Involvement of hydrogen peroxide in the differentiation of clonal HD-11EM cells into osteoclast-like cells

Abstract

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 Res., 10(Suppl 1):S424; Hsia, and Hauschka, 1997, unpublished data).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.

Fig. 1

Histochemical staining for tartrate-resistant acid phosphatase (TRAP) activity and multinucleated cell counts 48 hr after 1α,25-(OH)₂D₃ stimulation. HD-11EM were plated at a density of 3 × 10⁴ cells/well of a six-well plate in 2.5 ml of Dulbecco’s modified Eagle’s medium/F12 media supplemented with 10% heat-inactivated fetal calf serum. After 3 days in culture, the cells were stimulated with vehicle [0.005% (v/v) final ethanol concentration], 1, or 10 nM 1α,25-(OH)₂D₃ in medium (no media change at time of stimulation) for 48 hr. The cells were then washed, lightly fixed, and stained for TRAP activity. The total number of cells, the percentage of TRAP stained cells, and the number of multinucleated cells were determined at 48 hr. A: In cell preparations receiving vehicle only, an occasional multinucleated cell (three or more nuclei) was observed, and a low number of the cells contained a red precipitate, indicating TRAP activity. B: Treatment of the cells with 10 nM 1α,25-(OH)₂D₃ dramatically increased the multinucleated cell formation three- to fourfold, and uniformly increased the intensity of staining and the number of TRAP stained cells fourfold. This figure contains representative data from one of three independent experiments.

Fig. 2

Quantitative measurements of tartrate-resistant acid phosphatase (TRAP) activity/cell over a 48-hr period in response to 1α,25-(OH)₂D₃. HD-11EM cells were plated and cultured as described in Figure 1. After 3 days in culture, the cells were stimulated with (A) vehicle, 1, or 10 nM 1α,25-(OH)₂D₃ in medium for 0, 6, 24, or 48 hr; or (B) vehicle, 1, or 10 nM 1α,25-(OH)₂D₃ in phosphate buffered saline (PBS) for 20 min, followed by the addition of fresh medium without 1α,25-(OH)₂D₃ for 0, 6, 24, or 48 hr. Arbitrary units of TRAP activity/cell represent the amount of reaction product measured by fluorescence intensity. Cell numbers were determined by counting cells in duplicate plates receiving the same stimulation media. Data are expressed as the mean of four experiments ± SEM. Results are significantly different from vehicle-only responses at that time point: **, P < 0.01; and *, P < 0.05.

Fig. 3

Expression of the unique cytochrome b₅₅₈ subunits of NADPH-oxidase by HD-11EM cells. After 3 days in culture, the cells were washed, and fixed in 2% paraformaldehyde. Immunocytochemical studies were done using the standard indirect avidin-biotin technique: (A) no immunostaining was observed in the absence of specific monoclonal antibody; (B) brown oxidized deposits of 3,3-diaminobenzidine (DAB) immunoperoxidase products were observed in HD-11EM cells immunostained with a mAb recognizing the gp91-phox subunit of cytochrome b₅₅₈.

Fig. 4

Superoxide and H₂O₂ production by HD-11EM in response to stimulation with 1α,25-(OH)₂D₃ or 4B-phorbol-12-myristate-13-acetate (PMA). A: After 3 days in culture, the HD-11EM were stimulated in medium for 20 min with vehicle, 1, or 10 nM 1α,25-(OH)₂D₃ or PMA 100 ng/ml. Cells were then incubated for 30 min at 37°C and the amount of superoxide produced was determined using the superoxide dismutase-inhibitable change in absorbance at 550 nm. Data are expressed as the mean ± SEM of four experiments. Results are significantly different from vehicle-only responses for each experiment at that time point: **, P < 0.01; and * P < 0.05. B: After 3 days in culture, the amount of H₂O₂ produced in response to vehicle, 1, or 10 nM 1α,25-(OH)₂D₃ at 37°C was measured continuously from 0 to 30 min. Representative data are presented from one experiment done in duplicate and performed three times.

Fig. 5

Tartrate-resistant acid phosphatase (TRAP) activity/cell over a 48-hr period in response to H₂O₂ generated by glucose oxidase. HD-11EM cells were stimulated for 2 hr in various media (2.5 ml/well of a six-well plate), the cells were washed, received fresh media, and at indicated time points measurements of TRAP activity/cell were done. A: To determine the amount of H₂O₂ produced at 30 min by 1.0 to 5.0 mU of glucose oxidase, and to evaluate the effects at 24 hr of increasing amounts of H₂O₂ on TRAP activity/cell, HD-11EM were incubated in phosphate buffered saline (PBS) containing glucose, superoxide dismutase (SOD) 241 U, 10 mM 3-amino-1,2,4-triazole (ATZ) (catalase inhibitor), and increasing units of glucose oxidase. B: To evaluate TRAP activity/cell at 6, 24, or 48 hr after treatment with amounts of H₂O₂ within the range produced by HD-11EM stimulated with 1 or 10 nM 1α,25-(OH)₂D₃, HD-11EM were incubated for 2 hr with glucose, SOD 241 U, and 10 mM ATZ (SOD/ATZ); no glucose, SOD, ATZ, and glucose oxidase 5.0 mU (No glucose + GO 5.0 mU + SOD/ATZ); glucose, SOD, ATZ, and glucose oxidase 2.5 mU (GO 2.5 mU + SOD/ATZ) or 5.0 mU (GO 5.0 mU + SOD/ATZ). C: To determine the effects of H₂O₂ without inhibiting the intracellular catalase activity with ATZ, HD-11EM were treated for 2 hr with glucose and SOD (SOD); no glucose, SOD, and glucose oxidase 5.0 mU (No glucose/SOD); or glucose, SOD, and glucose oxidase 2.5 mU (GO 2.5 mU + SOD) or 5.0 mU (GO 5.0 mU + SOD). Arbitrary units of TRAP activity/cell represent the amount of product generated measured by fluorescence. Cell numbers were determined by counting cells in duplicate plates receiving the same stimulation media. Data are expressed as the mean ± SEM of four experiments. Results are significantly different from SOD ± ATZ responses at that time point: **, P < 0.01; and *, P < 0.05.

Fig. 6

1α,25-(OH)₂D₃ or H₂O₂ increase tartrate-resistant acid phosphatase (TRAP) mRNA and decrease the amounts of v-myc mRNA. HD-11EM were grown for 3 days in Dulbecco’s modified Eagle’s medium/F12 medium supplemented with 10% heat-inactivated fetal calf serum, then stimulated in (A) conditioned medium with 10 nM 1α,25-(OH)₂D₃ or (C) with glucose/glucose oxidase 2.5 mU, 241 U SOD, and 10 mM ATZ in PBS for 2 hr, and then fresh medium supplemented with 10% heat-inactivated FCS. Total RNA was isolated at the indicated time points and analyzed by Northern blot procedures; filters were analyzed for TRAP mRNA, washed, and reprobed for v-myc mRNA. B: The amount of mRNA relative to 28S rRNA over time in HD-11EM treated with 10 nM 1α,25-(OH)₂D₃ or (D) with glucose/glucose oxidase 2.5 mU, SOD, and ATZ. These figures contain representative data from one of three independent experiments.