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Abstract
In order to investigate the effect of ascorbic acid (AsA) and ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C
derivative, on the growth and differentiation of human osteoblast-like cells, we supplemented the culture medium of MG-63 cells
with various concentrations (0.25 to 1 mM) of these factors. Asc 2-P significantly stimulated nascent cell growth at all concentrations
in the presence of fetal bovine serum (FBS). On the other hand, AsA showed a growth repressive effect depending on its
concentration, and that of FBS. Asc 2-P also increased expression of osteoblast differentiation markers, such as collagen synthesis
and alkaline phosphatase (ALP) activity. These stimulative activities of Asc 2-P were attenuated by inhibitors of collagen synthesis,
indicating that these effects were dependent on collagen synthesis. Electron micrographs of the cells showed the formation of a
three-dimensional tissue-like structure endowed with a mature extracellular matrix in the presence of Asc 2-P.
! 2004 Elsevier Ltd. All rights reserved.
Keywords: Ascorbic acid 2-phosphate; Ascorbic acid; Long-acting vitamin C; Human osteoblasts; Osteoblast differentiation
1. Introduction
Multifarious activities of -ascorbic acid (vitamin C,
AsA) have been demonstrated in many biological systems and in various scientific fields (Burns et al., 1987;
Hata, 1996). One of the essential functions of AsA may
be its role as a cofactor for the hydroxylation of proline
and lysine residues in collagen, which is the most abundant protein in the body (Kielty et al., 1993; Prockop
and Kivirikko, 1995). It is also an essential supplement
for the differentiation of various kinds of cells in culture
(Franceschi, 1992). One difficulty with the handling of
AsA is its instability in solution, especially under the
* Corresponding author. Tel.: +81-46-822-8840;
fax: +81-46-822-8839
E-mail address: ryuhata@kdcnet.ac.jp (R. Hata).
Abbreviations: Asc 2-P, ascorbic acid 2-phosphate; AsA, ascorbic
acid.
1065-6995/$ - see front matter ! 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cellbi.2004.01.010
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Fig. 1. Schematic representation of metabolism of -ascorbic acid 2-Phosphate (Asc 2-P) and -ascorbic acid (AsA). Bold arrows indicate rapid
reactions.
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Fig. 2. Effect of various concentrations of serum, AsA and Asc 2-P on the morphology of MG-63 cells. The cells were plated (1"105/35 mm dish)
and cultured in DMEM-10 for 24 h. Then the culture medium was changed to fresh DMEM containing various concentrations of FBS (0, 1, 10%)
in the absence (control, A, B, C) or presence of AsA (+AsA: 0.25 [D, E, F], 0.5 [G, H, I], or 1.0 mM [J, K, L]), or Asc 2-P (+Asc 2-P; 0.25 [d, e,
f], 0.5 [g, h, I], or 1.0 mM [j, k, l]). Phase-contrast photomicrographs were taken 3 days after the addition of AsA or Asc 2-P. Magnification "200.
and 1% FBS (Fig. 2K). In fact, dose-dependent inhibition of cell growth was indicated by the decrease in
DNA content, when the medium was supplemented with
AsA under serum-free culture conditions (Fig. 3B).
Similar apparent growth inhibition was observed by
supplementation of the culture medium with 1.0 mM
AsA and 1% FBS (Fig. 2K, Fig. 3B). On the other hand,
no apparent growth inhibition was observed when Asc
2-P was the supplement, whether FBS was present or not
(Fig. 2d to i). Instead, significant growth stimulation was
observed by supplementation with 1.0 mM Asc 2-P,
irrespective of the FBS concentration (Fig. 3C). Growth
stimulative effects of Asc 2-P were also observed at
lower concentrations, when FBS was present in the
culture medium (Fig. 3C).
3.2. Effects of AsA and Asc 2-P on ALP activity and
mRNA levels of MG-63 cells
AsA at 0.25 mM, the concentration normally used for
culture, significantly stimulated ALP activity in MG-63
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Fig. 3. Effect of various concentrations of serum, AsA and Asc 2-P on the growth of MG-63 cells. The cells were plated (1"105/35 mm dish) and
cultured in DMEM-10 for 24 h. Then the medium was exchanged for fresh medium containing FBS at 0%, 1%, and 10% without (A, control) or with
AsA (B, +AsA: 0.25, 0.5, 1.0 mM) or Asc 2-P (C, +Asc 2-P: 0.25, 0.5, 1.0 mM). Values are the means of quadruplicate assays$S.D. Significantly
different from respective controls: *P<0.05, **P<0.01.
