First published online 1 April 2003
doi: 10.1242/jcs.00393
Anti-membrane-bound transferrin-like protein antibodies induce cell-shape change and chondrocyte differentiation in the presence or absence of concanavalin A
Ryo Oda1,*,
Ketut Suardita2,*,
Katsumi Fujimoto2,
Haiou Pan2,
Weiqun Yan2,
Atsushi Shimazu3,
Hideaki Shintani1 and
Yukio Kato2,
1 Department of Operative Dentistry, Faculty of Dentistry, Hiroshima University,
1-2-3 Kasumi, Hiroshima 734-8553, Japan
2 Department of Biochemistry, Faculty of Dentistry, Hiroshima University, 1-2-3
Kasumi, Hiroshima 734-8553, Japan
3 Department of Preventive Dentistry, Faculty of Dentistry, Hiroshima
University, 1-2-3 Kasumi, Hiroshima 734-8553, Japan
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Fig. 1. SDS-PAGE and immunoblotting of the chondrocyte membrane treated with
various concentrations of retinoic acid, and the effect of retinoic acid
pretreatment on MTf expression by chondrocyte cultures. (A) Chondrocytes were
exposed or not to 10–6 M retinoic acid 4 days before the end
of incubation. The protein in the crude membrane fraction (6 µg) was
analysed by SDS-PAGE and stained with silver. (B) Chondrocytes were exposed to
retinoic acid at 0 M, 10–8 M, 10–7 M and
10–6 M 4 days before the end of incubation. The protein in
the ConA-bound membrane fraction (2 µg) was analysed by SDS-PAGE and
stained with silver. (C) Chondrocytes were exposed to retinoic acid at
10–6 M 0 hours, 24 hours, 48 hours and 72 hours before the
end of the incubation. The proteins in the ConA-bound fraction (2 µg) were
resolved by SDS-PAGE and stained with silver. (D) Chondrocytes were exposed to
retinoic acid at 10–6 M 0 hours, 24 hours, 48 hours and 72
hours before the end of the incubation. The MTf level in the chondrocyte
cultures was analyzed by immunoblotting. (E) Chondrocytes were exposed, or not
exposed, to retinoic acid at 10–6 M for 4 days (left) and
then incubated in the absence of retinoic acid for 3 days (right). The MTf
level in the chondrocyte cultures was analysed by immunoblotting. (F)
Chondrocytes in confluent cultures were incubated with retinoic acid at
10–6 M for 4 days. The MTf and GAPDH mRNA levels in the
chondrocytes were determined by northern blot analysis.
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Fig. 2. Effects of retinoic acid pretreatment on the shape of cultured chondrocytes
in the presence or absence of ConA. Poorly differentiated chondrocytes were
not exposed to retinoic acid for 4 days and then incubated in the absence (A)
or presence (B) of 5 µg ml–1 ConA for 24 hours. Poorly
differentiated chondrocytes were exposed to retinoic acid at
10–6 M for 4 days and then incubated in the absence (C) or
presence (D) of 5 µg ml–1 ConA for 24 hours. Poorly
differentiated chondrocytes were exposed to retinoic acid at
10–6 for 4 days and then incubated in the absence (E) or
presence (F) of 5 µg ml–1 ConA for 72 hours. Bar, 30
µm.
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Fig. 3. Specificity of anti-MTf antibodies and the appearance of chondrocytes not
exposed (none) or exposed to ConA or ConA plus anti-MTf antibodies for 24
hours. (A) The proteins in the chondrocyte cultures were resolved by SDS-PAGE.
MTf in the samples was analysed by immunoblotting with pAb1 (lane 1), pAb2
(lane 2), pAb3 (lane 3), pAb4 (lane 4) or mAb2 (lane 5). (B) Poorly
differentiated chondrocytes were incubated with 1% control serum in the
absence (control) or presence of 5 µg ml–1 ConA for 24
hours. Alternatively, these cells were incubated with 1% pAb1-4 serum in the
presence of 5 µg ml–1 ConA for 24 hours (ConA+pAb1-4). In
the other studies, these cells were incubated with control mouse IgG (40 µg
ml–1) or mAb2 (10-40 µg ml–1) in the
presence of 5 µg ml–1 ConA for 24 hours. (C) Chondrocytes
were incubated with the F(ab')2 fragment of mAb2 (40 µg
ml–1) in the presence of 5 µg ml–1 ConA
for 24 hours. Bar, 30 µm.
