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Endocrinology Vol. 141, No. 8 3006-3011
Copyright © 2000 by The Endocrine Society

ARTICLES

Role of Ascorbic Acid in the Osteoclast Formation: Induction of Osteoclast Differentiation Factor with Formation of the Extracellular Collagen Matrix1

Eri Otsuka, Yoshihisa Kato, Shigehisa Hirose and Hiromi Hagiwara

Research Center for Experimental Biology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan

Address all correspondence and requests for reprints to: Hiromi Hagiwara, Ph.D., Research Center for Experimental Biology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. E-mail: hhagiwar{at}bio.titech.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteoclasts are bone-resorbing multinucleated cells. Tartrate-resistant acid phosphatase-positive (TRAP-positive) mononuclear and multinucleated cells, which are osteoclast-like cells (OCLs), were formed as a result of the coculture of mouse bone marrow cells and clonal stromal ST2 cells in the presence of 1{alpha},25-dihydroxyvitamin D3. Removal of ascorbic acid from the culture medium prevented the formation of TRAP-positive OCLs. Addition of ascorbic acid to the medium formed TRAP-positive OCLs, and the effect of ascorbic acid was dose-dependent. When we examined the level of messenger RNA (mRNA) for osteoclast differentiation factor (RANKL/ODF) in ST2 cells, we found that ascorbic acid caused an approximately 5-fold increase in the level of this mRNA. The half-life of the mRNA was unaffected by ascorbic acid. To characterize the mechanism of action of ascorbic acid, we investigated the relationship between formation of TRAP-positive OCLs and formation of the collagen matrix. Inhibitors of the formation of collagen triple helices blocked both the formation of TRAP-positive OCLs and the expression of the mRNA for RANKL/ODF in response to ascorbic acid. Our findings suggest that ascorbic acid might be essential for osteoclastogenesis and might induce the formation of TRAP-positive OCLs via induction of the synthesis of RANKL/ODF that is somehow mediated by the extracellular matrix.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOCLASTS ARE multinucleated cells that are responsible for the resorption of bone. They are formed from hematopoietic cells of the monocyte/macrophage lineage. The processes of differentiation and fusion can be reproduced in a system in which mouse bone marrow cells are cocultured with mouse osteoblasts/osteogenic stromal cells (1). Several osteotropic hormones and cytokines affect osteoclastogenesis at distinct stages of the development of osteoclasts. These factors include 1{alpha},25-dihydroxyvitamin D3 [1{alpha},25(OH)2D3], macrophage colony-stimulating factor (M-CSF), calcitonin, PTH, interleukin 1, and interleukin 6. However, gene-disruption experiments have demonstrated that none of these factors is essential for the formation of osteoclasts in vivo.

Recently, genes for ODF (osteoclast differentiation factor) [RANKL/ODF; receptor activator of NF-{kappa}B ligand (RANKL)/ODF] (2, 3, 4) have been cloned and characterized (5, 6). RANKL/ODF is produced by osteoblast/osteogenic stromal cells and plays important roles in osteoclastogenesis. It is involved in the formation of osteoclast-like cells (OCLs), the fusion of osteoclastic precursor cells, and the resorption of bone (5, 6). Antibodies against RANKL/ODF abolish the 1{alpha},25(OH)2D3-induced, PTH-induced, and PG E2-induced resorption of bone in mouse fetal long-bone cultures (7). In addition, Odf-deficient mice exhibit severe osteopetrosis and defects in tooth eruption (4). Furthermore, osteoprotegerin [OPG; also known as osteoclastogenesis-inhibitory factor (OCIF)], which is a decoy receptor for RANKL/ODF, inhibits osteoclastogenesis by interfering with cell-to-cell signaling between osteoclast progenitors and osteoblast/osteogenic stromal cells (8, 9). Thus, RANKL/ODF seems to be essential for the development of osteoclasts both in vivo and in vitro. By contrast, a recent study showed that ascorbic acid induces the differentiation of osteoclasts in a coculture system in which differentiation is activated by 1{alpha},25(OH)2D3 and dexamethasone (10). Ascorbic acid is required as a cofactor for enzymes that are involved in the posttranslational modification of collagens; for example, prolyl and lysyl hydroxylases (11). Ascorbic acid deficiency caused a decrease in the corporation of sulfate into proteoglycans of guinea pig costal cartilage (12). Ascorbic acid is also involved in myogenesis, chondrogenesis, and osteogenesis via formation of the collagen matrix (11). However, the mechanism of action of ascorbic acid in osteoclastogenesis is not fully understood. In this report, we describe a correlation between the formation of OCLs and the expression of messenger RNA (mRNA) for RANKL/ODF in response to ascorbic acid.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
L-ascorbic acid and 1{alpha},25(OH)2D3 were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 3,4-Dehydro-L-proline (DHP) and ethyl-3,4-dihydroxybenzoate (EDHB) were obtained from Sigma (St. Louis, MO). 32P-labeled nucleotides were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). FBS and penicillin/streptomycin antibiotic mixture were obtained from Life Technologies (Grand Island, NY). Ascorbic acid-free {alpha}-MEM was purchased from Kojin Bio, Ltd. (Sakado, Japan).

