REGULATION OF GENE EXPRESSION BY cGMP and PKG IN BONE

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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.