Determination of the Mechanisms by which IGFBP-2 Stimulates Bone Remodeling

描述（由申请人提供）：本提案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 - 1协同作用调节骨重塑的机制。骨中的IGF调控系统包括高度保守的IGF结合蛋白（igfbp）家族、蛋白酶、受体和IGF。IGFBP-2在成骨细胞中高表达，可刺激间充质间质细胞（MSC）增殖。Igfbp2-/-小鼠骨转换低，成骨细胞和破骨细胞分化受损，骨髓脂肪生成加速。为了确定IGFBP-2独立于igf - 1的作用机制，我们合成了一个短的13个氨基酸的肽，它不结合igf，但在IGFBP-2中含有一个独特的肝素结合域（即HBD1），并表明HBD1和完整的IGFBP-2一样刺激OB分化。每天给Igfbp2 -/-小鼠注射聚乙二醇形式的HBD1肽，持续3周，可挽救其低小梁骨量，增加OB数量，增强造血祖细胞。来自Igfbp2-/-小鼠的成骨细胞增加了PTEN（磷酸酶和紧张素同源物）转录，这可以通过添加外源性IGFBP-2、IGF-I或HBD来抑制。添加igf - 1 + IGFBP-2时，Igfbp2-/-小鼠MSCs中AKT的磷酸化程度最大。综上所述，我们假设igf - 1诱导的IGFBP-2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塑。提出的两个具体目标是：1)确定IGFBP-2对骨重塑的作用及其在MSCs， hsc和破骨细胞前体谱系分配中的作用。2)描述IGFBP-2作用于骨细胞的分子机制，并确定HBD1是否足以通过多营养因子受体介导这些作用。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我们更清楚地了解IGFBP-2在成人骨骼中的功能。

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.