Formation of OCLs in vitro
Bone marrow cells were collected from the femurs and tibias of 6-week-old male ddY mice, as described by Takahashi et al. (13). Mouse bone marrow-derived stromal ST2 cells were supplied by the RIKEN Cell Bank (Tsukuba, Japan) and were cocultured (2 x 104 cells/well) with bone marrow cells (5 x 104 cells/well) in {alpha}-MEM that contained 10% FBS and 10-8 M 1{alpha},25(OH)2D3 in 48-well plates (0.98 cm2/well; IWAKI, Tokyo, Japan). The cultures were maintained at 37 C in a humidified atmosphere of 5% CO2 in air. Fresh medium was supplied at 3-day intervals, and fresh ascorbic acid was added in the medium every time.

Localization of tartrate-resistant acid phosphatase (TRAP)
After coculture for 6–8 days, adherent cells were fixed in 10% formalin for 5 min and then in a mixture of ethanol and acetone (1:1, vol/vol) for 1 min. Then they were stained for TRAP activity, as described by Udagawa et al. (14). TRAP-positive mononuclear cells and TRAP-positive multinucleated cells (three or more nuclei) were counted under a microscope (IX70; Olympus Corp., Tokyo, Japan).

Semiquantitative RT-PCR
We detected mRNAs for RANKL/ODF, OPG, and M-CSF in ST2 cells by semiquantitative RT-PCR. RNA was extracted from ST2 cells that had been exposed to ascorbic acid by the acid guanidinium-phenol-chloroform method (15). Total RNA (5 µg) was reverse transcribed by Moloney murine leukemia virus reverse transcriptase, Superscript (200 U; Life Technologies), with random primers (50 ng) in a 20-µl reaction mixture. The complementary DNA (cDNA) was amplified in 20 µl Taq DNA polymerase mixture (Takara, Tokyo, Japan) that contained 1 µM sense primer, 5'-CAGGTTTGCAGGACTCGAC-3', and antisense primer, 5'-AGCAGGGAAGGGTTGGACA-3', for mouse RANKL/ODF (accession number AF013170, 434–1,034: 601 bp) (3); 1 µM sense primer, 5'-CCACTCTTATACGGACAGCT-3', and antisense primer, 5'-TCTCGGCATTCACTTTGGTC-3', for mouse OPG (accession number U94331, 291–796: 506 bp); 1 µM sense primer, 5'-TTGCCAAGGAGGTGTCAGAA-3', and antisense primer, 5'-TATTGGAGAGTTCCTGGAGC-3', for mouse M-CSF (accession number M21952, 251–511: 261 bp) (16); or 1 µM sense primer, 5'-ACTTTGTCAAGCTCATTTCC-3', and antisense primer, 5'-TGCAGCGAACTTTATTGATG-3', for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(accession number M32599, 957-1223: 267 bp). Each reaction cycle (performed 25, 25, 30, and 25 times for amplification of the cDNA for RANKL/ODF, OPG, M-CSF, and GAPDH, respectively) consisted of incubation at 94 C for 30 sec, at 60 C for 30 sec, and at 72 C for 30 sec. Products of PCR were subjected to electrophoresis on a 2% agarose gel and visualized by staining with ethidium bromide. DNA marker fragments (molecular weight marker V; Roche Molecular Biochemicals, Tokyo, Japan) were used as size markers.

