LMP and Bone Healing

描述(申请人提供)：骨愈合是一个复杂的过程，涉及许多不同的因素，包括骨形态发生蛋白(BMPs)、信号/转录因子、核转录因子以及细胞外基质成分等可溶性因素。虽然BMPs作为重组蛋白可以诱导局部骨形成和骨缺损的愈合，但我们和其他人之前已经证明，在兔和大鼠临界大小的股骨和颅骨缺损部位进行BMP-2局部基因转移可以更快、更有效地促进骨愈合。最近，我们已经证明了LIM矿化蛋白(LMP)的基因转移，一种新的成骨细胞分化计划的细胞内正调控因子，可以诱导有效的骨形成。在人类中，已经鉴定出三种不同的LMP剪接变体，分别称为LMP-1、LMP-2和LMP-3。人LMP-1和LMP-3基因转移诱导骨形成相关基因的表达，包括某些骨形态发生蛋白(BMPs)，在体外促进骨结节形成，在体内促进异位骨形成，促进大鼠节段性和下颌骨缺损的愈合，并能促进胸腰椎后段融合愈合。我们还表明，在某些条件下，LMP可以在培养中诱导肌肉生成。我们已经证明，LMP-1的至少四个不同区域和LMP-3中的一个额外结构域可以促进成骨。在局部，LMP-3的一个20个氨基酸的区域被称为骨诱导结构域-1(OD-1)，能够在细胞培养中诱导矿化和骨特异基因的表达，并在体内诱导异位成骨。人工合成的OD-1肽融合到蛋白转导结构域也能够诱导矿化和骨特异基因。因此，这项建议的目标是利用细胞培养分析确定成骨和肌肉形成所需的最小以及最佳的LMP-1和LMP-3结构域，研究LMP中最小的结构域能够诱导成骨的途径(BMP信号、Runx2/OSX转录和/或MAP激酶)，并检测LMP-1和LMP-3衍生的结构域在蛋白质转导介导的传递后在体内诱导高效和适当的骨形成的能力。建议实验的成功完成将有助于更好地理解LMP诱导成骨的重要途径，并将导致使用LMP衍生多肽刺激新骨形成的临床相关方法。公共卫生相关性：骨愈合是一个复杂的过程，涉及许多不同的因素。虽然BMPs作为重组蛋白可以诱导局部骨形成和骨缺损的愈合，但我们已经证明局部基因转移BMPs可以更快、更有效地促进骨愈合。我们已经证明，LIM矿化蛋白(LMP)的基因转移可以与BMP-2一样或更有效地诱导成骨和骨形成。该方案的目的是确定LMP中成骨所需的结构域，开始研究LMP诱导成骨的机制，并检测基于LMP的结构域在基因和蛋白质转导介导的转导下在体内诱导骨形成的能力。建议实验的成功完成将有助于更好地理解LMP诱导成骨的重要途径，并将导致使用LMP多肽刺激新骨形成的临床相关方法。

DESCRIPTION (provided by applicant): Bone healing is complex process involving a number of different factors including soluble factors such as bone morphogenetic proteins (BMPs), signaling / transcription factors, nuclear transcription factors as well as extracellular matrix components. Although the delivery of BMPs as recombinant proteins can induce local bone formation and healing of bone defects, we and others have demonstrated previously that local gene transfer of BMP-2 at the site of critical size femoral and cranial defects in the rabbit and rat resulted in more rapid and efficient bone healing. More recently, we have shown that gene transfer of the LIM Mineralization Protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation. In humans, three different LMP splice variants have been identified, termed LMP-1, LMP-2, and LMP-3. Gene transfer of human LMP-1 and LMP-3 induces expression of genes involved in bone formation including certain bone morphogenetic proteins (BMPs), promotes bone nodule formation in vitro and ectopic bone formation in vivo, facilitates healing of rat segmental and mandibular bone defects critical size defects and can facilitate posterior thoracic and lumbar spine fusion healing. We also have shown that LMP can induce myogenesis, under certain conditions, in culture. We have demonstrated that at least four different regions of LMP-1 and an additional domain in LMP-3 can contribute to osteogenesis. In partiular, a 20 amino acid region in LMP-3, termed Osteoinductive Domain-1 (OD-1), is able to induce mineralization and bone specific gene expression in cell culture and confer induction of ectopic bone formation in vivo. A synthetic OD-1 peptide fused to a protein transduction domain also was able to induce mineralization and bone specific genes. Thus the goals of this proposal are to identify the minimal as well as optimal LMP-1 and LMP-3 domains required for osteogenesis and myogenesis using cell culture assays, to examine the pathways through which the minimal, domains in LMP are able to induce osteogenesis (BMP signaling, RunX2/OSX transcription and/or MAP kinase) and to examine the ability of the LMP-1 and LMP-3-derived domains to induce efficient and appropriate bone formation in vivo following protein-transduction mediated delivery. The successful completion of the proposal experiments will lead to a better understanding of the pathways important for induction of osteogenesis by LMP and will result in clinically relevant approaches to stimulate new bone formation using LMP-derived peptides. PUBLIC HEALTH RELEVANCE: Bone healing is complex process involving a number of different factors. Although the delivery of BMPs as recombinant proteins can induce local bone formation and healing of bone defects, we have demonstrated previously that local gene transfer of BMPs results in more rapid and efficient bone healing. We have shown that gene transfer of the LIM Mineralization Protein (LMP) can induce osteogenesis and bone formation as or more efficiently than BMP-2. The goals of this proposal are to identify the domains in LMP required for osteogenesis, to begin to examine the mechanisms through which LMP is able to induce osteogenesis and to examine the ability of the LMP-based domains to induce bone formation in vivo following gene and protein- transduction mediated delivery. The successful completion of the proposal experiments will lead to a better understanding of the pathways important for induction of osteogenesis by LMP and will result in clinically relevant approaches to stimulate new bone formation using LMP peptides.