also examine whether the effects of TGFb are mediated through TGFb receptor binding, PKC and the MAP kinase pathway; (3) to determine the mechanisms by which TGFb acts synergistically with 24,25D to regulate chondrocyte maturation. The applicants will characterize the mechanisms by which TGFb exerts its effects on RC chondrocytes by measuring alkaline phosphatase mRNA production and activity and the acquisition of a 1,25D-responsive phenotype characteristic of the more mature growth zone cells. How these TGFb-mediated effects are modulated by 24,25D will be determined, including changes in TGFb type I and type II receptors. It is suggested that these results will provide insight into the role these factors play in bone development and repair.

描述（根据申请人的摘要改编）：这项研究将 检查转化增长作用的基本机制 内侧软骨骨形成的因子β-1（TGFB）通过重点 TGFB与1,25（OH）2d3（1,25d）和24,25（OH）D3之间的相互关系 （24,25d），使用特征良好的细胞培养模型，该模型允许 在两个不同的成熟阶段进行比较：静息区（RC） 和生长区（GC）软骨细胞。 以下假设将是 解决：TGFB的生产和激活受维生素D调节 通过基因组和非基因组机制代谢物；基质囊泡酶 激活细胞外基质中的TGFB； TGFB调节生产 1,25d和24,25d以细胞成熟依赖性方式和某些TGFB的方式 影响是由于这些激素的TGFB依赖性产生。 蛋白质介导了软骨细胞成熟的TGFB依赖性调节 激酶C（PKC）;并且，TGFB通过信号调节内软骨的发育 与1,25D和24,25D使用的途径不同。 测试 这些假设，将追求以下具体目标：（1） 将主动TGFB产生的调节表征为1,25d和24,25d。 研究人员将确定1、25D和24,25D是否调节TGFB 通过调节LTGFB基因表达，蛋白质合成或 激活或通过改变LTGFB结合蛋白（LTBP）mRNA水平，蛋白 合成或掺入细胞外基质中。 是否维生素 D代谢物通过传统维生素D受体（VDR）或快速作用 膜介导的效果将使用特定的类似物进行评估 维生素D对VDR的亲和力低。 矩阵的机制 囊泡将通过确定哪些酶是激活LTGFB 负责，以及它们是否受维生素D代谢物调节 基因组或非基因组途径。 一旦确定了LTGFB激活剂， 他们将启动实验以纯化和测序蛋白质和克隆 它的基因； （2）表征维生素D的外部调节 TGFB的代谢产物生产。 申请人将检查直接 通过测量1α-和24-的调节25D羟基化 TGFB处理的RC软骨细胞中的羟化酶mRNA水平和活性。 TGFB是否通过调节羟化酶co因子施加其影响， 细胞色素P450将使用特定抑制剂确定。 这 研究人员还将检查TGFB的影响是否介导 通过TGFB受体结合，PKC和MAP激酶途径； （3）到 确定TGFB与24,25D协同作用的机制 调节软骨细胞的成熟。 申请人将表征 TGFB通过测量TGFB对RC软骨细胞发挥影响的机制 碱性磷酸酶mRNA的产生和活性以及A的获取 1,25D响应性表型的特征更成熟生长区 细胞。 这些TGFB介导的效果如何由24,25D调节 确定，包括TGFB I型和II型受体的变化。 这是 建议这些结果将为这些角色提供深入的了解 骨骼发育和修复的因素。