For quantitative analysis of mRNAs for RANKL/ODF, OPG, M-CSF, and GAPDH, the products of PCR were blotted onto nylon membranes (MagnaGraph; Micron Separation Inc., Westborough, MA) after electrophoresis. Blots were prehybridized at 42 C for 2 h in 6 x SSPE (1 x SSPE consists of 0.15 M NaCl, 8.65 mM NaH2PO4·2H2O, and 1.25 mM EDTA, pH 7.4) that contained 2 x Denhardt’s solution (1 x Denhardt’s solution consists of 0.1% each of BSA, polyvinylpyrrolidone, and Ficoll), 50% formamide, 100 µg herring sperm DNA, and 0.5% SDS. Then blots were allowed to hybridize, at 42 C for 16 h, in the same solution with a 32P-labeled cDNA probe specific for RANKL/ODF, OPG, M-CSF or GAPDH at 106 cpm/ml. Blots were washed twice in 0.1 x SSC and 0.1% SDS at 60 C for 1 h. Washed blots were analyzed with a Bioimage Analyzer (BAS 2000; Fuji Photo Film Co., Ltd., Tokyo, Japan).

For analysis of stability of RANKL/ODF mRNA, ST2 cells were cultured for 2 days, with or without 50 µg/ml ascorbic acid, before the addition of 5 µg/ml actinomycin D, for the indicated times. Total RNA was extracted from ascorbic acid-treated and untreated cultures. RNA was analyzed by PCR and Southern blot analysis with 32P-labeled cDNA for RANKL/ODF as probe. The stability of mRNA for GAPDH is also shown and was used to normalize the results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1Go shows that TRAP-positive mononuclear and multinucleated cells were formed in response to the addition of ascorbic acid to the medium, and the effect of ascorbic acid was dose-dependent. Induction of formation of TRAP-positive OCLs was observed upon inclusion of ascorbic acid, at 1–5 µg/ml, in the medium. The effects of ascorbic acid on osteoclastogenesis could reproduce by using mouse calvarial primary cells instead of ST2 cells. The effects of the slowly hydrolyzable form 2-P-ascorbic acid (Wako Pure Chemical Industries, Ltd.) on the formation of OCLs was also similar to those of ascorbic acid.



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  		Figure 1. Formation of TRAP-positive OCLs requires ascorbic acid. Mouse bone marrow cells and ST2 cells were cocultured in 48-well plates for 8 days in ascorbic acid-free {alpha}-MEM that contained 10% FBS and 10-8 M 1{alpha},25(OH)2D3, plus ascorbic acid at the indicated concentrations. Fresh medium was supplied every 3 days. The cells were stained for TRAP activity. TRAP-positive mononuclear cells (A) and TRAP-positive multinucleated cells (B) were counted under a microscope. Data are means ± SE of results from three determinations. The photographs in C show typical staining for TRAP of OCLs in cocultures of cells, grown in the presence and in the absence of 50 µg/ml ascorbic acid, for 8 days. Arrows and asterisks indicate TRAP-positive mononuclear cells and TRAP-positive multinucleated cells, respectively. Bars, 100 µm.

