The Journal of Cell Biology
The Journal of Cell Biology, Volume 160, Number 4, February 17, 2003 487–493
The Rockefeller University Press, 0021-9525/2003/02/487/7 $8.00
Cyclic AMP induces integrin-mediated cell adhesion
through Epac and Rap1 upon stimulation of the
and Johannes L. Bos
Jorrit M. Enserink,
H. Bea Kuiperij,
Johan de Rooij,
Leo S. Price,
Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht,
Universiteitsweg 100, 3584 CG Utrecht, Netherlands
BIOLOG Life Science Institute, D-2807 Bremen, Germany
AMP controls many cellular processes mainly through
the activation of protein kinase A (PKA). However, more
recently PKA-independent pathways have been established
through the exchange protein directly activated by cAMP
(Epac), a guanine nucleotide exchange factor for the small
GTPases Rap1 and Rap2. In this report, we show that
cAMP can induce integrin-mediated cell adhesion through
Epac and Rap1. Indeed, when Ovcar3 cells were treated
with cAMP, cells adhered more rapidly to fibronectin. This
cAMP effect was insensitive to the PKA inhibitor H-89. A
similar increase was observed when the cells were trans-
fected with Epac. Both the cAMP effect and the Epac effect
on cell adhesion were abolished by the expression of
Rap1–GTPase-activating protein, indicating the involvement
of Rap1 in the signaling pathway. Importantly, a recently
characterized cAMP analogue, 8-(4-chloro-phenylthio)-2
-cyclic monophosphate, which spe-
cifically activates Epac but not PKA, induced Rap-dependent
cell adhesion. Finally, we demonstrate that external stimuli
of cAMP signaling, i.e., isoproterenol, which activates the
-adrenergic receptor can induce integrin-
mediated cell adhesion through the Epac-Rap1 pathway.
From these results we conclude that cAMP mediates receptor-
induced integrin-mediated cell adhesion to fibronectin
through the Epac-Rap1 signaling pathway.
cAMP is a common second messenger controlling many
cellular processes. Protein kinase A (PKA)* is a general receptor
for cAMP, resulting in the phosphorylation of a large variety
of cellular targets. Specificity is regulated by A kinase anchor-
ing proteins that target PKA to specific regions in the cell. A
few years ago we discovered an additional cAMP target,
exchange protein directly activated by cAMP (Epac)1. This
protein and its close relative Epac2 contain cAMP-binding
domains very similar to the cAMP-binding domains in the
regulatory subunit of PKA and are exchange factors of the
small GTPases Rap1 and Rap2 (de Rooij et al., 1998, 2000;
Kawasaki et al., 1998).
Rap1 is a GTPase of the Ras superfamily, which functions
as a molecular “switch,” cycling between inactive GDP- and
active GTP-bound forms. Specific guanine nucleotide exchange
factors are the “on switches,” and GTPase-activating proteins
(GAPs) are the “off switches” (for review see Bos et al.,
2001). Rap1 was initially identified in a screen for proteins
that can suppress the transformed phenotype of fibroblasts
transformed by oncogenic K-Ras (Kitayama et al., 1989),
providing a model in which Rap1 functions as an antagonist
of Ras signaling mainly by trapping Ras effectors (Raf-1) in
an inactive complex. However, from numerous reports accu-
mulated so far it is evident that Rap1 signaling is important
in itself and independently of Ras regulates several important
cellular processes (Bos et al., 2001). One of the most consistent
findings is the involvement of Rap1 in integrin-mediated
S. Rangarajan and J.M. Enserink contributed equally to this work.
Address correspondence to Johannes L. Bos, Dept. of Physiological
Chemistry and Centre for Biomedical Genetics, University Medical Center
Utrecht, Universiteitsweg 100, 3584 CG Utrecht, Netherlands. Tel.: 31-
30-2538977. Fax: 31-30-2539035. E-mail: J.L.Bos@med.uu.nl
*Abbreviations used in this paper: CMV, cytomegalovirus; 8CPT-
monophosphate; Epac, exchange protein directly activated by cAMP;
GAP, GTPase-activating protein; PKA, protein kinase A; RalGDS, Ral
guanine nucleotide dissociation stimulator; RBD, ras-binding domain;
RGD, arginine, glycine, aspartic acid; TK, thymidine kinase.
