Regulation of Osteoblast Function by Megakaryocytes: Key Signaling Proteins

描述（由申请人提供）：越来越多的证据表明，巨核细胞（MK）在调节骨骼稳态中起关键作用。为了支持这一点，缺乏加塔-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，这些小鼠表现出小梁骨矿物质密度增加300%。这种骨量增加的细胞和组织水平机制尚不清楚。然而，我们对加塔-1和NF-E2缺陷小鼠的组织学评价显示，小梁表面上成骨细胞（OB）的数量更高。重要的是，增加的骨表型可以使用来自突变小鼠的脾细胞过继转移到照射的野生型小鼠中，这表明造血细胞（最可能是在这些小鼠中升高的MK）在该机制中的作用。与这些体内实验一致，我们的体外数据显示MK通过涉及整联蛋白接合的直接细胞与细胞接触增强OB增殖（高达6倍）。 虽然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）和鼠双微体-2（Mdm 2）（一种调节蛋白体介导的降解的E3泛素连接酶）的表达瞬时降低。最近，我们发现MK调节富含脯氨酸的酪氨酸激酶2（Pyk 2）的两种亚型的时间表达，Pyk 2是参与活化整合素下游信号传导的关键蛋白激酶，并且Pyk 2与Rb和Mdm 2形成复合物。此外，MK介导的OB数量增加在来自Pyk 2-/-小鼠的OB中被消除。因此，我们的中心假设是，MK通过Pyk 2介导的上游和下游信号蛋白的调节途径调节OB中的细胞周期进程，对骨具有合成代谢作用。在目的1中，我们将通过在OB中异位表达Pyk 2或Pyk 2-S并评估细胞周期调节和分化来证明Pyk 2亚型在MK调节的OB中的功能作用。在目标2中，我们将确定Pyk 2的磷酸化和活性在调节其与Rb和Mdm 2的相互作用及其降解中的作用。最后，在目标3中，我们将通过移植富含MK的造血前体的Pyk 2-/-小鼠来证明Pyk 2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Pyk 2在OB细胞周期调节中的作用。确定导致体内骨体积增加的途径将导致开发新的治疗方法，刺激骨形成以治疗骨质疏松症和其他骨丢失疾病。 公共卫生相关性：巨核细胞，血小板生成细胞，可以增加小鼠和人类的骨量。我们已经发现巨核细胞增加成骨细胞增殖（形成骨骼的细胞），并试图了解这是如何发生的。我们希望这项工作最终能为治疗骨质疏松症等骨丢失疾病提供新的策略。

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. PUBLIC HEALTH RELEVANCE: Megakaryocytes, the platelet producing cells, can increase bone mass in mice and humans. We have found that megakaryocytes increase osteoblast proliferation (the cells that form bone) and are trying to understand how this occurs. We hope that this work will eventually offer new strategies to treat bone loss diseases such as osteoporosis.