BBP (Bone Morphogenetic Protein Binding Peptide) and Bone Healing

描述(由申请人提供)： 许多转化生长因子/骨形态发生蛋白细胞因子刺激多能间充质细胞增殖和分化为软骨细胞和成骨细胞，形成软骨和骨。重组人骨形态发生蛋白(RhBMPs)用于骨科手术，但价格昂贵，易溶于水，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数量要大得多。Wozney等人推测，脱钙骨基质(DBM)含有某种东西，“有助于BMP的缓慢释放/固定特性”，从而“减少了刺激成骨所需的BMP的量”。DBM含有分泌型磷蛋白-24 kDa(Spp24)，这是一种蛋白水解性蛋白质，含有矿物质结合域和转化生长因子-β/BMP细胞因子结合域，这可能是导致释放缓慢的原因。FL(全长)-spp24及其降解产物(即spp18.1、spp16和spp14.5)与BMP-2和-7结合。只有FL-spp24(但不是其截断的衍生物)与转化生长因子-β2结合，这表明spp24蛋白降解导致不同的结合BMP和转化生长因子-b的产物。差异结合可以调节转化生长因子-b与骨形态发生蛋白的相对利用率，并可能有助于骨代谢的调节。FL-spp24含有2个BMP结合域(TRH1或TGF-b受体II同源-1区和一个新的C末端结构域)，而spp24降解产物只含有1个BMP结合域(TRH1域)，这可能是BMP和TGF-b结合不同的原因。与spp24的TRH1结构域相对应的Cys1到Cys19二硫键合成肽称为环骨形成蛋白结合肽(CBBP)。CBBP增加BMP-2的组织滞留，刺激BMP-2和BMP-7介导的脊柱融合和BMP-2介导的长骨愈合。从理论上讲，CBBP与BMP的结合特性使其成为合成骨移植替代品(SBGS)中BMP载体的理想候选者。我们研究的长期目标是：(1)确定spp24及其降解产物在调节骨骼中转化生长因子-β细胞因子超家族活性中的作用；(2)设计用于SBGS的改良CBBP衍生物。我们将检验假设：(A)FL-spp24与转化生长因子-β结合抑制转化生长因子-β与其受体之间的相互作用，并减弱下游、受体后Smad依赖和非依赖的信号和基因调节，以及(B)化学修饰CBBP，降低其对BMP的Kd(增加其对BMP的亲和力)或增加CBBP上的负电荷，使其能够更有效地结合带正电荷的BMP，从而促进BMP在SBGS中的滞留，并增强细胞因子的生物活性。在特定的目标1中，我们将通过SPR(表面等离子共振)和竞争受体结合实验来确定spp24的C端降解对其与转化生长因子-b结合的动力学的影响。在特定的目标2中，我们将确定spp24降解对Smad依赖的信号转导途径介导的转化生长因子-b信号转导的影响，评估为R-Smad磷酸化和核转位。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降解对转化生长因子-b处理的细胞中Smad非依赖性MAPK、JNK、ERK和p38激酶通路的影响。结果将通过对转化生长因子-b介导的基因表达的定量聚合酶链式反应来验证。在具体目标4中，我们将通过定量组织学和免疫组织学方法，通过I型和II型胶原和骨钙素来确定spp24降解在转化生长因子-b诱导的膜内骨形成和软骨形成中的作用。在具体目标5中，我们将合成与BMP亲和力降低的CBBP衍生物或通过化学磷酸化来增加其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成骨生物测定中确定，并在啮齿类动物的骨愈合模型中得到证实，例如脊柱融合。

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.