Involvement of hydrogen peroxide in the differentiation of clonal HD-11EM cells into osteoclast-like cells.
The present study uses the osteoclast precursor clonal line, HD-11EM, to study the potential of hydrogen peroxide (H2O2) in mediating the differentiation of HD-11EM into osteoclast-like cells. HD-11EM cells are a newly established clonal cell line that, in response to 1alpha,25-(OH)2D3, differentiate into osteoclast-like cells that are multinucleated (more than three nuclei), express tartrate-resistant acid phosphatase (TRAP), and excavate resorption pits when cultured on dentin slices in the presence of osteoblasts (Hsia et al., 1995, J. Bone Miner. Res., 10(Suppl 1):S424; Hsia, and Hauschka, 1997, unpublished data). Here we demonstrate that HD-11EM express the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase specific cytochrome b558 subunits, and that stimulation of HD-11EM with 1 or 10 nM 1alpha,25-(OH)2D3 increases the extracellular release of H2O2 within 5-10 min. Ours is the first report that stimulation of a cell with 1alpha,25-(OH)2D3 enhances the activation of NADPH-oxidase and increases the basal release of superoxide and the formation of its dismutation product, H2O2. To determine the possible involvement of H2O2 in the differentiation of HD-11EM, these cells were exposed to glucose/glucose oxidase. This enzyme system was used to deliver a pure and continuous source of H2O2 in nanomole amounts consistent with quantities produced by HD-11EM in response to 1alpha,25-(OH)2D3. Both 1alpha,25-(OH)2D3 and the exogenously generated H2O2 stimulated a dose- and time-dependent increase in TRAP activity/cell and the number of multinucleated cells 24-48 hr after treatment. Northern analysis confirmed an increase in expression of TRAP mRNA in response to either 1alpha,25-(OH)2D3 or H2O2. Decreases in cell proliferation and v-myc mRNA were also observed in response to these agents. Taken together, our findings indicate that production of H2O2 by HD-11EM is an important local factor involved in differentiation of HD-11EM into osteoclast-like cells, and suggest that H2O2 may play a role in native osteoclast differentiation.
Steinbeck
M J
MJ
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115-5701, USA. mjstein@warren.med.harvard.edu
Kim
J K
JK
Trudeau
M J
MJ
Hauschka
P V
PV
Karnovsky
M J
MJ
D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism; D015152:Blotting, Northern; D002117:Calcitriol / Q000494:pharmacology; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002467:Cell Nucleus / Q000502:physiology; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D003573:Cytochrome b Group / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D016259:Genes, myc / Q000187:drug effects; D005949:Glucose Oxidase / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D000071681:Tartrate-Resistant Acid Phosphatase
D003573:Cytochrome b Group; D007527:Isoenzymes; D012333:RNA, Messenger; D013481:Superoxides; C036888:cytochrome b558; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D019255:NADPH Oxidases; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D002117:Calcitriol