REGULATION OF GENE EXPRESSION BY cGMP and PKG IN BONE

描述（由申请人提供）：一氧化氮(NO)/cGMP/cGMP依赖性蛋白激酶（PKG）信号通路在调节成骨细胞和成软骨细胞的生长和分化中起重要作用。NO合酶m缺失小鼠在机械刺激下的骨重塑减少，PKG ii缺失小鼠表现出软骨内成骨缺陷，但对PKG在骨中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，并且这种协同作用需要PKG II调节的转录因子C/ ebp - β和CREB之间的合作，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在骨稳态和发育中起重要作用。本提案的具体目的是：(1)确定cGMP/PKG如何调节成骨细胞中C/ ebp - β磷酸化；（II）确定C/ ebp - β磷酸化对cGMP/ ca++刺激成骨细胞的功能影响；（III）研究NO/cGMP/PKG在机械刺激成骨细胞c-fos诱导中的作用。我们将绘制cgmp依赖的C/ ebp - β磷酸化位点，并使用药理学和遗传学方法定义介导PKG作用的途径。我们将利用C/EBP-（突变体）和sirna进行电泳迁移、报告基因、染色质免疫沉淀和体外蛋白结合实验，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启动子的共激活子募集的影响。我们将研究NO/cGMP/PKG等途径对流体剪切应力诱导成骨细胞c-fos和cox-2 mRNA诱导的贡献，并确定c-fos在机械刺激诱导成骨细胞增殖中的作用。这些研究将为cGMP/PKG在骨中的作用提供新的见解；更好地了解NO/cGMP信号在机械诱导的骨重塑过程中可能会改善骨质疏松症的治疗方法。

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.