Transcriptional regulation of tendon differentiation and matrix formation

说明(申请人提供)：肌腱将肌肉收缩产生的力传递到骨骼，通过牢固和高度组织化的胶原纤维束建立肌腱的生物力学特性。在肌腱中，细胞延伸吞噬了一束束的胶原纤维，并为纤维生长创造了微环境。肌腱中胶原纤维的生长是决定肌腱生物力学性能和抗拉强度的最重要因素。胶原原纤维的生长有两个阶段，胚胎阶段的缓慢纤维组装和较快的纤维生长速度，这可能是在出生后阶段通过纤维融合发生的。然而，人们对调控这些过程的遗传程序一无所知。我们先前已经证明，bHLH转录因子硬化轴在肌腱早期分化中是必不可少的，它可能在胶原纤维形成中也起着重要作用。在第二个肌腱转录因子莫霍克的突变中，我们现在发现了出生后后期胶原纤维生长的失败。本项目的重点是硬化轴和莫霍克在胶原纤维形成和肌腱成熟中的作用。第一个特定的目标是在胚胎肌腱中组装肌腱，第二个目标是在出生后阶段肌腱成熟和胶原纤维的快速生长的调节。这些进程将在两个阶段采取类似的办法加以处理。正常的肌腱生长和肌腱表型最初将通过一套增强的结构参数进行评估，并将检查SCX或MKX过表达对肌腱生长的影响。通过微阵列分析鉴定Skeraxis和Mohawk功能的分子介体，并通过对SCX或MKX表型的转基因挽救将单个或多个靶基因的表达重新引入突变背景来确定少数靶基因的调节作用。我们最近确定了ZFP185，一种锌指转录因子，是我们计划进行转基因救援的第一个有希望的候选基因。确定控制原纤维生长的调节途径可能有助于在临床环境中增强和调节这些过程的能力。

DESCRIPTION (provided by applicant): Tendons transmit the force generated by muscle contraction to the skeleton, through robust and highly organized bundles of collagen fibrils that establish the biomechanical properties of tendons. In the tendons, cellular extensions engulf bundles of collagen fibrils and generate the microenvironment for fibril growth. Growth of the collagen fibrils in tendons is the single most significant factor that determines biomechanical properties and tensile strength of tendons. Collagen fibril growth occurs in two phases, slow fibril assembly in embryonic stages and a much faster pace of fibril growth that likely occurs through fibril fusion in postnatal stages. Nothing, however, is known about the genetic program that regulates these processes. We have previously shown that the bHLH transcription factor Scleraxis is essential for early tendon differentiation and that it likely also plays an important role in collagen fibrillogenesis. In mutants of a second tendon transcription factor, Mohawk, we now find a failure of the later postnatal phase of collagen fibril growth. This project focuses on the role of Scleraxis and Mohawk in collagen fibrillogenesis and tendon maturation. The first specific aim focuses on tendon assembly in embryonic tendons and the second aim looks at the regulation of tendon maturation and rapid collagen fibril growth in postnatal stages. These processes will be addressed following a similar approach in both stages. Normal tendon growth and tendon phenotypes will initially be evaluated with an enhanced set of structural parameters and the effects of overexpression of Scx or Mkx on tendon growth will be examined. Molecular mediators of Scleraxis and Mohawk functions will be identified by microarray profiling and the regulatory roles of a small number of target genes will be determined by a transgenic rescue of the Scx or Mkx phenotypes reintroducing the expression of a single or multiple target genes into the mutant background. We recently identified ZFP185, a Zinc Finger Transcription factor as the first promising candidate for which we plan to proceed with a transgenic rescue. Identifying regulatory pathways that control fibril growth will likely contribute to the ability to enhance and regulate these processes in clinical settings.