REGULATION OF GENE EXPRESSION BY cGMP and PKG IN BONE

说明书(申请人提供)：一氧化氮(NO)/cGMP/cGMP依赖的蛋白激酶(PKG)信号通路在调节成骨细胞和软骨细胞的生长和分化中发挥重要作用。在NO合成酶m缺陷的小鼠中，机械刺激下的骨重建减少，PKG II缺陷的小鼠表现出软骨内成骨缺陷，但对骨中PKG的下游靶点知之甚少。在之前的资助期间，我们确定了PKG I和II调节基因表达的几种机制。我们发现，NO/cGMP和钙(Ca)协同激活成骨细胞中的c-fos启动子，这种协同作用需要PKG II调节转录因子C/EBP-β和CREB之间的协同作用，而PKG II间接调节C/EBP-β的磷酸化和c-fos启动子的募集。C/EBP-β、CREB和c-Fos在骨的动态平衡和发育中起重要作用。本研究的具体目的是：(I)确定cGMP/PKG如何调节成骨细胞中C/EBP-β的磷酸化；(Ii)确定c/EBP-β磷酸化在cGMP/Ca刺激的成骨细胞中的功能后果；(Iii)研究NO/cGMP/PKG在机械刺激的成骨细胞c-fos诱导中的作用。我们将绘制依赖于cGMP的C/EBP-β磷酸化位点图，并使用药理学和遗传学方法确定介导PKG效应的途径(S)。我们将使用C/EBP-(突变体和siRNAs)在凝胶迁移率改变、报告基因、染色质免疫沉淀和体外蛋白质结合实验中，确定C/EBP-β磷酸化对DNA结合、反式激活、与CREB的相互作用以及FOS启动子上共激活子招募的影响。我们将检测NO/cGMP/PKG等通路在流体切应力刺激的成骨细胞c-fos和COX-2mRNA诱导中的作用，并确定c-fos在机械刺激诱导的成骨细胞增殖中的作用。这些研究将为cGMP/PKG在骨骼中的作用提供新的见解；更好地理解机械诱导的骨重建过程中的NO/cGMP信号可能有助于改进骨质疏松的治疗方法。

DESCRIPTION (provided by applicant): The nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) signaling pathway plays an important role in regulating osteoblast and chondroblast growth and differentiation. Bone remodeling in response to mechanical stimulation is reduced in NO synthase m-deficient mice, and PKG II-deficient mice show defective endochondral ossification, but little is known about the down-stream targets of PKG in bone. During the previous grant period, we identified several mechanisms whereby PKG I and II regulate gene expression. We showed that NO/cGMP and calcium (Ca++) synergistically activate the c-fos promoter in osteoblasts, and that the synergism requires PKG II-regulated cooperation between the transcription factors C/EBP-beta and CREB, with PKG II indirectly regulating C/EBP-beta phosphorylation and recruitment to the c-fos promoter. C/EBP-beta, CREB, and c-Fos play important roles in bone homeostasis and development. The Specific Aims of this proposal are: (I) to determine how cGMP/PKG regulate C/EBP-beta phosphorylation in osteoblasts; (II) to determine functional consequences of C/EBP-beta phosphorylation in cGMP/Ca++-stimulated osteoblasts; and (III) to study the role of NO/cGMP/PKG in c-fos induction in mechanically stimulated osteoblasts. We will map cGMP-dependent C/EBP-beta phosphorylation sites and define the pathway(s) mediating PKG's effects using pharmacologic and genetic approaches. We will determine the effects of C/EBP-beta phosphorylation on DNA binding, transactivation, interaction with CREB, and co-activator recruitment at the fos promoter, using C/EBP-( mutants and siRNAs in electrophoretic mobility shift, reporter gene, chromatin immunoprecipitation, and in vitro protein binding experiments. We will examine the contribution of NO/cGMP/PKG and other pathways to c-fos and cox-2 mRNA induction in fluid shear stress stimulated osteoblasts, and determine the role of c-fos in osteoblast proliferation induced by mechanical stimulation. These studies should provide new insights into cGMP/PKG actions in bone; a better understanding of NO/cGMP signaling during mechanically-induced bone remodeling may lead to improved therapies for osteoporosis.