title: Homocysteine alters the osteoprotegerin/RANKL system in the osteoblast to promote bone loss: pivotal role of the redox regulator forkhead O1.
abstract: In this study we determined the molecular mechanisms of how homocysteine differentially affects receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) synthesis in the bone. The results showed that oxidative stress induced by homocysteine deranges insulin-sensitive FOXO1 and MAP kinase signaling cascades to decrease OPG and increase RANKL synthesis in osteoblast cultures. We observed that downregulation of insulin/FOXO1 and p38 MAP kinase signaling mechanisms due to phosphorylation of protein phosphatase 2A (PP2A) was the key event that inhibited OPG synthesis in homocysteine-treated osteoblast cultures. siRNA knockdown experiments confirmed that FOXO1 is integral to OPG and p38 synthesis. Conversely homocysteine increased RANKL synthesis in osteoblasts through c-Jun/JNK MAP kinase signaling mechanisms independent of FOXO1. In the rat bone milieu, high-methionine diet-induced hyperhomocysteinemia lowered FOXO1 and OPG expression and increased synthesis of proresorptive and inflammatory cytokines such as RANKL, M-CSF, IL-1α, IL-1β, G-CSF, GM-CSF, MIP-1α, IFN-γ, IL-17, and TNF-α. Such pathophysiological conditions were exacerbated by ovariectomy. Lowering the serum homocysteine level by a simultaneous supplementation with N-acetylcysteine improved OPG and FOXO1 expression and partially antagonized RANKL and proresorptive cytokine synthesis in the bone milieu. These results emphasize that hyperhomocysteinemia alters the redox regulatory mechanism in the osteoblast by activating PP2A and deranging FOXO1 and MAPK signaling cascades, eventually shifting the OPG:RANKL ratio toward increased osteoclast activity and decreased bone quality.
authors.affiliation: Molecular Sciences Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Viji
authors.initials: V
authors.lastname: Vijayan
authors.affiliation: Molecular Sciences Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Mayuri
authors.initials: M
authors.lastname: Khandelwal
authors.affiliation: Molecular Sciences Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Kapil
authors.initials: K
authors.lastname: Manglani
authors.affiliation: Molecular Sciences Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Rajiv Ranjan
authors.initials: RR
authors.lastname: Singh
authors.affiliation: Molecular Sciences Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India. Electronic address: sarika@nii.ac.in.
authors.forename: Sarika
authors.initials: S
authors.lastname: Gupta
authors.affiliation: Molecular Sciences Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India; Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560 012, India. Electronic address: surolia@mbu.iisc.ernet.in.
authors.forename: Avadhesha
authors.initials: A
authors.lastname: Surolia
mesh_terms: D000111:Acetylcysteine; D000818:Animals; D002478:Cells, Cultured; D005260:Female; D051858:Forkhead Transcription Factors / Q000502:physiology*; D006710:Homocysteine / Q000494:pharmacology*; D020138:Hyperhomocysteinemia / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000502:physiology*; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D010024:Osteoporosis / Q000209:etiology*; D053244:Osteoprotegerin / Q000032:analysis / Q000502:physiology*; D054648:Protein Phosphatase 2 / Q000378:metabolism; D053245:RANK Ligand / Q000032:analysis / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology; D000071161:Forkhead Box Protein O1
chemical_list: D000111:Acetylcysteine; D051858:Forkhead Transcription Factors; D006710:Homocysteine; D009419:Nerve Tissue Proteins; D053244:Osteoprotegerin; D054648:Protein Phosphatase 2; D053245:RANK Ligand; D048051:p38 Mitogen-Activated Protein Kinases; C074639:Foxo1 protein, rat; D000071161:Forkhead Box Protein O1
keywords: Forkhead box; Free radicals; Osteoprotegerin; Protein phosphatase 2A; Receptor activator of nuclear factor-κB ligand; c-Jun MAP kinase; p38