Determination of the mechanisms by which IGFBP-2 stimulates bone remodeling

描述(申请人提供)：这项建议的长期目标是确定胰岛素样生长因子结合蛋白-2(IGFBP-2)在骨骼获得和维持中的作用，并确定该分子与IGF-I协同作用以调节骨重建的机制。骨组织中的胰岛素样生长因子调节系统包括一系列高度保守的胰岛素样生长因子结合蛋白(IGFBPs)、蛋白水解酶、受体和IGFS。IGFBP-2在成骨细胞中高表达，并能刺激间充质基质细胞(MSC)增殖。全球IGFBP2-/-小鼠骨转换低，成骨细胞和破骨细胞分化受损，骨髓脂肪生成加快。为了确定IGFBP-2独立于IGF-I的作用机制，我们合成了一个不与IGF结合但在IGFBP-2中含有一个独特的肝素结合域的13个氨基酸的短肽(即HBD1)，结果表明HBD1和完整的IGFBP-2一样能促进OB的分化。每天给IGFBP2-/-小鼠服用聚乙二醇化形式的HBD1多肽3周，可以挽救它们的低骨小梁质量，增加OB数量，并增强造血祖细胞。来自IGFBP2-/-小鼠的成骨细胞增加了PTEN(磷酸酶和张力蛋白同源)的转录，这可以被外源IGFBP-2、IGF-I或HBD抑制。当加入IGF-I+IGFBP-2时，IGFBP2-/-小鼠骨髓间充质干细胞AKT的磷酸化程度最高。综上所述，我们假设IGFBP-2是由IGF-I诱导的，通过HBD1刺激MSCs和OBS。此外，我们假设IGF-I和IGFBP-2通常通过不同的细胞表面受体协同作用，以互补的方式促进骨重建。提出的两个具体目标是：1)确定IGFBP-2在骨重建中的作用及其在MSCs、HSCs和破骨细胞前体的谱系分配中的作用。2)阐明IGFBP-2对骨细胞作用的分子机制(S)，并确定HBD_1是否足以通过多营养素受体介导这种作用。利用HBD1肽，我们将研究IGFBP-2如何影响促进细胞增殖的细胞内信号通路。这些跨学科的研究将使人们更清楚地了解IGFBP-2在成人骨骼中的功能。 与公共健康相关：这项建议具有重大的公共健康影响，因为它将有助于描述IGF监管系统在骨骼中的作用。重要的是，对肝素结合域的研究将确定这种小分子是否可以用于治疗代谢性骨骼疾病。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to define the role of insulin like growth factor binding protein-2 (IGFBP-2) in skeletal acquisition and maintenance, and to determine the mechanisms whereby this molecule acts synergistically with IGF-I to regulate bone remodeling. The IGF regulatory system in bone includes a family of highly conserved IGF binding proteins (IGFBPs), proteases, receptors and the IGFs. IGFBP-2 is highly expressed in osteoblasts, and it stimulates mesenchymal stromal cell (MSC) proliferation. Global Igfbp2-/- mice have low bone turnover, impaired osteoblast and osteoclast differentiation, and accelerated marrow adipogenesis. To determine the mechanism of action of IGFBP-2 independent of IGF-I, we synthesized a short 13 amino acid peptide that does not bind IGFs but contains a unique heparin binding domain within IGFBP-2 (i.e. HBD1), and showed that HBD1 stimulated OB differentiation as did intact IGFBP-2. Daily administration of a pegylated form of the HBD1 peptide to Igfbp2 -/- mice for 3 weeks rescued their low trabecular bone mass, increased OB number, and enhanced hematopoietic progenitor cells. Osteoblasts from Igfbp2-/- mice have increased PTEN (phosphatase and tensin homolog) transcripts which can be suppressed by addition of exogenous IGFBP-2, IGF-I or HBD. Phosphorylation of AKT in MSCs from Igfbp2-/- mice was greatest when IGF-I + IGFBP-2 were added. Taken together, we hypothesize that IGFBP-2, which is induced by IGF-I, stimulates MSCs and OBs through HBD1. Furthermore we postulate that IGF-I and IGFBP-2 normally act in a complementary manner to promote bone remodeling by acting synergistically through distinct cell surface receptors. The two specific aims proposed are: 1) to determine IGFBP-2 actions on bone remodeling and its role in lineage allocation of MSCs, HSCs, and osteoclast precursors. 2) To delineate the molecular mechanism(s) of IGFBP-2 actions on bone cells, and determine if HBD1 is sufficient to mediate these effects through the pleiotrophin receptor. Using the HBD1 peptide we will study how IGFBP-2 affects the intracellular signaling pathways that promote cell proliferation. These interdisciplinary studies will lead to a clearer understanding of the function of IGFBP-2 in the adult skeleton. PUBLIC HEALTH RELEVANCE: This proposal has significant public health implications since it will help delineate the role of the IGF regulatory system in bone. Importantly, studies of the heparin binding domain will determine whether this small molecule can be used for the treatment of metabolic bone disorders.