Regulation of Osteoblast Function by Megakaryocytes: Key Signaling Proteins

巨核细胞对成骨细胞功能的调节：关键信号蛋白

基本信息

批准号：
8302194
负责人：
ANGELA BRUZZANITI
金额：
$ 34.65万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8302194
关键词：
Actins Acute Adoptive Transfer Binding Biological Assay Blood Clot Blood Platelets Blood coagulation Bone Density Bone Marrow Cells Caspase Cell Count Cell Cycle Arrest Cell Cycle Progression Cell Cycle Regulation Cell Proliferation Cells Coculture Techniques Complex Data Development Disease Double Minutes Evaluation Exhibits Hematopoietic Homeostasis Human In Vitro Integrins Knock-out Lead Luciferases Mediating Megakaryocytes Molecular Mus Mutant Strains Mice Osteoblasts Osteogenesis Osteoporosis Pathway interactions Phase Phenotype Phosphorylation Phosphotransferases Physiological Platelet Count measurement Play Process Production Protein Isoforms Protein Kinase RNA Splicing Regulation Retinoblastoma Retinoblastoma Protein Role Signal Pathway Signal Transduction Signaling Protein Spleen Surface Testing Tissues Transplantation Wild Type Mouse Work base bone bone loss bone mass human GATA1 protein in vivo multicatalytic endopeptidase complex novel therapeutic intervention nuclear factor-erythroid 2 osteoblast differentiation overexpression promoter protein complex protein degradation protein tyrosine kinase PYK2 research study response skeletal substantia spongiosa ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): A growing body of evidence suggests that megakaryocytes (MKs) play a key role in regulating skeletal homeostasis. In support of this, mice deficient in GATA-1 or NF-E2, transcription factors required for normal MK development, exhibit an increase in immature MKs as well as a dramatic decrease in platelet numbers. Importantly, these mice exhibit a 300% increase in trabecular bone mineral density. The cellular and tissue-level mechanisms underlying this increase in bone mass remain unclear. However, our histological evaluation of GATA-1 and NF-E2 deficient mice reveals higher numbers of osteoblasts (OBs) on trabecular surfaces. Importantly, the increased bone phenotype can be adoptively transferred into irradiated wild-type mice using spleen cells from mutant mice suggesting a role for hematopoietic cells, most likely MKs which are elevated in these mice, in this mechanism. Consistent with these in vivo experiments, our in vitro data show that MKs enhance OB proliferation (up to 6-fold) by direct cell-to-cell contact which involves integrin engagement. Although the exact mechanisms by which MKs enhance OB proliferation remain to be determined, these observations suggest that the interaction of MKs with OBs results in increased osteogenesis. Furthermore, in OBs co-cultured with MKs, the expression of the cell cycle arrest protein Retinoblastoma (Rb), and murine double minute-2 (Mdm2), an E3 ubiquitin ligase that regulates proteosome mediated degradation, are transiently decreased. Recently, we discovered that MKs regulate the temporal expression of two isoforms of the proline-rich tyrosine kinase 2 (Pyk2), a key protein kinase involved in signaling downstream of activated integrins, and that Pyk2 forms a complex with Rb and Mdm2. Moreover, the MK-mediated increase in OB number was abolished in OBs from Pyk2-/- mice. Therefore, our central hypothesis is that MKs have a anabolic effect on bone by regulating cell cycle progression in OBs via a pathway involving the Pyk2-mediated regulation of upstream and downstream signaling proteins. In Aim 1 we will demonstrate the functional role of Pyk2 isoforms in MK-regulated OBs by ectopically expressing either Pyk2 or Pyk2-S in OBs and assessing cell cycle regulation and differentiation. In Aim 2 we will determine the role of Pyk2's phosphorylation and activity in regulating its interaction with Rb and Mdm2 and its degradation. Finally, in Aim 3 we will demonstrate the role of Pyk2 in MK-induced bone formation by transplanting Pyk2-/- mice with hematopoietic precursors enriched with MKs. Successful accomplishment of these Aims will demonstrate the importance of MKs in regulating anabolic bone formation and the role of Pyk2 in OB cell cycle regulation. Identifying the pathways that lead to enhanced bone volume in vivo will lead to the development of novel therapeutic approaches that stimulate bone formation for the treatment of osteoporosis and other bone loss diseases.

描述（由申请人提供）：越来越多的证据表明巨核细胞（MK）在调节骨骼稳态中发挥着关键作用。为了支持这一点，缺乏 GATA-1 或 NF-E2（正常 MK 发育所需的转录因子）的小鼠表现出未成熟 MK 的增加以及血小板数量的急剧减少。重要的是，这些小鼠的小梁骨矿物质密度增加了 300%。骨量增加的细胞和组织水平机制仍不清楚。然而，我们对 GATA-1 和 NF-E2 缺陷小鼠的组织学评估显示，小梁表面的成骨细胞 (OB) 数量较多。重要的是，增加的骨表型可以使用突变小鼠的脾细胞过继转移到受辐射的野生型小鼠中，这表明造血细胞（最有可能是在这些小鼠中升高的 MK）在此机制中发挥作用。与这些体内实验一致，我们的体外数据表明，MK 通过涉及整联蛋白接合的直接细胞间接触来增强 OB 增殖（高达 6 倍）。尽管 MK 增强 OB 增殖的确切机制仍有待确定，但这些观察结果表明 MK 与 OB 的相互作用导致骨生成增加。此外，在与 MK 共培养的 OB 中，细胞周期阻滞蛋白视网膜母细胞瘤 (Rb) 和鼠双分钟-2 (Mdm2)（一种调节蛋白体介导的降解的 E3 泛素连接酶）的表达暂时降低。最近，我们发现 MK 调节富含脯氨酸的酪氨酸激酶 2 (Pyk2) 的两种亚型的时间表达，Pyk2 是参与激活整联蛋白下游信号传导的关键蛋白激酶，并且 Pyk2 与 Rb 和 Mdm2 形成复合物。此外，MK 介导的 OB 数量增加在 Pyk2-/- 小鼠的 OB 中被消除。因此，我们的中心假设是，MK 通过涉及 Pyk2 介导的上游和下游信号蛋白调节的途径调节 OB 中的细胞周期进程，从而对骨产生合成代谢作用。在目标 1 中，我们将通过在 OB 中异位表达 Pyk2 或 Pyk2-S 并评估细胞周期调节和分化来证明 Pyk2 同工型在 MK 调节的 OB 中的功能作用。在目标 2 中，我们将确定 Pyk2 的磷酸化和活性在调节其与 Rb 和 Mdm2 相互作用及其降解中的作用。最后，在目标 3 中，我们将通过向 Pyk2-/- 小鼠移植富含 MK 的造血前体细胞来证明 Pyk2 在 MK 诱导的骨形成中的作用。这些目标的成功实现将证明 MK 在调节合成代谢骨形成中的重要性以及 Pyk2 在 OB 细胞周期调节中的作用。确定导致体内骨量增加的途径将导致开发刺激骨形成的新治疗方法，以治疗骨质疏松症和其他骨质流失疾病。