 
We examined the expression of the mRNA for RANKL/ODF in ST2 cells that had been treated with 50 µg/ml ascorbic acid, in the presence and in the absence of 10-8 M 1{alpha},25(OH)2D3, an inducer of RANKL/ODF, by semiquantitative RT-PCR. We used this technique because of the relatively low levels of this mRNA in ST2 cells. As shown in Fig. 2AGo, no mRNA for RANKL/ODF was detectable in ST2 cells grown in the absence of 1{alpha},25(OH)2D3. By contrast, 1{alpha},25(OH)2D3 markedly increased the level of mRNA for RANKL/ODF. Furthermore, the combination of ascorbic acid and 1{alpha},25(OH)2D3 increased the level of mRNA for RANKL/ODF to 4.7 times the level obtained with 1{alpha},25(OH)2D3 alone. The level of mRNA for OPG also doubled after treatment of cells with 50 µg/ml ascorbic acid (Fig. 2BGo). However, ascorbic acid had no effect on the level of mRNA for M-CSF in ST2 cells (Fig. 2BGo).



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  		Figure 2. The effects of ascorbic acid on the expression of mRNAs for RANKL/ODF, OPG, and M-CSF in ST2 cells. Total RNA was isolated from ST2 cells that had been cultured for 3 days in the presence and in the absence of 10-8 M 1{alpha},25(OH)2D3 (VD3) and 50 µg/ml ascorbic acid, as indicated (A). We also examined the effects of ascorbic acid on the expression of mRNAs for RANKL/ODF, OPG, and M-CSF in the presence of 1{alpha},25(OH)2D3 on day 2 (B). The products of RT-PCR were subjected to electrophoresis in a 2% agarose gel and were allowed to hybridize with 32P-labeled cDNAs for RANKL/ODF, OPG, M-CSF, and GAPDH. The results shown are representative of the results of four experiments.

 
To examine whether the effects of ascorbic acid on the level of mRNA for RANKL/ODF were attributable to enhanced stability of the transcript, we examined the half-life of the mRNA for RANKL/ODF in ST2 cells in which transcription was inhibited. ST2 cells were incubated for 2 days in medium with and without 50 µg/ml ascorbic acid. Then cells were incubated with 5 µg/ml actinomycin D for 0, 3, 6, and 12 h. There were no consistent or significant differences in the half-life of the mRNA for RANKL/ODF between ascorbic acid-treated and untreated cells (Fig. 3Go).



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  		Figure 3. Effects of ascorbic acid on the stability of the mRNA for RANKL/ODF in ST2 cells in which transcription was blocked by actinomycin D. ST2 cells were cultured for 2 days, with or without 50 µg/ml ascorbic acid, before the addition of 5 µg/ml actinomycin D for the indicated times. Total RNA was extracted from ascorbic acid-treated and untreated cultures. RNA was analyzed by PCR and Southern blot analysis with 32P-labeled cDNA for RANKL/ODF as probe (–{circ}–, untreated cells; –•–, ascorbic acid-treated cells). The inset shows results of Southern blot analysis of PCR products obtained after treatment with actinomycin D for 0, 3, 6, and 12 h. The stability of mRNA for GAPDH is also shown and was used to normalize the results.

 
Ascorbic acid is required for the formation of the collagen matrix. We reported previously that ascorbic acid induces the osteoblastic differentiation of ST2 cells by inducing the synthesis of the collagen matrix (17). Therefore, we examined the effects of inhibitors of the formation of the collagen matrix, such as the proline analog DHP and a structural analog of {alpha}-ketoglutarate and ascorbic acid, EDHB (18), on the induction by ascorbic acid of the formation of OCLs (Fig. 4Go) and on the effect of ascorbic acid on the level of mRNA for RANKL/ODF in ST2 cells (Fig. 5Go). As shown in Fig. 4Go, DHP and EDHB inhibited the ascorbic acid-induced formation of TRAP-positive OCLs in a dose-dependent manner. DHP at 0.5 mM and EDHB at 0.05 mM each completely inhibited the formation of TRAP-positive OCLs. The ascorbic acid-induced increase in the level of the mRNA for RANKL/ODF was also prevented by 0.5 mM DHP and by 0.05 mM EDHB (Fig. 5Go).



