title: Homocysteine enhances apoptosis in human bone marrow stromal cells.
abstract: BACKGROUND
  High plasma homocysteine (Hcy) levels have been associated with increased risk of fracture. Since Hcy has been shown to induce apoptosis in many cell types, including vascular endothelial cells, we hypothesized that Hcy would have a similar apoptotic effect on osteoblasts, leading to osteoporosis by reducing bone formation.
  
  
  METHODS
  Using primary human bone marrow stromal cells (hBMSC) and HS-5 cell line (human bone marrow stromal cell line), we investigated the effects of Hcy on these cells by cell viability assay and analysis of cytoplasmic histone-associated DNA fragments. Caspase activity assay, Western blots, and electrophoresis mobility shift assay (EMSA) were performed to find the mechanism of apoptosis. Intracellular reactive oxygen species (ROS) were measured by spectrometry using dichlorofluorescein diacetate, and cellular total glutathione level was determined by a commercially available kit. N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) were used as tools for investigating the role of ROS and nuclear factor-kappaB (NF-kappaB), respectively.
  
  
  RESULTS
  Hcy induced apoptosis in primary human bone marrow stromal cells and the HS-5 cell line, and this apoptotic effect was caspase-dependent. In addition, Hcy increased cytochrome c release into the cytosol, and activated caspase-9 and caspase-3, but not caspase-8, indicating that Hcy induces apoptosis via the mitochondria pathway. Hcy increased ROS, and NAC inhibited the apoptotic effect of Hcy. Western blot and EMSA showed that Hcy activated the NF-kappaB pathway. PDTC blocked Hcy-induced caspase-3 activation and apoptosis.
  
  
  CONCLUSIONS
  These results suggest that Hcy induces apoptosis via the ROS-mediated mitochondrial pathway and NF-kappaB activation in hBMSCs, and that Hcy may contribute to the development of osteoporosis by reducing bone formation. Antioxidants may have a role in preventing bone loss in individuals with hyperhomocysteinemia.
authors.affiliation: Division of Endocrinology and Metabolism, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Poongnap-Dong, Songpa-Gu, Seoul 138-736, Republic of Korea.
authors.forename: Duk Jae
authors.initials: DJ
authors.lastname: Kim
authors.forename: Jung-Min
authors.initials: JM
authors.lastname: Koh
authors.forename: Oksun
authors.initials: O
authors.lastname: Lee
authors.forename: Na Jung
authors.initials: NJ
authors.lastname: Kim
authors.forename: Young-Sun
authors.initials: YS
authors.lastname: Lee
authors.forename: Yang Soon
authors.initials: YS
authors.lastname: Kim
authors.forename: Joong-Yeol
authors.initials: JY
authors.lastname: Park
authors.forename: Ki-Up
authors.initials: KU
authors.lastname: Lee
authors.forename: Ghi Su
authors.initials: GS
authors.lastname: Kim
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001853:Bone Marrow / Q000187:drug effects* / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D006710:Homocysteine / Q000494:pharmacology*; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism
chemical_list: D016328:NF-kappa B; D017382:Reactive Oxygen Species; D006710:Homocysteine; D045304:Cytochromes c; D020169:Caspases