First published online 22 July 2003
doi: 10.1242/jcs.00680
VE-cadherin simultaneously stimulates and inhibits cell proliferation by altering cytoskeletal structure and tension
Celeste M. Nelson1 and
Christopher S. Chen1,2,*
1 Department of Biomedical Engineering, Johns Hopkins School of Medicine, 720
Rutland Avenue, Baltimore, MD 21205, USA
2 Department of Oncology, Johns Hopkins School of Medicine, 720 Rutland Avenue,
Baltimore, MD 21205, USA
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Fig. 1. Role of VE-cadherin in cell spreading and proliferation. (A,B) Phase
contrast images of bovine pulmonary artery endothelial cells treated for 96
hours with anti-VE-cadherin (A) and control (B) antibodies. Scale bar: 50
µm. (C) Graph of mean projected area of endothelial cells with time after
treatment with anti-VE-cadherin and control antibodies. (D) Graph of number of
cells after treatment with anti-VE-cadherin and control antibodies, normalized
to number of cells at 24 hours after plating. (E) Flow cytometry diagrams of
cell-cycle distribution of unsynchronized cells (left) and synchronized cells
(right). Quantification of cell-cycle distribution is shown for each
condition. (F) Graph of percentage of cells in S phase (BrdU incorporation)
and mitotic index with time after replating of synchronized cells. (G) Graph
of mean projected area of endothelial cells as a function of seeding density
after treatment with anti-VE-cadherin and control antibodies. (H) Graph of
percentage of endothelial cells entering S phase (incorporating BrdU) as a
function of seeding density after treatment with anti-VE-cadherin and control
antibodies. Error bars represent the s.d., with (*)
P<0.05 relative to controls as calculated by t-test.
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Fig. 2. Effect of cell-cell contact on proliferation when spreading is controlled.
(A) Schematic outline of method used to pattern substrates to control cell
spreading and cell-cell contact simultaneously. (B) Differential interference
contrast images of single cells or pairs of cells in agarose wells of 750
µm2/half (left two images) and 1000 µm2/half
(right two images). Bovine pulmonary artery and adrenal microvascular
endothelial cells were G0-synchronized and cultured on arrays of
wells for 24 hours and fixed for analysis. Cells distributed randomly as
single cells and pairs of cells in the wells. (C) Immunofluorescence images of
pairs of cells in wells of 750 µm2/half (top images) or
monolayers (bottom images) stained for VE-cadherin (VEcad) or ß-catenin
(ßcat). Both VE-cadherin and ß-catenin specifically localized to the
zone of contact. Broken lines (white) indicate the borders of the wells. (D)
Graph of percentage of cells entering S phase (incorporating BrdU) for single
cells and pairs of cells in both sizes of wells. (E) Graph of percentage of
cells entering S phase for cells in wells of 750 µm2/half
treated with VE-cadherin antibody. Similar results were seen with both types
of endothelial cells analyzed. Error bars represent the s.d., with
(*) P<0.05 relative to single cells (D) or controls (E)
as calculated by Student's t-test. (F) Immunofluorescence images of pairs of
cells in wells treated with control (top) and anti-VE-cadherin (bottom)
antibodies and stained for VE-cadherin (VEcad) or ß-catenin (ßcat).
(G) Phase contrast images of endothelial cells treated with control (left) or
anti-VE-cadherin (right) antibodies. Scale bars: 25 µm.
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Fig. 3. Effect of VE-cadherin engagement on endothelial cell proliferation.
Immunofluorescence images of co-cultures of endothelial cells and
VE+ cells (A) and null cells (B) stained for VE-cadherin (VEcad),
connexin 43 (Cx43), PECAM-1 or occludin. Each image shows portions of three
cells within a monolayer, with an endothelial cell in the center contacted by
another endothelial cell on the right and a VE+ cell or null cell
on the left. Open triangles denote location of heterotypic contact; closed
triangles denote location of homotypic endothelial cell contact. (C) Graph of
percentage of endothelial cells entering S phase when co-cultured with null
cells or VE+ cells in wells of 750 µm2/half. Error
bars represent the s.d., with (*) P<0.05 relative to
null cell co-cultures as calculated by Student's t-test.
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Fig. 4. Role of cell spreading in contact-mediated inhibition of proliferation. (A)
Graph of percentage of fully spread cells for single cells and pairs of cells
cultured in wells of 1500 µm2/half, 2000 µm2/half
and 2500 µm2/half (left); percentage of fully spread endothelial
cells when co-cultured with null or VE+ cells in wells of 1500
µm2/half (right). (B) Graph of percentage of cells entering S
phase for single cells and pairs of cells cultured in all three sizes of
wells. (C,D) Graph of percentage of cells entering S phase for single cells
and pairs of cells cultured in all three sizes of wells when cells were
separated into (C) fully or (D) partially spread populations. Error bars
indicate the s.d. of four experiments, with (*) P<0.05
relative to single cells, as calculated by the paired Student's
t-test.
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Fig. 5. Role of MEK and PKC pathways in cell-cell contact-mediated proliferation.
Graphs of percentage of cells in S phase (A-C) and immunofluorescence images
of actin (left) or ß-catenin (right) staining (D-G) in cells on agarose
patterns either treated with U0126 (A,D), Ro-31-7549 (B,E), or H-7 (C,F), or
untreated (G). Scale bar: 25 µm. Actin and ß-catenin images were
acquired on different samples. Error bars indicate s.d. of three experiments,
with (*) P<0.05 relative to untreated controls, as
calculated by Student's t-test.
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Fig. 6. Role of the cytoskeleton in cell-cell contact-mediated proliferation.
Immunofluorescence images of actin (left) or ß-catenin (right) staining
and graphs of percentage of cells in S phase on agarose patterns treated with
cytochalasin D (A-B) or latrunculin B (C-D) to disrupt the actin cytoskeleton
and BDM (E-F) or ML-7 (G-H) to inhibit actomyosin dynamics. Broken lines
(white) indicate the borders of the wells. Scale bar: 25 µm. Actin and
ß-catenin images were acquired on different samples. Error bars indicate
s.d. of three experiments, with (*) P<0.05 relative to
untreated controls, as calculated by Student's t-test.
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Fig. 7. Role of the cytoskeletal regulator Rho in cell-cell contact-mediated
proliferation. Immunofluorescence images of actin (left) or ß-catenin
(right) staining and graphs of percentage of cells in S phase on agarose
patterns treated with Y-27632 (A-B) to inhibit ROCK, or infected with Ad-GFP
control or Ad-RhoN19 (C-D) to inhibit Rho. Broken lines (white) indicate the
borders of the wells. Scale bar: 25 µm. Actin and ß-catenin images
were acquired on different samples. Error bars indicate s.d. of three
experiments, with (*) P<0.05 relative to controls, as
calculated by Student's t-test.
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Fig. 8. Model proposing how VE-cadherin mediates simultaneous opposing signals for
proliferation. VE-cadherin inhibits cell spreading, leading to proliferation
arrest. When cells are already physically constrained, VE-cadherin engagement
leads to an increase in proliferation by signaling through Rho-dependent
changes in cytoskeletal tension.
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© The Company of Biologists Ltd 2003