Key words: integrins; cyclic nucleotides; GTPases; guanine nucleotide
exchange factor; cell adhesion
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488 The Journal of Cell Biology
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cell adhesion (Caron et al., 2000; Katagiri et al., 2000;
Reedquist et al., 2000; Arai et al., 2001; Ohba et al., 2001;
de Bruyn et al., 2002; Sebzda et al., 2002). Integrins are het-
erodimeric cell adhesion molecules consisting of one of sev-
chains and one of at least five different
chains. One of the first indications was that in 32D cells
granulocyte colony stimulating factor-induced cell adhesion
could be abolished by the introduction of Spa1, a GAP for
Rap proteins (Tsukamoto et al., 1999). This finding was fol-
lowed by three independent observations showing a role for
Rap1 in the inside-out signaling to integrins. First, in Jurkat
cells introduction of Rap1 induced integrin
mediated adhesion to the intercellular adhesion molecule.
Importantly, adhesion induced by ligation of the T cell re-
ceptor was inhibited by introduction of an interfering mu-
tant of Rap1 (Katagiri et al., 2000). Second, also in Jurkat
cells ligation of the adhesion molecule CD-31 induced acti-
2, which was inhibited by blocking Rap1 sig-
naling (Reedquist et al., 2000). Finally, in a macrophage cell
line, complement-mediated phagocytosis, which requires ac-
2, was abolished by inhibition of Rap1 signal-
ing (Caron et al., 2000). Other studies reached the same
conclusion for integrins with a
al., 2001) and for integrins with a
(Bertoni et al., 2002). Recently, it was shown that in mice
expressing active Rap1 in their T cell compartment both the
thymocytes and mature T cells exhibited increased integrin-
mediated cell adhesion. In addition, these cells showed en-
hanced T cell receptor–mediated responses (Sebzda et al.,
1 chain, i.e.,
3 chain, i.e.,
1 (Arai et
2002). From the above results we hypothesized that cAMP
or signals that raise cAMP levels may regulate integrin-medi-
ated cell adhesion through Epac and Rap1. We have tested
this model and found that indeed cAMP was able to induce
integrin-mediated cell adhesion to fibronectin.
It has been reported previously that PKA was a key part in
a signaling pathway activated by mAb 12G10, an antibody
that can activate
1 integrins and induce integrin-mediated
cell–cell and cell–substrate adhesion in human fibrosarcoma
cells (Whittard and Akiyama, 2001). To distinguish which
of the two independent cAMP signaling pathways, mediated
by either PKA or Epac, is involved in integrin regulation in
Ovcar3 cells, we used a novel analogue of cAMP, 8-(4-
phosphate (8CPT-2Me-cAMP) that specifically targets Epac
and not PKA. We demonstrate that an Epac-Rap1 pathway
mediates this effect independently of PKA. We also impli-
cate physiological consequences of intracellular increases in
cAMP in vivo by demonstrating that agonist stimulation of
-adrenergic receptor is linked to increased cell adhe-
sion via Rap1.
Results and discussion
To investigate whether cAMP could induce integrin-
mediated cell adhesion, we used ovarian carcinoma cells
(Ovcar3), since these cells express the
chains 1–6 and
integrins mediating binding to fibronectin (Cannistra
, with the
1 integrin chain in
fibronectin in a PKA-independent manner.
(A) Treatment with 8-Br-cAMP induces adhesion to
fibronectin. Ovcar3 cells were transiently transfected
with CMV-luciferase plasmid, and cells adhering
to fibronectin (2 ?g/ml) in the presence of increasing
concentrations of 8-Br-cAMP were quantified as
described in Materials and methods. (B) 8-Br-cAMP
induces Rap1 activation. (Top) Ovcar3 cells were
treated with increasing concentrations 8-Br-cAMP
for 15 min. Cells were lysed, and equal amounts of
cell lysate were analyzed for activation of Rap1
(top blot) and CREB (bottom blot). Total levels of
Rap1 in cell lysates are shown (middle blot).
