BBP (Bone Morphogenetic Protein Binding Peptide) and Bone Healing

描述（由申请人提供）： 许多TGF-β（转化生长因子）/BMP（骨形态发生蛋白）细胞因子刺激多能间充质细胞增殖并分化成软骨细胞和成骨细胞，从而形成软骨和骨。重组人骨形成蛋白（rhBMP）用于骨科手术，但它们价格昂贵，水溶性，并且保留时间短。皮质骨含有启动骨折愈合所需的微量BMP，但诱导骨形成所需的rhBMP量要大得多。Wozney等人，假设脱矿骨基质（DBM）含有“有助于BMP的缓慢释放/固定特性”的物质，从而“减少刺激骨生成所需的BMP的量”。DBM含有分泌的磷蛋白-24 kDa（spp 24），这是一种蛋白水解不稳定的蛋白质，含有矿物质和TGF-β/BMP丝氨酸结合结构域，这可能是缓慢释放的原因。FL（全长）-spp 24及其降解产物（即，spp 18.1、spp 16和spp 14.5）结合BMP-2和BMP-7。只有FL-spp 24（而不是其截短衍生物）结合TGF-β 2，表明spp 24蛋白水解导致差异结合BMP和TGF-β的产物。差异结合可以调节TGF-β相对于BMP的相对可用性，并可能有助于调节骨代谢。FL-spp 24含有2个BMP结合结构域（TRH 1或TGF-b受体II同源-1结构域和一个新的C-末端结构域），但spp 24降解产物仅含有1个BMP结合结构域（TRH 1结构域），这可能是BMP和TGF-b结合差异的原因。对应于spp 24的TRH 1结构域的Cys 1-至-Cys 19二硫键结合的合成肽被称为环状BMP结合肽（cBBP）。cBBP增加BMP-2的组织保留并刺激BMP-2和BMP-7介导的脊柱融合和BMP-2介导的长骨愈合。理论上，cBBP的BMP结合特性使其成为SBGS（合成骨移植替代品）中BMP载体的理想候选物。 我们的研究的长期目标是：（1）确定spp 24及其降解产物在骨中调节TGF-β细胞因子超家族活性中的作用，以及（2）设计用于SBGS的改进的cBBP衍生物。我们将测试以下假设：（a）FL-spp 24与TGF-β的结合抑制TGF-β与其受体之间的相互作用，并减弱下游、受体后Smad依赖性和非依赖性信号传导和基因调控，和（B）降低其KD的cBBP的化学修饰（增加其“亲和力”）或增加cBBP上的负电荷以允许其正结合BMP-2。更有效地荷电的BMP将促进BMP保留并增强SBGS中的细胞因子生物活性。在具体目标1中，我们将通过SPR（表面等离子体共振）和竞争性受体结合测定来确定spp 24的C末端降解对其与TGF-β结合的动力学的影响。在特定目标2中，我们将确定spp 24降解对由Smad依赖性途径介导的TGF-β信号转导的影响，评估为R-Smad磷酸化和核转位。在特定目标3中，我们将基于磷酸化特异性抗体阵列的定量分析，确定TGF-β处理的细胞中spp 24降解对Smad非依赖性MAPK、JNK、ERK和p38激酶途径的影响。将通过TGF-β介导的基因表达的qPCR验证结果。在具体目标4中，我们将通过I型和II型胶原和骨钙素的定量组织学和免疫组织学来确定spp 24降解对TGF-β诱导的膜内骨形成和软骨形成的影响。在具体目标5中，我们将合成对BMP具有降低的KD（增加的亲和力）或被化学磷酸化以增加其BMP结合能力的cBBP衍生物。将通过SPR评估它们与BMP的结合，并且将在异位骨形成生物测定中确定它们对BMP-2和BMP-7生物活性的影响，并在骨愈合的啮齿动物模型（例如脊柱融合）中进行确认。 公共卫生相关性： 该项目的临床相关性是直接的，因为目标是生产可以转化为治疗的材料。复杂骨折、骨不连和因创伤或癌症手术导致的需要广泛重建的大骨缺损是退伍军人和战斗受伤人员的常见问题。我们的长期目标是开发更便宜，更有效的合成骨移植替代品用于骨科应用。我们希望开发一种骨生长因子或BMP/TGF-β（骨形态发生蛋白/转化生长因子-β）结合材料家族，其可用于需要将生长因子保留在其植入的骨骼部位的临床情况，以增强其活性并促进骨生长以及骨移植物或人工关节成功整合到骨骼中。我们还将确定天然骨蛋白spp 24（分泌磷蛋白-24 kDa）如何抑制BMP/TGF-β依赖性肿瘤细胞生长。

DESCRIPTION (provided by applicant): Many TGF-b (transforming growth factor)/BMP (bone morphogenetic protein) cytokines stimulate pluripotent mesenchymal cells to proliferate and differentiate into the chondrocytes and osteoblasts that elaborate cartilage and bone. Recombinant human BMPs (rhBMPs) are used in orthopedic surgery, but they are expensive, water-soluble, and exhibit short retention times. Cortical bone contains the tiny amount of BMP required to initiate fracture healing, but the amount of rhBMP required to induce bone formation is much greater. Wozney, et al., postulated that demineralized bone matrix (DBM) contains something that "contributes slow release/immobilization characteristics to BMP", thus "reducing the amount of BMP needed" to stimulate osteogenesis. DBM contains secreted phosphoprotein-24 kDa (spp24), a proteolytically-labile protein that contains both mineral- and TGF-b/BMP cytokine-binding domains, which may account for slow release. FL (full-length)-spp24 and its degradation products (i.e., spp18.1, spp16, and spp14.5) bind BMP-2 and -7. Only FL-spp24 (but not its truncated derivatives) binds TGF-b2, suggesting that spp24 proteolysis results in products that differentially bind BMP and TGF-b. Differential binding could modulate the relative availability of TGF-bs vs. BMPs and may contribute to the regulation of bone metabolism. FL-spp24 contains 2 BMP-binding domains (the TRH1 or TGF-b receptor II homology-1 domain and a novel C-terminal domain), but spp24 degradation products contain only 1 BMP-binding domain (the TRH1 domain), which may account for the difference in BMP and TGF-b binding. The Cys1-to- Cys19 disulfide-bonded synthetic