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  		Figure 4. Inhibition of the ascorbic acid-induced formation of OCLs acid by inhibitors of formation of the collagen matrix. Mouse bone marrow cells and ST2 cells were cocultured in 48-well plates for 8 days in {alpha}-MEM that contained 10% FBS, 10-8 M 1{alpha},25(OH)2D3, 50 µg/ml ascorbic acid, and either DHP (A) or EDHB (B) at the indicated concentrations. Fresh medium was supplied every 3 days. The cells were stained for TRAP activity. TRAP-positive mononuclear cells and TRAP-positive multinucleated cells were counted under a microscope. Data are means ± SD of results from three determinations.

 


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  		Figure 5. Inhibition of the expression of mRNA for RANKL/ODF by inhibitors of formation of the collagen matrix. Total RNA was isolated from ST2 cells that had been cultured for 2 days in {alpha}-MEM that contained 10% FBS, 10-8 M 1{alpha},25(OH)2D3, with and without 50 µg/ml ascorbic acid, and with and without DHP or EDHB. The products of RT-PCR were subjected to electrophoresis in a 2% agarose gel and were allowed to hybridize with 32P-labeled cDNA for RANKL/ODF. Results are representative of results of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results suggest that ascorbic acid might induce the formation of OCLs in cocultures of mouse bone marrow cells and ST2 cells through its effects on the synthesis of RANKL/ODF in ST2 cells. Usually, cocultures for the formation of TRAP-positive OCLs are maintained in {alpha}-MEM, which contains 50 µg/ml ascorbic acid. As a result, the potential importance of ascorbic acid in osteoclastogenesis has been overlooked. We found that ascorbic acid can induce the formation of TRAP-positive OCLs at concentrations from 0.5–5 µg/ml. The normal concentration of ascorbic acid in human plasma ranges from approximately 5–10 µg/ml. Therefore, our results suggest that the effect of ascorbic acid on osteoclastogenesis might be physiological.

RANKL/ODF is a membrane-bound protein that is expressed on osteoblast/osteogenic stromal cells (e.g. ST2 cells) and that induces osteoclastogenesis in combination with M-CSF (5, 6). Antibodies against RANKL/ODF completely abolish the 1{alpha},25(OH)2D3-induced resorption of bone (7). Conversely, 1{alpha},25(OH)2D3 raises the level of mRNA for RANKL/ODF (6, 7, 19). These results suggest that the stimulation of osteoclastogenesis by 1{alpha},25(OH)2D3 might occur via enhancement of the expression of RANKL/ODF. However, these effects of 1{alpha},25(OH)2D3 were recognized when cells were cultured in medium that contained ascorbic acid. In this study, 1{alpha},25(OH)2D3 in the absence of ascorbic acid and, similarly, ascorbic acid in the absence of 1{alpha},25(OH)2D3 were unable to induce the formation of TRAP-positive multinucleated cells in a coculture system. By contrast, the combination of ascorbic acid and 1{alpha},25(OH)2D3 resulted in the formation of TRAP-positive OCLs. Furthermore, the combination of ascorbic acid and 1{alpha},25(OH)2D3 increased the level of mRNA for RANKL/ODF to 4.7 times the level obtained with 1{alpha},25(OH)2D3 alone. All our findings together suggest that a high level of mRNA for RANKL/ODF, expressed in response to exposure to both ascorbic acid and 1{alpha},25(OH)2D3, might promote the formation of OCLs in our coculture system.

The RANKL/ODF and OPG produced by osteoblast/osteogenic stromal cells are key extracellular regulators of osteoclastogenesis (5, 6). OPG, which is a decoy receptor for RANKL/ODF, inhibits formation of OCLs both in vivo and in vitro. The ratio of levels of RANKL/ODF and OPG in the microenvironment is critical in the regulation of the formation of OCLs. In the present study, the level of mRNA for OPG doubled after treatment of ST2 cells with ascorbic acid at 50 µg/ml. However, the increase in the level (4.7-fold) of the mRNA for RANKL/ODF induced by ascorbic acid was considerably greater than that (2-fold) in the level of the mRNA for OPG. Thus, coculture in the presence of ascorbic acid might be expected to result in the formation of OCLs.