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Fig. 4. Effects of AsA and Asc 2-P on the growth and ALP activity of MG-63 cells. The cells were plated (1"105/35 mm dish) and cultured in
DMEM-10 in the absence (Control) or presence of AsA (0.25 mM) or Asc 2-P (0.25 mM) for 6 days. The medium was changed 3 days after seeding.
A: DNA content per 35 mm dish. B: ALP activity shown as femtomoles of phenol released per minute per g DNA. Values are the means of
quadruplicate assays $S.D. Significant differences are indicated by brackets: *P<0.05, **P<0.01.
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Fig. 5. RTPCR analysis of ALP mRNA levels of MG-63 cells cultured in the absence or presence of 0.25 mM AsA or Asc 2-P for 7 days. The cells
were plated (1"105/35 mm dish) and cultured in DMEM-10 in the absence (Control) or presence of AsA (0.25 mM) or Asc 2-P (0.25 mM) for
7 days. Total RNAs were prepared and used for RTPCR as described in the text. A: Aliquots of RTPCR product were separated by 2% agarose
gel electrophoresis and visualized by staining with ethidium bromide. B: Relative amounts of ALP mRNA expression were determined using a Fluor
Imager. Values are means$S.D. of triplicate determinations using two different preparations of total RNAs. Significant differences are indicated by
brackets: *P<0.05, **P<0.01.
repressed the cell growth and stimulated the differentiation of these human osteoblast-like cells. We are
interested in this work, because these cells respond to
active vitamin D3 by expressing ALP, an early osteoblast differentiation marker, and osteocalcin, a marker
of terminally differentiated osteoblasts (Franceschi and
Young, 1990; Hata et al., 2002; Lian et al., 1999;
Maehata et al., 2002). When we used Asc 2-P, a longacting ascorbic acid derivative, as a supplement in the
culture medium of MG-63 cells, it stimulated the nascent
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Fig. 6. Effects of Asc 2-P and inhibitors of collagen synthesis on collagen synthesis, cell proliferation and ALP activity of MG-63 cells. The cells were
inoculated (1"105/35 mm dish) and cultured in DMEM-10 in the absence (Control, open bars) or presence of 0.25 mM Asc 2-P. Azetizine
2-carboxylic acid (Az-C, 0.5 or 1.0 mM) or ethyl-3,4-dihydroxybenzoate (EDHBA, 0.2 or 0.4 mM), inhibitors of collagen synthesis were added to
some of the Asc 2-P-containing cultures, and the cells were cultured for a further 7 days. A: Relative rate of collagen synthesis was determined by
a method using purified bacterial collagenases. B: DNA content was determined by fluorometric assay. C: ALP activity indicated as picomoles of
phenol released per minute per dish. D: ALP activity expressed as femtomoles of phenol released per minute per g DNA. Significantly different
from control values: *P<0.05, **P<0.01 and significantly different from Asc 2-P-supplemented cultures, aP<0.05, bP<0.01.
AsA, i.e. its stimulatory effect on growth, and its inhibitory side effects under in vitro culture conditions, where
there is an excess of O2 (Makino et al., 1999; Oda et al.,
2001).
Ascorbyl radicals produced from AsA and H2O2 are
generated by self-reduction of oxidized AsA, due to an
excessive amount of it in the presence of excess O2. In
fact, 10 mM AsA and 0.25 mM H2O2 killed nearly 100%
of MG-63 cells (data not shown). Asc 2-P is resistant to
oxidation by O2 and releases free AsA by the action of
ALP in the cell membranes of most animal cells, including osteoblasts. There was no inhibitory effect on growth
by Asc 2-P up to 10 mM (Figs. 2 and 3, and data not
shown), suggesting that free AsA is formed from Asc 2-P
only at the cell surface and the AsA thus formed is
immediately incorporated by the cells. These data indicate that Asc 2-P is superior to AsA as a supplement
for in vitro cultures, so we used only the former in
subsequent experiments.
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Fig. 7. Effect of Asc2-P on the ultrastructure of MG-63 cells in culture. The cells were cultured in the absence (A and C) or presence (B and D) of
0.25 mM Asc2-P for 6 weeks. For electron microscopy, the dishes were processed as described in the text. The electron photomicrographs are of
cultures sectioned perpendicular to the dish surface. G, Golgi apparatus; M, mitochondrion; rER, rough endoplasmic reticulum. Bars indicate 1 m
(A and B), 200 nm (inserts of A and B), and 500 nm (C and D), respectively.
and ALP activity were also observed in normal osteoblastic cell cultures, even though their relationship was
not examined (Owen et al., 1990).
We have previously observed that Asc 2-P stimulates
the synthesis and maturation of collagen molecules
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