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

骨中的 PKA-Wnt 串扰是由 β-连环蛋白核复合物形成介导的

• 批准号: 8113254
• 负责人: Lawrence S Kirschner
• 金额: $ 16.47万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-20 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8113254
• 关键词: Acute Promyelocytic Leukemia; Affect; Albers-Schonberg disease; Applications Grants; Atrial myxoma with lentigines; Binding; Biological; Biology; Bone Development; Bone neoplasms; CREB1 gene; Cell Nucleus; Cell physiology; Co-Immunoprecipitations; Code; Colon Carcinoma; Complex; Coronary Arteriosclerosis; Coupled; Cyclic AMP Response Element; Cyclic AMP-Dependent Protein Kinases; DNA Binding; Data; Deposition; Development; Drosophila genus; EP300 gene; Exhibits; Family; Family member; Forskolin; GNAS gene; Gene Mutation; Genes; Genetic Transcription; Growth; Heterotrimeric G Protein Subunit; Homeostasis; Hormones; Human Biology; Hyperlipidemia; Hypertension; Immunofluorescence Immunologic; Individual; Inherited; Laboratories; Maintenance; Malignant Neoplasms; McCune-Albright Syndrome; Mediating; Mediator of activation protein; Membrane; Modeling; Molecular; Molecular Profiling; Multiprotein Complexes; Mus; Mutation; Nature; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Nuclear Protein; Online Mendelian Inheritance In Man; Osteoblasts; Osteoporosis; Parathyroid Hormone Receptor; Pathway Analysis; Pathway interactions; Patients; Pattern; Penetrance; Phenotype; Phosphorylation; Phosphorylation Site; Physiology; Play; Polyostotic fibrous dysplasia; Proteins; Proteomics; Pseudohypoparathyroidism; Receptor Gene; Role; Signal Pathway; Signal Transduction; Site; Testing; Travel; Tumor-Derived; Up-Regulation; base; beta catenin; bone; bone cell; bone loss; bone mass; cell growth; cell type; insight; interest; member; osteoporosis-pseudoglioma syndrome; parathyroid hormone-related protein; programs; promoter; protein complex; public health relevance; receptor; research study; response; transcription factor; transcription factor PML; tumor

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.

描述（由申请人提供）：Wnt信号通路是一种高度保守的发育模式，在细胞分化、增殖和稳态中发挥作用。该途径已被证明对骨生理学特别重要，因为Wnt信号传导的失活导致骨丢失，而过量的信号传导导致骨量增加。蛋白激酶A（PKA，cAMP依赖性蛋白激酶）信号传导在骨生理学中也是重要的，因为成骨细胞增殖和分化可以通过由甲状旁腺激素受体的活化介导的PKA信号传导来控制。我们以前已经证明，由于PKA调节亚基Prkar 1a突变而表现出整体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。使用全局表达谱分析肿瘤鉴定了Prkar 1a +/-小鼠骨肿瘤中Wnt信号通路成员的上调。我们随后确定，这些肿瘤表现出增强的Wnt依赖性转录，虽然没有观察到的β-连环蛋白丰度或核质定位模式的改变。然而，肿瘤中<$-连环蛋白亚细胞定位的免疫荧光分析显示<$-连环蛋白经历了核重新分布，其可以通过用毛喉素刺激PKA途径在野生型细胞中重现。共定位实验表明，<$-连环蛋白与早幼粒细胞白血病（PML）体相关，响应PKA激活。此外，对Prkar 1a +/-肿瘤中上调的基因启动子的分析表明，这些基因中的大多数在其启动子中含有Wnt和cAMP反应元件。基于这些初步的数据，我们假设PKA磷酸化的<$-catenin增强其结合转录活性的多蛋白核复合物，也包含磷酸化CREB。在这项拨款申请中，我们建议1）研究这种核内再分布对连环蛋白内PKA磷酸化位点的需求; 2）确定PKA和/或Wnt激活如何改变转录改变的基因启动子处的连环蛋白和含CREB的核复合物的分布，以及3）确定这些蛋白的全部性质。- 含有连环蛋白-PML的复合物，以鉴定存在的其他蛋白质（包括CREB）。这些研究将为<$-连环蛋白在骨细胞中发挥其生物学作用的机制提供新的见解，并提供描述<$-连环蛋白和PKA信号通路串扰的新机制细节。这些研究不仅对骨生理学有影响，而且可能为介导复杂信号级联的多蛋白转录复合物的形成提供更大的见解。 公共卫生相关性：蛋白激酶A（PKA）和Wnt/β-连环蛋白信号通路对于正常骨的发育和维持都是必不可少的，因为它们参与细胞功能和生长的控制。使用一种新的骨肿瘤模型，我们发现PKA刺激Wnt/<$-catenin通路的功能，并提出描述这种相互作用发生的不寻常机制。这些研究的结果可能不仅对于确定可以调节骨量的新方法（例如，治疗骨质疏松症），但也可能对理解PKA如何调节肿瘤中Wnt/β-连环蛋白的活性具有更大的意义。