ANABOLIC ACTION OF Wnt IN THE ADULT SKELETON

Wnt 在成人骨骼中的合成代谢作用

• 批准号: 7350178
• 负责人: DIANE M CULLEN
• 金额: $ 41.14万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-10 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7350178
• 关键词: Acute; Adult; Agonist; Alkaline Phosphatase; Anabolic Agents; Andro-Diane; Apoptosis; Bromodeoxyuridine; Cell Proliferation; Cells; Cultured Cells; Cyclin D1; DNA biosynthesis; DNA chemical synthesis; Daily; Dinoprostone; Disruption; Dose; End Point; Gene Expression; Gene Targeting; Genes; Genotype; Histocytochemistry; Human; In Vitro; Knock-out; Label; Leg; Measures; Mechanical Stimulation; Mechanics; Mediating; Messenger RNA; Mus; Mutation; Nuclear; Organ; Osteoblasts; Osteogenesis; PTGS2 gene; Parathyroid Hormones; Pathway interactions; Phosphorylation; Primary Cell Cultures; Prostaglandin-Endoperoxide Synthase; Prostaglandins; Proteins; Purpose; Range; Rate; Regulation; Research Personnel; Role; Second Messenger Systems; Shapes; Signal Pathway; Signal Transduction; Skeletal Development; Skeleton; Small Interfering RNA; Specificity; Staining method; Stains; Stimulus; System; Testing; Transcriptional Activation; Transgenic Organisms; Treatment Effectiveness; Up-Regulation; Week; Work; bone; bone turnover; cyclooxygenase 1; fluid flow; human PTH protein; in vivo; inhibitor/antagonist; long bone; mRNA Expression; prevent; programs; protein expression; proto-oncogene protein Wnt-2; receptor; response; second messenger; tibia; ulna

Lrp5 works through the canonical Wnt/p-catenin signaling pathway during skeletal development. The HBM mutation (G171V) in Lrp5 results in up-regulation of bone mass such that the bones are over adapted to daily loads, but normal in shape and turnover rates. Our general hypothesis is that in the adult skeleton, the LRP5/Wnt pathway is directly involved in mechanical regulation of bone mass such that there is a dose response relationship between LRP5 expression level with mechanical stimulation and that the HBM mutation further increases the response. Our alternate hypothesis is that the HBM mutation increases mechano-sensitivity independent of Lrp5 expression level. The specific hypotheses we will test are: 1) increased LRP5 gene expression, and especially the HBM mutation, increase the bone response to mechanical loading, 2) Lrp5 and the HBM mutation modulate the osteogenic response to mechanical stimulation through the canonical Wnt pathway, 3) while parathyroid hormone (PTH) will be additive with loading, there will be no HBM or LRP5 dose effect with PTH, and 4) prostaglandins are important in the LRP5 (HBM) response to mechanical loading such that suppression will inhibit the anabolic response and addition of PGE2 will stimulate a dose response. To establish a dose response and test our hypotheses we will compare four mouse genotypes with a range of Lrp5 expression: the Lrp5 heterozygous knockout (Lrp5+/-), wild type C57BI/6, the human LRP5tg transgenic, and the HBMtg transgenic with the human WT or G171V mutation inserted. We will use in vitro primary cell cultures to specifically test the Writ canonical pathway and in vivo studies to determine the organ level regulation of bone turnover. The specific aims are to 1) establish the threshold, dose response, and half-maximal effective dose (EDso) for loading in vivo and in vitro; 2) in vitro compare the effects of fluid flow to a Wnt agonist on the Wnt canonical pathway for GSK 3P ser9 phosphorylation, nuclear localization of p-catenin, and response to disruption of the pathway by DKK1 or siRNA for p-catenin; 3) in vivo examine the interaction of loading and PTH 1-34 to determine the specificity of the LRP5 and HBM mutation response to anabolic stimulation; and 4) in vivo combine loading with a Cox2 inhibitor or PGE2, to examine the role of prostaglandins in mediating Lrp5 responses to loading. In vivo studies will examine long term (4 wk) adaptation to treatment with histomorphometry and then acute responses via mRNA and histochemistry (BrDU labeling, apoptosis, p-catenin expression, alkaline phosphatase, and TRAP).The in vitro studies will examine primary osteoblast cell cultures for mRNAand protein expression along with cell proliferation. Both systems will examine genes known to be up regulated by mechanical loading in HBMtg that are Wnt target genes - WISP-2 (Wnt induced secreted protein 2), Wnt related -Cox-2, eNOS, Cyclin-D1, and non-target genes - Lrp5, OPGandWnt 10b. These studies provide a unique opportunity to test the role of LRP5 in mechanical regulation of bone mass through Wnt signaling pathways.