peptide corresponding to the TRH1 domain of spp24 is called cyclic BMP binding peptide (cBBP). cBBP increases the tissue retention of BMP-2 and stimulates BMP-2 and BMP-7- mediated spinal fusion and BMP-2-mediated long bone healing. In theory, the BMP-binding properties of cBBP make it an ideal candidate for a BMP carrier in SBGS (synthetic bone graft substitutes). The long-term objectives of our research are to: (1) determine the roles of spp24 and its degradation products in the regulation of TGF-b cytokine superfamily activity in bone, and (2) design improved cBBP derivatives for use in SBGS. We will test hypotheses that: (a) FL-spp24 binding to TGF-bs inhibits the interaction between TGF-bs and their receptors and attenuates down-stream, post-receptor Smad- dependent and -independent signaling and gene regulation, and (b) chemical modifications of cBBP that decrease its KD (increase its "affinity") for BMP or increase the negative charge on cBBP to permit it to bind the positively-charged BMPs more effectively will promote BMP retention and enhance cytokine bioactivity in SBGS. In Specific Aim 1, we will determine the effects of C-terminal degradation of spp24 on the kinetics of its binding to TGF-bs by SPR (surface plasmon resonance) and competitive receptor binding assay. In Specific Aim 2, we will determine the effects of spp24 degradation on TGF-b signal transduction mediated by Smad-dependent pathways, assessed as R-Smad phosphorylation and nuclear translocation. In Specific Aim 3, we will determine the effects of spp24 degradation on Smad-independent MAPK, JNK, ERK and p38 kinase pathways in TGF-b-treated cells, based on quantitative analysis of phospho-specific antibody arrays. The results will be validated by qPCR of TGF-b-mediated gene expression. In Specific Aim 4, we will determine the effects of spp24 degradation on TGF-b-induced intramembranous bone formation and chondrogenesis by quantitative histology and immunohistology of types I and II collagen and osteocalcin. In Specific Aim 5, we will synthesize cBBP derivatives with decreased KDs (increased affinities) for BMPs or which are chemically phosphorylated to increase their BMP-binding capacity. Their binding to BMPs will be assessed by SPR, and their effects on BMP-2 and -7 bioactivity will be determined in the ectopic bone forming bioassay, with confirmation in a rodent model of bone healing, such as spinal fusion. PUBLIC HEALTH RELEVANCE: The clinical relevance of this project is direct because the object is to produce materials that can be translated into therapeutics. Complex fractures, non-unions, and large bone defects due to trauma or cancer surgery that require extensive reconstruction are common problems among both veterans and combat injured individuals. Our long-term objective is to develop less expensive, more effective synthetic bone graft substitutes for use in orthopedic applications. We hope to develop a family of bone growth factor or BMP/TGF-b (bone morphogenetic protein/transforming growth factor-beta)-binding materials that can be employed in clinical situations where there is a need to retain the growth factors at the skeletal site where they are implanted in order to enhance their activity and promote bone growth and the successful integration of bone grafts or artificial joints into the skeleton. We will also determine how the native bone protein spp24 (secreted phosphoprotein-24 kDa) inhibits BMP/TGF-b-dependent tumor cell growth.