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（由适用提供）：Wnt信号通路是保存完好的发育范式，在细胞分化，增殖和稳态中作用。由于Wnt信号的失活会导致骨骼损失，而超过信号导致骨骼质量增加，因此该途径已被证明对骨骼生理尤为重要。蛋白激酶A（PKA，cAMP依赖性蛋白激酶）信号传导在骨骼生理学中也很重要，因为成骨细胞增殖和分化可以通过甲状旁腺赛替酮受体的激活介导的PKA信号传导来控制。我们以前已经证明，由于PKA调节亚基PRKAR1A的突变，表现出全球PKA激活的小鼠经常出现骨肿瘤。这些肿瘤源自成骨细胞谱系，表现出对PKA生长促进作用的反应性。使用全局表达分析对肿瘤进行分析确定了PRKAR1A +/-小鼠骨肿瘤中Wnt信号通路成员的上调。随后，我们确定了这些肿瘤暴露了增强的Wnt依赖性转录，尽管未观察到 - - 卡宁丰度或核质质定位模式的改变。然而，肿瘤中的 - 加度蛋白亚细胞定位的免疫荧光分析表明，`` - 卡丁蛋白经过了核分布，可以通过刺激野生型细胞中的PKA途径在野生型细胞中概括。共定位实验表明，与PKA激活相关的与前胞素白血病（PML）体相关的 - 钙蛋白。此外，对PRKAR1A +/-肿瘤中更新的基因启动子的分析表明，这些基因中的大多数都包含启动子中的Wnt-和CAMP响应元素。基于此初步数据，我们假设 - 帕宁蛋白的PKA磷酸化增强了其与转录活性多蛋白质核复合物的结合，该核复合物还含有磷酸化的Creb。在此赠款应用中，我们建议1）研究 - 帕宁蛋白内的PKA磷酸化位点的要求； 2）确定PKA和/或Wnt激活如何改变» - 蛋白蛋白和含CREB的核复合物在具有转录变化的基因的启动子上的分布，以及3），以确定这些»catenin -Pml的全部性质，以鉴定其他蛋白质（包括CREB）。这些研究将提供有关� -catenin在骨细胞中执行其生物学作用的机制的新见解，并提供了描述� -Catenin和PKA信号传导途径的串扰的新机械细节。这些研究不仅对骨骼生理有影响，而且可以提供更大的见解，以介导介导复合信号级联的多蛋白转录复合物的形成。 公共卫生相关性：蛋白激酶A（PKA）和Wnt/¿-Catenin信号通路对于正常骨的发育和维持至关重要，因为它们参与了细胞功能和生长的控制。使用新的骨肿瘤模型，我们发现PKA刺激了Wnt/® -Catenin途径的功能，并提出了这种相互作用的异常机制的建议。这些研究的结果不仅对于确定可以调节骨骼质量的新含义（例如，治疗骨质疏松症）的结果可能很重要，而且对于理解PKA如何调节肿瘤中Wnt/® -Catenin的活性也可能具有更大的影响。