Lrp5 在骨骼发育过程中通过经典的 Wnt/p-catenin 信号通路发挥作用。这 Lrp5 中的 HBM 突变 (G171V) 导致骨量上调，导致骨骼过度适应 日常负载，但形状和周转率正常。我们的一般假设是，在成人骨骼中， LRP5/Wnt 通路直接参与骨量的机械调节，因此存在剂量 LRP5表达水平与机械刺激的反应关系与HBM 突变进一步增强了反应。我们的替代假设是 HBM 突变增加 机械敏感性与 Lrp5 表达水平无关。我们将测试的具体假设是：1） LRP5 基因表达增加，尤其是 HBM 突变，增加骨对 机械负荷，2) Lrp5 和 HBM 突变调节对机械负荷的成骨反应 通过经典 Wnt 通路的刺激，3) 而甲状旁腺激素 (PTH) 将与 负载，不会有 HBM 或 LRP5 与 PTH 的剂量效应，4) 前列腺素在 LRP5 (HBM) 对机械负荷的反应，抑制会抑制合成代谢反应， 添加 PGE2 将刺激剂量反应。为了建立剂量反应并测试我们的假设，我们 将比较具有一系列 Lrp5 表达的四种小鼠基因型：Lrp5 杂合敲除 (Lrp5+/-)、野生型 C57BI/6、人 LRP5tg 转基因以及人 WT 或 HBMtg 转基因 插入 G171V 突变。我们将使用体外原代细胞培养物来专门测试令状规范 途径和体内研究以确定骨转换的器官水平调节。具体目标是 1) 建立体内负荷的阈值、剂量反应和半最大有效剂量 (EDso) 体外; 2) 体外比较 GSK 的 Wnt 激动剂流体流动对 Wnt 经典途径的影响 3P ser9 磷酸化、p-连环蛋白的核定位以及对通路破坏的反应 DKK1 或 p-catenin 的 siRNA； 3) 体内检查负载和 PTH 1-34 的相互作用以确定 LRP5 和 HBM 突变对合成代谢刺激反应的特异性； 4) 体内联合加载 与 Cox2 抑制剂或 PGE2 一起使用，以检查前列腺素在介导 Lrp5 对负荷反应中的作用。 体内研究将检查对组织形态计量学治疗的长期（4周）适应，然后检查急性适应 通过 mRNA 和组织化学反应（BrDU 标记、细胞凋亡、p-连环蛋白表达、碱性 磷酸酶和 TRAP）。体外研究将检查原代成骨细胞培养物中的 mRNA 和 蛋白质表达与细胞增殖有关。两个系统都会检查已知上调的基因 通过 HBMtg 中的机械负载，Wnt 靶基因 - WISP-2（Wnt 诱导分泌蛋白 2）、Wnt 相关 -Cox-2、eNOS、Cyclin-D1 和非靶基因 - Lrp5、OPG 和 Wnt 10b。这些研究提供了 通过 Wnt 信号传导测试 LRP5 在骨量机械调节中的作用的独特机会 途径。