Abstract

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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR051300-06A2
Application #
6869033
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Sharrock, William J
Project Start
1998-09-01
Project End
2009-08-31
Budget Start
2004-09-30
Budget End
2005-08-31
Support Year
6
Fiscal Year
2004
Total Cost
$300,032
Indirect Cost

  Institution

Name
University of California San Diego
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093

  Publications

Ramdani, Ghania; Schall, Nadine; Kalyanaraman, Hema et al. (2018) cGMP-dependent protein kinase-2 regulates bone mass and prevents diabetic bone loss. J Endocrinol 238:203-219
Kalyanaraman, Hema; Ramdani, Ghania; Joshua, Jisha et al. (2017) A Novel, Direct NO Donor Regulates Osteoblast and Osteoclast Functions and Increases Bone Mass in Ovariectomized Mice. J Bone Miner Res 32:46-59
Saha, Arindam; Connelly, Stephen; Jiang, Jingjing et al. (2014) Akt phosphorylation and regulation of transketolase is a nodal point for amino acid control of purine synthesis. Mol Cell 55:264-76
Joshua, Jisha; Schwaerzer, Gerburg K; Kalyanaraman, Hema et al. (2014) Soluble guanylate cyclase as a novel treatment target for osteoporosis. Endocrinology 155:4720-30
Schwappacher, Raphaela; Kilic, Ana; Kojonazarov, Baktybek et al. (2013) A molecular mechanism for therapeutic effects of cGMP-elevating agents in pulmonary arterial hypertension. J Biol Chem 288:16557-66
Casteel, Darren E; Turner, Stephanie; Schwappacher, Raphaela et al. (2012) Rho isoform-specific interaction with IQGAP1 promotes breast cancer cell proliferation and migration. J Biol Chem 287:38367-78
Rangaswami, Hema; Schwappacher, Raphaela; Tran, Trish et al. (2012) Protein kinase G and focal adhesion kinase converge on Src/Akt/ýý-catenin signaling module in osteoblast mechanotransduction. J Biol Chem 287:21509-19
Marathe, Nisha; Rangaswami, Hema; Zhuang, Shunhui et al. (2012) Pro-survival effects of 17?-estradiol on osteocytes are mediated by nitric oxide/cGMP via differential actions of cGMP-dependent protein kinases I and II. J Biol Chem 287:978-88
He, Zhao; Zhang, Sharon S; Meng, Qingyuan et al. (2012) Shp2 controls female body weight and energy balance by integrating leptin and estrogen signals. Mol Cell Biol 32:1867-78
Casteel, Darren E; Smith-Nguyen, Eric V; Sankaran, Banumathi et al. (2010) A crystal structure of the cyclic GMP-dependent protein kinase I{beta} dimerization/docking domain reveals molecular details of isoform-specific anchoring. J Biol Chem 285:32684-8

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