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Fig. 4. Effects of mAb2 on proteoglycan synthesis and DNA synthesis in chondrocyte
cultures, and on DNA synthesis in lymphocyte cultures in the presence or
absence of ConA or sConA. (A-C) Poorly differentiated chondrocytes were
incubated in the absence or presence of 5 µg ml–1 ConA or
10 µg ml–1 sConA with or without mAb2 or control mouse IgG
for 24 hours. Alternatively, these cells were exposed to DBcAMP or insulin in
the presence or absence of mAb2 at 40 µg ml–1. (D)
Lymphocytes were incubated in the presence or absence of 3 µg
ml–1 ConA with or without mAb2 or control IgG at 40 µg
ml–1 for 72 hours. The values are averages ± s.d. for
the four cultures. (A: ConA/sConA versus ConA/sConA + mAb2;
*P<0.01; **P<0.005; ***P<0.0001. B:
*P<0.01; **P<0.001.)
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Fig. 5. Effects of anti-MTf-antibodies on cell shape and proteoglycan synthesis by
chondrocytes in the absence of ConA. (A) Poorly differentiated chondrocytes
were incubated with 1% control serum, 1% pAb2-4, 100 µg
ml–1 control mouse IgG or 100 µg ml–1 IgG
purified from the pAb4 serum for 24 hours. (B) Poorly differentiated
chondrocytes were incubated with the control serum or pAb4 serum at
concentrations of 0.05-1% for 24 hours. (C) Poorly differentiated chondrocytes
were incubated with 1% control serum or 1% pAb4 for 7 days. Bar, 30 µm. The
values are averages ± s.d. for the four cultures.
*P<0.01.
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Fig. 6. Effects of MTf overexpression on chondrogenic differentiation of ATDC5
cells in response to ConA. ConA (5-20 µg ml–1) or sConA
(35 µg ml–1) was added to the cultures in the presence of
5% serum, transferrin and selenite with no other growth factors from day 10,
every four days or every other day, respectively. (A) Rabbit MTf levels in the
cell layer of Pc1 (lane 1), Pc2 (lane 2), MTf1 (lane 3) and MTf5 (lane4) were
analysed by immunoblotting. The proportion of spherical/polygonal chondrocytes
among total cells (B) and the uronic acid content (C) were determined on day
23. (D) The mRNA levels of aggrecan, collagen type IIA, collagen type IIB and
GAPDH were determined on day 23 by RT-PCR and Southern-blot analysis. (E) The
appearance of ATDC5-MTf5 cells exposed to 15 µg ml–1 ConA
in the presence or absence of mAb2 (50 µg ml–1)
(Experiment A) or pAb1 serum (1%) (Experiment B) on day 12. The lectin and/or
antibodies were added to the cultures on day 10. Bar, 30 µm.
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Fig. 7. Effects of MTf overexpression on chondrogenic differentiation of C3H10T1/2
cells in response to ConA. C3H10T1/2 cells were transfected with
MTf-expression or empty vector. (A) MTf levels in the cells transfected with
the empty vector (lane 1) or MTf-expression vector (lane 2) were analyzed by
immunoblotting. (B) ConA (5 µg ml–1) was added to
confluent cultures of these cells for 48 hours. (C) The proportion of
spherical/polygonal/spindle-like cells among total cells was calculated. (D)
The antibody mAb2 or control IgG (100 µg ml–1) was added
to confluent cultures of MTf-overexpressing C3H10T1/2 cells for 4 days. ConA
(5 µg ml–1) was added to these cultures for 48 hours. (E)
The proportion of spherical/polygonal/spindle-like cells among total cells was
calculated. Bar, 30 µm.
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© The Company of Biologists Ltd 2003
