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(MKs)在调节骨骼动态平衡方面发挥着关键作用。为了支持这一点，缺乏正常MK发育所需的转录因子GATA-1或NF-E2的小鼠，表现出未成熟MK的增加和血小板数量的急剧减少。重要的是，这些小鼠的骨小梁密度增加了300%。这种骨量增加背后的细胞和组织水平的机制尚不清楚。然而，我们对GATA-1和NF-E2缺陷小鼠的组织学评估显示，骨小梁表面有更多的成骨细胞(OB)。重要的是，增加的骨表型可以使用突变小鼠的脾细胞过继地转移到受辐射的野生型小鼠体内，这表明造血细胞在这一机制中发挥了作用，很可能是这些小鼠体内升高的MKs。与这些体内实验一致，我们的体外数据显示，MKs通过细胞与细胞之间的直接接触(涉及整合素参与)促进OB的增殖(高达6倍)。尽管MKs促进OB增殖的确切机制尚不清楚，但这些观察表明MKs与OBS的相互作用导致成骨增加。此外，在与MKs共同培养的OBS中，细胞周期停滞蛋白视网膜母细胞瘤(RB)和调节蛋白酶体介导的降解的E3泛素连接酶小鼠双分钟2(MDM2)的表达一过性降低。最近，我们发现MKs调节两种富含Pro的酪氨酸激酶2(PYK2)的时间表达，PYK2是参与激活整合素下游信号传递的关键蛋白激酶，并且PYK2与Rb和MDM2形成复合体。此外，在PYK2-/-小鼠的OBS中，MK介导的OB数量的增加被取消。因此，我们的中心假设是，MKs通过参与Pyk2介导的上下游信号蛋白的调节途径来调节OBS的细胞周期进程，从而对骨骼产生合成代谢作用。在目标1中，我们将通过在OBS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异构体在MK调节的OBS中的功能作用。在目标2中，我们将确定PYK2‘S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，我们将通过将富含MKs的造血祖细胞移植到PYK2-/-小鼠身上来证明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MKs在调节合成代谢骨形成中的重要性，以及Pyk2在OB细胞周期调控中的作用。识别体内导致骨体积增加的途径将导致开发新的治疗方法，刺激骨形成，用于治疗骨质疏松症和其他骨丢失疾病。 与公共健康相关：巨核细胞是产生血小板的细胞，可以增加小鼠和人类的骨量。我们已经发现，巨核细胞促进成骨细胞(形成骨的细胞)的增殖，并试图了解这是如何发生的。我们希望这项工作最终将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.