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Involvement of hydrogen peroxide in the differentiation of clonal HD‐11EM cells into osteoclast‐like cells
Author(s) -
Steinbeck Marla J.,
Kim JungKeun,
Trudeau Mathew J.,
Hauschka Peter V.,
Karnovsky Morris J.
Publication year - 1998
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/(sici)1097-4652(199809)176:3<574::aid-jcp14>3.0.co;2-#
Subject(s) - osteoclast , multinucleate , acid phosphatase , nicotinamide adenine dinucleotide phosphate , chemistry , cellular differentiation , cell culture , extracellular , hydrogen peroxide , oxidase test , biochemistry , stimulation , microbiology and biotechnology , enzyme , biology , endocrinology , in vitro , genetics , gene
The present study uses the osteoclast precursor clonal line, HD‐11EM, to study the potential of hydrogen peroxide (H 2 O 2 ) 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 1α,25‐(OH) 2 D 3 , 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 Res., 10(Suppl 1) :S424; Hsia, and Hauschka, 1997, unpublished data).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 b 558 subunits, and that stimulation of HD‐11EM with 1 or 10 nM 1α,25‐(OH) 2 D 3 increases the extracellular release of H 2 O 2 within 5–10 min. Ours is the first report that stimulation of a cell with 1α,25‐(OH) 2 D 3 enhances the activation of NADPH‐oxidase and increases the basal release of superoxide and the formation of its dismutation product, H 2 O 2 . To determine the possible involvement of H 2 O 2 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 H 2 O 2 in nanomole amounts consistent with quantities produced by HD‐11EM in response to 1α,25‐(OH) 2 D 3 . Both 1α,25‐(OH) 2 D 3 and the exogenously generated H 2 O 2 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 1α,25‐(OH) 2 D 3 or H 2 O 2 . Decreases in cell proliferation and v‐myc mRNA were also observed in response to these agents. Taken together, our findings indicate that production of H 2 O 2 by HD‐11EM is an important local factor involved in differentiation of HD‐11EM into osteoclast‐like cells, and suggest that H 2 O 2 may play a role in native osteoclast differentiation. J. Cell. Physiol. 176:574–587, 1998. © 1998 Wiley‐Liss, Inc.