Ascorbic acid did not affect the stability of the mRNA for RANKL/ODF. Our results suggest that ascorbic acid might regulate osteoclast differentiation via induction of the synthesis of the mRNA for RANKL/ODF in ST2 cells. However, the mechanism by which ascorbic acid controls the expression of the mRNA for RANKL/ODF by ascorbic acid remains unclear. Ascorbic acid is necessary for the expression of osteoblastic markers and for mineralization in a variety of osteoblast culture systems, acting via induction of the formation of a collagen-containing extracellular matrix (20, 21, 22, 23, 24, 25, 26). We found previously that ascorbic acid induces the differentiation of ST2 cells into osteoblast-like cells (OCLs) (17). We also showed that the formation of a collagen matrix, in response to ascorbic acid, is essential for the differentiation of ST2 cells (17). Type I collagen is detected in the extracellular matrix of ST2 cells in response to ascorbic acid (data not shown). Therefore, we postulated that ascorbic acid might induce ST2 cells to proceed to a particular stage of differentiation through the formation of a collagen-containing extracellular matrix that has the capacity to support the differentiation of osteoclasts. We examined the effects of the collagen-containing extracellular matrix on the formation of OCLs, using two inhibitors of the formation of the collagen matrix, namely, the proline analog DHP and a structural analog of {alpha}-ketoglutarate and ascorbic acid, EDHB. Both compounds inhibited the formation of OCLs in cocultures, as well as the expression of the mRNA for RANKL/ODF in ST2 cells. These results suggest that induction of the formation of the collagen matrix by ascorbic acid might be essential for the formation of OCLs that involves the expression of RANKL/ODF. Type I collagen interacts with various types of cells, through binding to {alpha}2ß1-integrin (27). Interactions between collagen and integrin induce the osteoblastic differentiation of ST2 cells (17) and MC3T3-E1 cells (25, 26). It seems possible that the formation of OCLs induced by ascorbic acid might be mediated by signal transduction pathways that involve collagen and integrin. In this context, it is of interest to note that the interaction between type I collagen and {alpha}2-integrin in mouse preosteoblastic MC3T3-E1 cells stimulates the binding of Cbfa1 to DNA without affecting levels of Cbfa1 mRNA (28). Cbfa1 is a mammalian transcription factor related to the runt protein of Drosophila. There is, moreover, a consensus binding site (AACCACT) for Cbfa1 in the 5'-flanking region of the mouse gene for RANKL/ODF (29). Furthermore, mRNA for RANKL/ODF is undetectable in calvarial cells from Cbfa1-deficient mice (30). All these findings together suggest that ascorbic acid might induce the formation of OCLs, at least in part, by regulating the expression of RANKL/ODF. This regulation involves regulation of the formation of the collagen matrix by ascorbic acid, which is supposedly followed by the activation of Cbfa1. Further investigations, including measurements of levels of RANKL/ODF itself during osteoclastogenesis, are needed to validate such a suggestion.

In summary, we have shown that ascorbic acid is essential for osteoclastogenesis and induces the formation of OCLs through the expression of RANKL/ODF and, moreover, that ascorbic acid acts in cooperation with 1{alpha},25(OH)2D3. Our findings suggest that ascorbic acid might play a key role in the regulation of the balance, in terms of differentiation and activation, between osteoclasts and osteoblasts.


   Acknowledgments
 
The authors thank Dr. Atsuto Inoue (Tokyo Institute of Technology) for helpful discussions.


   Footnotes
 
1 This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and by grants from the Smoking Research Foundation and the Ground Research Announcement for the Space Utilization, promoted by NASDA and Japan Space Forum. Back

Received December 23, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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