Differential regulation of Rho GTPases by ß1 and ß3 integrins: the role of an extracellular domain of integrin in intracellular signaling
Hui Miao1,
Song Li1,*,
Ying-Li Hu1,
Suli Yuan1,
Yihua Zhao1,
Benjamin P. C. Chen1,
,
Wilma Puzon-McLaughlin2,
Takehiko Tarui2,
John Y.-J. Shyy1,
,
Yoshikazu Takada2,
Shunichi Usami1 and
Shu Chien1,¶
1 Department of Bioengineering and The Whitaker Institute of Biomedical
Engineering, University of California, San Diego, La Jolla CA 92093-0427,
USA
2 Department of Vascular Biology VB-1, The Scripps Research Institute, La Jolla,
CA 92037, USA
* Present address: Department of Bioengineering, University of California,
Berkeley, Berkeley, CA, USA
Present address: Department of Biomedical Sciences, University of California,
Riverside, Riverside, CA, USA
Present address: Lawrence Berkeley National Laboratory, One Cyclotron Road, MS
74-157, Berkeley, CA, USA
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Fig. 1. Morphological changes in different types of CHO cells after plating on
fibronectin and fibrinogen. CHO (A), ß1 (B), ß3 (C) and ß1-3-1
(D) cells were plated for 4 hours on slides coated with fibronectin. ß3
(E) and ß1-3-1 (F) cells were also plated for 4 hours on slides coated
with fibrinogen. Staining of actin was observed by confocal microscopy. In G,
the percentage of cells with central stress fibers was determined in CHO,
ß1, ß3 and ß1-3-1 cells seeded on fibronectin-coated slides for
2, 4 and 16 hours. Error bars represent standard deviations from three
separate experiments (*P<0.05 compared with CHO
cells).
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Fig. 2. Regulation of Rho activity by cell adhesion to fibronectin. After
starvation of cells in 0.5% serum for 20 hours, CHO (A), ß1 (B), ß3
(C) and ß1-3-1 (D) cells were trypsinized and kept in suspension in DMEM
for 1 hour and then plated on culture dishes coated with fibronectin for
various durations in the absence of serum. The amount of activated RhoA was
determined by RBD pull down assay. The top panels in A-D show RBD-bound RhoA
from cell lysates. The bottom immunoblot shows RhoA in whole cell lysates. For
each cell type, the results of densitometric analysis of RBD-bound RhoA were
normalized by the RhoA in whole cell lysates and plotted as a ratio of the
results (mean±s.d. from three experiments). Asterisks indicate
significant difference (P<0.05) from the ratio of 1. (E) RhoA
activities of CHO, ß1, ß3 and ß1-3-1 cells in suspension,
normalized for whole cell lysates and expressed as a ratio to CHO cells
(mean±s.d. from three experiments). There was no significant difference
among the four groups of cells in suspension.
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Fig. 3. Regulation of Rac1 activity by cell adhesion to fibronectin. 0.5%
serum-starved CHO, ß1, ß3 and ß1-3-1 cells were detached and
kept in suspension in DMEM for 1 hour and then either plated on
fibronectin-coated dishes for 4 hours (A) or used as suspension controls (B).
Rac1 activity was determined by a PBD assay followed by immunoblotting with an
anti-Rac1 antibody. The bottom immunoblot shows Rac1 in whole cell lysates.
The amount of activated Rac1 was normalized by the amount of Rac1 in whole
cell lysates. The bar graphs are densitometric analyses of mean±s.d.
from three separate experiments. The asterisk indicates significant
differences (P<0.05) compared with CHO cells.
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Fig. 4. Regulation of PI 3-kinase and JNK activities by cell adhesion to
fibronectin, and the effect of PI 3-kinase inhibitor on the adhesion induction
of Rac1 activation. 0.5% serum-starved CHO, ß1, ß3 and ß1-3-1
cells were detached and kept in suspension in DMEM for 1 hour and then plated
on fibronectin-coated dishes for 4 hours. In A, cell lysates from the various
samples following adhesion to fibronectin were immunoprecipitated with anti-PI
3-kinase p85 antibody followed by the PI 3-kinase activity assay. Shown at the
bottom is the amount of PI 3-kinase p85 immunoprecipitated from 200 µg cell
lysates, indicating that comparable amounts of PI 3-kinase p85 were
immunoprecipitated in these samples. (B) The effect of a PI 3-kinase
inhibitor, LY294002, on adhesion induction of Rac1 activity is shown in the
top panel. The bottom immunoblot shows Rac1 in whole cell lysates. The amount
of activated Rac1 was normalized by the amount of Rac1 in whole cell lysates.
(C) The top panel shows JNK kinase activity using GST-c-Jun as the substrate,
and the bottom panel is the immunoblot using anti-JNK1, which indicates equal
loading. The bar graphs in A-C are the densitometric analyses, representing
the mean±s.d. of three separate experiments. The asterisks in A and C
indicate significant differences (P<0.05) compared with CHO cells,
and the asterisk in B indicates a significant difference (P<0.05)
compared with cells without the PI 3-kinase inhibitor.
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Fig. 5. Attachment and detachment of cells on fibronectin. (A) Cells were seeded on
fibronectin-coated wells and the percentage of bound cells was quantified by
the adhesion assay described in the Materials and Methods. (B) Cells were
seeded on fibronectin-coated slides and then subjected to a dislodging shear
stress of 250 dyn/cm2. The percentage of detached cells was
quantified. Bars represent mean±s.d. of three separate experiments.
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