LMP and Bone Healing

描述（由申请人提供）：骨愈合是一个复杂的过程，涉及许多不同的因子，包括可溶性因子，如骨形态发生蛋白（BMP）、信号传导/转录因子、核转录因子以及细胞外基质成分。尽管BMP作为重组蛋白的递送可以诱导局部骨形成和骨缺损的愈合，但是我们和其他人先前已经证明，在兔和大鼠中的关键尺寸股骨和颅骨缺损部位处的BMP-2的局部基因转移导致更快速和有效的骨愈合。最近，我们已经表明，基因转移的LIM矿化蛋白（LMP），一种新的细胞内成骨细胞分化程序的正调控因子，可以诱导有效的骨形成。在人类中，已经鉴定了三种不同的LMP剪接变体，称为LMP-1、LMP-2和LMP-3。人LMP-1和LMP-3的基因转移诱导参与骨形成的基因（包括某些骨形态发生蛋白（BMP））的表达，促进体外骨结节形成和体内异位骨形成，促进大鼠节段性和下颌骨缺损临界尺寸缺损的愈合，并且可以促进后路胸腰椎融合愈合。我们还表明，在一定条件下，在培养中，LMP可以诱导肌发生。我们已经证明LMP-1的至少四个不同区域和LMP-3中的另外一个结构域可以促进骨生成。特别是LMP-3中的20个氨基酸区域，称为骨诱导结构域-1（Osteoinductive Domain-1，OD-1），能够在细胞培养中诱导矿化和骨特异性基因表达，并在体内诱导异位骨形成。与蛋白质转导结构域融合的合成OD-1肽也能够诱导矿化和骨特异性基因。因此，本发明的目的是使用细胞培养测定来鉴定骨生成和肌生成所需的最小以及最佳LMP-1和LMP-3结构域，以检查LMP中的最小结构域能够诱导骨生成的途径（BMP信号传导，RunX 2/OSX转录和/或MAP激酶），并检查LMP-1和LMP-3-T1的能力。衍生的结构域，以在蛋白转导介导的递送后在体内诱导有效和适当的骨形成。该实验的成功完成将导致更好地理解LMP诱导成骨的重要途径，并将导致使用LMP衍生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BMP作为重组蛋白的递送可以诱导局部骨形成和骨缺损的愈合，但是我们先前已经证明BMP的局部基因转移导致更快速和有效的骨愈合。我们已经表明，LIM矿化蛋白（LMP）的基因转移可以诱导骨生成和骨形成，或比BMP-2更有效。该提议的目的是鉴定成骨所需的LMP中的结构域，开始检查LMP能够诱导成骨的机制，并检查基于LMP的结构域在基因和蛋白转导介导的递送后体内诱导骨形成的能力。该实验的成功完成将使我们更好地了解LMP诱导成骨的重要途径，并将导致临床相关的方法来刺激新骨形成使用LMP肽。

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.