(Bottom) Ovcar3 cells were treated with 1 mM
8-Br-cAMP for the indicated times. Cells were
lysed as above and analyzed for activation of Rap1
(top blot) and CREB (bottom blot). Total Rap1
levels are shown (middle blot). (C) 8-Br-cAMP–
induced adhesion is independent of PKA. Ovcar3
cells transiently transfected with CMV-luciferase
plasmid were either preincubated at 37?C for 30
min with the PKA inhibitor H-89 (10 ?M) 30 min
before seeding onto the wells (Short), or H-89 was
added 30 min before trypsinization and during the
recovery period (Long) and seeded onto wells with
or without 8-Br-cAMP. Cells were allowed to adhere
for 1 h, and nonadherent cells were removed. The
percentage of adherent cells was quantified and plotted relative to unstimulated cells (range from 2–10%). The plot shown is representative
of two (long pretreatment) and five (short pretreatment) experiments each in triplicate. Error bars represent SD. (D) Activation of CREB and
ERK but not Rap1 is blocked by H-89. Ovcar3 cells were pretreated with either H-89 or carrier for 30 min followed by stimulation with
8-Br-cAMP for 15 min. Cells were lysed, and equal amounts of cell lysates were incubated with precoupled GST-RalGDS-RBD, and activation
of Rap1 was analyzed by immunoblotting using a Rap1 antibody. Phosphorylation of CREB and ERK was assayed by Western blotting using
cAMP induces cell adhesion to
The Journal of Cell Biology
A cAMP-Epac-Rap1 pathway induces cell adhesion |
Rangarajan et al. 489
et al., 1995; Buczek-Thomas et al., 1998). Cytomegalovirus-
luciferase–transfected cells were detached with trypsin and
allowed to reexpress cell surface markers. The cells were
seeded onto fibronectin-coated multiwell plates in the pres-
ence or absence of 8-Br-cAMP, and the amount of cells that
adhered after a certain period of time was quantified. We
observed that 8-Br-cAMP augmented cell adhesion and acti-
vated Rap1 in a concentration-dependent manner to fi-
0.2–0.5 mM) (Fig. 1, A and B). Rap1
was activated rapidly and remained active for at least 3 h.
8-Br-cAMP–induced adhesion was also observed using a dif-
ferent promoter (thymidine kinase [TK]–luciferase) driving
luciferase expression and a direct method of measuring adhe-
sion by counting cells (unpublished data). Cell adhesion in-
duced by cAMP was insensitive to the PKA inhibitor H-89
when cells were pretreated for a short time just before adhe-
sion (Fig. 1 C, Short). It has been reported that detachment
of cells rapidly and transiently activates PKA, one of the
well-established targets of cAMP (Howe and Juliano, 2000),
raising the possibility that if a potential PKA substrate with a
sustained phosphorylation profile was involved, addition of
H-89 at a later time (post-PKA activation) may falsely imply
a PKA-independent mechanism. However, when cells were
treated with H-89 before trypsinization and throughout the
recovery period, we found that cAMP-induced adhesion was
not blocked (Fig. 1 C, Long), indicating that indeed PKA
was not involved. As a control for H-89 activity, we mea-
sured cAMP-induced phosphorylation of the direct PKA
target CREB (Gonzalez and Montminy, 1989) and ERK,
which is also PKA-dependent (Fig. 1 D). Activation of
Rap1, which is independent of PKA (de Rooij et al., 1998;
Kawasaki et al., 1998; Enserink et al., 2002) was measured
also. From these results we conclude that in Ovcar3 cells
cAMP can induce cell adhesion to fibronectin indepen-
dently of PKA.
Our finding that the induction of integrin-mediated cell
adhesion by cAMP is independent of PKA suggested that
Epac-Rap1 might be mediating this effect. To further test
this idea, Ovcar3 cells were transiently transfected with
Epac1. This resulted in an increase in basal adhesion to fi-
bronectin, which was further increased by stimulation with
8-Br-cAMP (Fig. 2 A), suggesting that Epac mediates cAMP-
cAMP-induced cell adhesion, which is Rap1GAPII
sensitive. (A) 8-Br-cAMP-Epac1–induced cell
adhesion is blocked by Rap1GAPII. Ovcar3 cells
were transiently transfected with TK-luciferase
plasmid and either mock DNA (vector), HA-tagged
Epac1, or HA-tagged Rap1GAPII where indicated.
