PKA-Wnt Crosstalk in Bone is Mediated by beta-Catenin Nuclear Complex Formation

描述(由申请人提供)：Wnt信号通路是一个保守的发育范例，在细胞分化、增殖和动态平衡中发挥作用。这一途径已被证明对骨生理学特别重要，因为Wnt信号失活会导致骨丢失，而过多的信号会导致骨量增加。蛋白激酶A(PKA，cAMP依赖的蛋白激酶)信号在骨生理学中也很重要，因为成骨细胞的增殖和分化可以通过激活甲状旁腺激素受体介导的PKA信号来调控。我们先前已经证明，由于PKA调节亚单位Prkar1a的突变而表现出全球PKA激活的小鼠经常发生骨肿瘤。这些肿瘤来源于成骨细胞谱系，对PKA的生长促进作用表现出过度的反应性。使用全球表达谱对肿瘤的分析发现，在Prkar1a/-鼠骨肿瘤中，Wnt信号通路的成员上调。我们随后确定，这些肿瘤表现出增强的Wnt依赖的转录，尽管没有观察到连接蛋白丰度或核质定位模式的变化。然而，对肿瘤细胞内β-catenin亚细胞定位的免疫荧光分析表明，β-catenin经历了核的重新分布，这种重新分布可以通过Forsklin刺激PKA途径在野生型细胞中重现。共定位实验表明，β-catenin与早幼粒细胞白血病(PML)小体对PKA激活的反应有关。此外，对Prkar1a/-肿瘤中上调的基因启动子的分析表明，大多数这些基因在其启动子中同时含有Wnt和cAMP反应元件。基于这些初步数据，我们假设-连环素的PKA磷酸化增强了它与转录活性的多蛋白核复合体的结合，这些复合体也包含磷酸化的CREB。在这项拨款申请中，我们建议1)研究连环蛋白中PKA磷酸化位点对核内再分布的要求；2)确定PKA和/或Wnt激活如何改变转录改变的基因启动子上连环蛋白和含CREB的核复合体的分布；以及3)确定这些连环蛋白-PML包含复合体的全部性质，以便识别存在的其他蛋白质(包括CREB)。这些研究将对连环蛋白在骨细胞中发挥生物学作用的机制提供新的见解，并提供新的机制细节来描述连环蛋白和PKA信号通路的串扰。这些研究不仅对骨生理学有影响，而且可能为多蛋白转录复合体的形成提供更大的洞察力，这些复合体介导复杂的信号级联。 与公共健康相关：蛋白激酶A(PKA)和Wnt/β-catenin信号通路对正常骨骼的发育和维持都是必不可少的，因为它们参与了细胞功能和生长的控制。利用一个新的骨肿瘤模型，我们发现PKA刺激Wnt/β-连环蛋白途径的功能，并建议描述这种相互作用发生的一种不寻常的机制。这些研究的结果可能不仅对于确定调节骨量的新方法(例如，治疗骨质疏松症)具有重要意义，而且可能对理解PKA如何调节肿瘤中Wnt/β-catenin的活性具有更大的意义。

DESCRIPTION (provided by applicant): The Wnt signaling pathway is a well-conserved developmental paradigm with roles in cellular differentiation, proliferation, and homeostasis. This pathway has been shown to be particularly important for bone physiology, as inactivation of Wnt signaling causes a loss of bone, whereas excess signaling causes increased bone mass. Protein Kinase A (PKA, cAMP-dependent protein kinase) signaling is also important in bone physiology, as osteoblast proliferation and differentiation can be controlled by PKA signaling mediated by activation of the parathyroid hormone receptor. We have previously demonstrated that mice exhibiting global PKA activation due to mutation of the PKA regulatory subunit Prkar1a frequently develop bone tumors. These tumors are derived from the osteoblast lineage and exhibit excess responsiveness to the growth promoting effects of PKA. Analysis of tumors using global expression profiling identified upregulation of members of the Wnt signaling pathway in Prkar1a+/- mouse bone tumors. We have subsequently determined that these tumors exhibited enhanced Wnt-dependent transcription, although no alterations in ¿-catenin abundance or nuclear-cytoplasmic localization pattern were observed. However, immunofluorescence analysis of ¿-catenin subcellular localization in the tumors revealed that ¿-catenin underwent a nuclear re-distribution which could be recapitulated in wild-type cells by stimulation of the PKA pathway with forskolin. Co-localization experiments indicated that ¿-catenin associated with promyelocytic leukemia (PML) bodies in response to PKA activation. Furthermore, analysis of the promoters of genes upregulated in the Prkar1a+/- tumors demonstrated that most of these genes contain both Wnt- and cAMP-response elements in their promoters. Based on this preliminary data, we hypothesize that PKA phosphorylation of ¿-catenin enhances its binding to transcriptionally active multi-protein nuclear complexes that also contain phosphorylated CREB. In this grant application, we propose 1) to study the requirement of PKA phosphorylation sites within ¿-catenin for this intranuclear redistribution; 2) to determine how PKA and/or Wnt activation alters the distribution of ¿-catenin and CREB-containing nuclear complexes at the promoters of genes with altered transcription, and 3) to determine the full nature of these ¿-catenin-PML containing complexes in order to identify other proteins (including CREB) present. These studies will provide new insights into the mechanism by which ¿-catenin exerts its biological effects in bone cells and provide new mechanistic details describing the crosstalk of the ¿-catenin and PKA signaling pathways. These studies not only have implications for bone physiology, but may provide larger insights into the formation of multiprotein transcriptional complexes mediating complex signaling cascades. PUBLIC HEALTH RELEVANCE: The Protein Kinase A (PKA) and Wnt/¿-catenin signaling pathways are both essential for the development and maintenance of normal bone, as they are involved in the control of cell function and growth. Using a new model of bone tumors, we have found that PKA stimulates the function of the Wnt/¿-catenin pathway and propose to characterize an unusual mechanism by which this interaction occurs. The results of these studies may be important not only for identifying new mean by which bone mass can be modulated (e.g., treatment for osteoporosis), but may also have larger implications for understanding how PKA may modulate the activity of Wnt/¿-catenin in tumors.