Homocysteine enhances apoptosis in human bone marrow stromal cells.
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.
Kim
Duk Jae
DJ
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.
Koh
Jung-Min
JM
Lee
Oksun
O
Kim
Na Jung
NJ
Lee
Young-Sun
YS
Kim
Yang Soon
YS
Park
Joong-Yeol
JY
Lee
Ki-Up
KU
Kim
Ghi Su
GS
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
D016328:NF-kappa B; D017382:Reactive Oxygen Species; D006710:Homocysteine; D045304:Cytochromes c; D020169:Caspases