BBP (Bone Morphogenetic Protein Binding Peptide) and Bone Healing

描述（由申请人提供）： 许多 TGF-b（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为软骨细胞和成骨细胞，从而形成软骨和骨骼。重组人 BMP (rhBMP) 用于骨科手术，但它们价格昂贵、水溶性且保留时间短。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 等人推测，脱矿骨基质 (DBM) 含有“为 BMP 提供缓慢释放/固定特性”的物质，从​​而“减少刺激成骨所需的 BMP 量”。 DBM 含有分泌型磷蛋白 24 kDa (spp24)，这是一种蛋白水解不稳定的蛋白质，含有矿物质和 TGF-b/BMP 细胞因子结合域，这可能是缓慢释放的原因。 FL（全长）-spp24 及其降解产物（即 spp18.1、spp16 和 spp14.5）结合 BMP-2 和 -7。只有 FL-spp24（但不是其截短的衍生物）结合 TGF-b2，表明 spp24 蛋白水解产生的产物与 BMP 和 TGF-b 结合有差异。差异结合可以调节 TGF-b 与 BMP 的相对可用性，并可能有助于骨代谢的调节。 FL-spp24包含2个BMP结合域（TRH1或TGF-b受体II同源1域和一个新的C端域），但spp24降解产物仅包含1个BMP结合域（TRH1域），这可能解释了BMP和TGF-b结合的差异。与 spp24 的 TRH1 结构域相对应的 Cys1 至 Cys19 二硫键合成肽称为环状 BMP 结合肽 (cBBP)。 cBBP 增加 BMP-2 的组织保留，并刺激 BMP-2 和 BMP-7 介导的脊柱融合以及 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 载体的理想候选者。 我们研究的长期目标是：(1) 确定 spp24 及其降解产物在骨中 TGF-b 细胞因子超家族活性调节中的作用，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-spp24 与 TGF-bs 结合抑制 TGF-bs 与其受体之间的相互作用，并减弱下游、受体后 Smad 依赖性和独立性信号传导和基因调节，以及 (b) cBBP 的化学修饰，降低其对 BMP 的 KD（增加其“亲和力”）或增加 cBBP 上的负电荷以允许其与带正电的电荷结合。 BMP 将更有效地促进 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（表面等离子共振）和竞争性受体结合测定来确定 spp24 C 端降解对其与 TGF-bs 结合动力学的影响。在具体目标 2 中，我们将确定 spp24 降解对 Smad 依赖性途径介导的 TGF-b 信号转导的影响，评估为 R-Smad 磷酸化和核易位。在具体目标 3 中，我们将基于磷酸化特异性抗体阵列的定量分析，确定 spp24 降解对 TGF-b 处理的细胞中不依赖于 Smad 的 MAPK、JNK、ERK 和 p38 激酶途径的影响。结果将通过 TGF-b 介导的基因表达的 qPCR 进行验证。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 spp24 降解对 TGF-b 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）或经过化学磷酸化以增加其 BMP 结合能力的 cBBP 衍生物。它们与 BMP 的结合将通过 SPR 进行评估，它们对 BMP-2 和 -7 生物活性的影响将在异位骨形成生物测定中确定，并在啮齿动物骨愈合模型（例如脊柱融合）中进行确认。 公共卫生相关性： 该项目的临床意义是直接的，因为其目标是生产可转化为治疗药物的材料。复杂的骨折、不愈合以及由于创伤或癌症手术而需要大量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。我们希望开发出一系列骨生长因子或BMP/TGF-b（骨形态发生蛋白/转化生长因子-β）结合材料，可用于需要在植入的骨骼部位保留生长因子的临床情况，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 spp24（分泌型磷蛋白 24 kDa）如何抑制 BMP/TGF-b 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.