Cells were stimulated with 8-Br-cAMP, and adhesion
of cells to fibronectin was quantified. The percentage
of adherent cells was plotted relative to unstimulated,
mock-transfected cells. Representative data
performed in triplicate are shown, and error bars
represent SD. The experiments were repeated at
least four times with identical results. (B) 8CPT-2Me-
cAMP-Epac1–induced cell adhesion is blocked by
Rap1GAPII. Ovcar3 cell were transiently transfected
as above. Cells were stimulated with 8CPT-2Me-
cAMP, and adhesion of cells to fibronectin was
quantified. The percentage of adherent cells was
plotted relative to unstimulated, mock-transfected
cells. Representative data performed in triplicate
are shown, and error bars represent SD. The
experiments were repeated at least four times
with identical results. (C) 8CPT-2Me-cAMP-Epac1–
induced Rap1 activation is blocked by Rap1GAPII.
Cells were treated with 50 ?M 8CPT-2Me-cAMP
for 15 min, lysed, and GTP-bound Rap1 levels
were determined as described in Materials and
methods (top). Rap1 protein levels were equal
(middle), and expression of transfected proteins
was confirmed with an anti-HA antibody (bottom).
(D) A ?1-integrin–blocking peptide containing the
RGD sequence present in fibronectin inhibits
8CPT-2Me-cAMP–induced cell adhesion. Ovcar3
cells were pretreated for 20 min with RGD peptide
(100 ?M) where indicated and seeded in wells
with or without 8CPT-2Me-cAMP. Cells were
allowed to adhere for 1 h, and nonadherent cells
were removed. The percentage of adherent cells
was measured and plotted relative to unstimulated
cells. Representative data from two experiments
Overexpression of Epac1 increases
performed in triplicate are shown with error bars representing SD. (E) 8CPT-2Me-cAMP does not increase the rate of cell adhesion to poly-
L-lysine. Ovcar3 cells were transfected with CMV-luciferase and seeded onto poly-L-lysine–coated plates. At various time points, nonadher-
ent cells were removed and adherent cells were quantified. A representative experiment in triplicate is shown.
The Journal of Cell Biology
490 The Journal of Cell Biology
Volume 160, Number 4, 2003
induced cell adhesion. This observation was further strength-
ened by the introduction of Rap1GAPII, an inhibitor of
Rap1 (Mochizuki et al., 1999), which attenuated Epac-
induced cell adhesion (Fig. 2 A). These results show that ec-
topic expression of Epac is sufficient to induce Rap1-depen-
dent cell adhesion to fibronectin, which can be enhanced by
additional stimulation with cAMP. It should be noted that
although Rap1GAPII is more effective on Rap1 than on
Rap2, we cannot exclude a role for Rap2 in this process.
To formally exclude the possibility that cAMP and Epac
are on parallel pathways, both of which would be required
for the induction of cell adhesion, we used a newly charac-
terized analogue of cAMP, 8CPT-2Me-cAMP, which spe-
cifically activates Epac but not PKA even at high concentra-
tions (Enserink et al., 2002). As observed with 8-Br-cAMP,
stimulation of Epac1-transfected cells with 8CPT-2Me-
cAMP further increased cell adhesion to fibronectin (Fig. 2
B) and raised Rap1GTP levels (Fig. 2 C). Expression of
Rap1GAPII inhibited adhesion of cells to fibronectin and
completely abolished Rap1GTP levels (Fig. 2, B and C), in-
dicating that Rap1 is critically involved in cAMP-induced
We next investigated whether activation of endogenous
Epac is sufficient to induce adhesion to fibronectin. Ovcar3
cells were treated with 8CPT-2Me-cAMP to activate endog-
enous Epac, which is abundantly expressed in ovary tissue
cell adhesion via Epac and Rap1.
(A) 8CPT-2Me-cAMP stimulates cell
adhesion. (Top) Ovcar3 cells were
transiently transfected with CMV-
luciferase plasmid and treated with
increasing concentrations of 8CPT-2Me-
cAMP. Cells adhering to fibronectin
(2 ?g/ml) were quantified as described in
Materials and methods. (Bottom) Ovcar3
cells were treated with increasing
concentrations of 8CPT-2Me-cAMP for
15 min, and cells were lysed and
analyzed for activation of Rap1 (top blot)
and CREB (bottom blot). Total Rap1 levels
are shown (middle blot). (B) 8CPT-2Me-
cAMP increases the rate of cell adhesion.
(Top) Ovcar3 cells were transfected with
TK-luciferase and seeded onto fibronectin-
coated plates. At various time points,
nonadherent cells were removed, and
adherent cells were quantified. (Bottom)
cells were treated with 60 ?M 8CPT-
2Me-cAMP for the indicated times. Cells
were lysed, and equal amounts of cell
lysates were analyzed for activation of
Rap1 (top blot) and CREB (bottom blot).
Total levels of Rap1 in cell lysates are
shown (middle blot). (C) Ovcar3 cells
were pretreated with H-89 as described
in the legend to Fig. 1 C and seeded
onto wells in the absence or presence
of 8CPT-2Me-cAMP (100 ?M). Cells
were allowed to adhere for 1 h, and
nonadherent cells were removed.
The percentage of adherent cells was
quantified and plotted relative to
unstimulated cells (range from 2–10%).
The plot shown is representative of
two (long pretreatment) and five (short
pretreatment) experiments, each in
triplicate. Error bars represent SD.
(D) cAMP-induced adhesion to fibronectin
is blocked by inhibitors of Rap1. (Left)
Ovcar3 cells were transiently transfected
with CMV-luciferase and either mock
DNA, increasing concentrations of
HA-Rap1GAP II (0.5, 1, 2, or 6 ?g,
respectively), HA-Rap1GAPI (6 ?g), or HA-RBD of RalGDS (6 ?g), respectively. Cells were treated with 8-Br-cAMP or 8CPT-2Me-cAMP,
and adhesion to fibronectin (5 ?g/ml) was determined and plotted relative to unstimulated, mock-transfected cells. Representative data from
experiments performed in triplicate are shown with error bars representing SD. The experiments were repeated (Rap1GAPII, at least four
times; Rap1GAPI and RBD, twice) with identical results. (Top right) Luciferase counts of total input cells per well in the above experiment are
shown with error bars representing SD of triplicates. (Bottom left panel) Expression of HA-Rap1GAPs in the above experiment is shown.
The Journal of Cell Biology
A cAMP-Epac-Rap1 pathway induces cell adhesion |
Rangarajan et al. 491
(Kawasaki et al., 1998). Indeed, 8CPT-2Me-cAMP signifi-
cantly induced cell adhesion to fibronectin (Fig. 2 D). To
investigate whether cAMP-induced cell adhesion is indeed
mediated by integrins, we pretreated Ovcar3 cells with the
1-integrin–binding arginine, glycine, aspartic acid (RGD)
peptide. Peptides containing the RGD amino acid sequence
motif bind to
1 integrins and have been shown to block fi-
bronectin binding in ovarian carcinoma cells (Buczek-Thomas
et al., 1998). As expected, 8CPT-2Me-cAMP–induced at-
tachment to fibronectin was abolished (Fig. 2 D). 8CPT-
2Me-cAMP did not increase the integrin-independent adhe-
sion of Ovcar3 cells to poly-
-lysine (Fig. 2 E). From these
results we conclude that activation of endogenous Epac in-
duces integrin-mediated cell adhesion to fibronectin.
8CPT-2Me-cAMP enhanced cell adhesion to fibronectin
and induced Rap1 activation at comparable concentrations
M) (Fig. 3 A). In a time-course analysis, we
noted that increased adhesion was already observed after 30
min, which correlated with a rapid and sustained Rap1 acti-
vation (Fig. 3 B). As expected, the induction of adhesion
and activation of Rap1 were insensitive to the PKA inhibitor
H-89 (Fig. 3 C). However, even low levels of Rap1GAPII
completely inhibited cAMP-induced adhesion of Ovcar3
cells to fibronectin (Fig. 3 D, left plot). Furthermore, the
Rap1-inhibitory proteins Rap1GAPI and Ras-binding do-
main (RBD) of Ral guanine nucleotide dissociation stimula-
(RalGDS) (Reedquist et al., 2000) also inhibited adhe-
sion to fibronectin (Fig. 3 D, left plot). Transfection of cells
with Rap1GAPs or RBD of RalGDS did not affect luciferase
expression (Fig. 3 D, right plot).
Our observations that cAMP analogs could induce adhe-
sion of Ovcar3 cells to fibronectin prompted us to test
whether cAMP-elevating receptors could also mirror the
same effect, thereby linking an in vivo cAMP signaling sys-
tem to integrin activation. The
2-AR) couples to G
type of heterotrimeric G proteins,
resulting in elevation of intracellular cAMP levels and subse-
quent activation of PKA and Epac1-Rap1 signaling cascades
(Marinissen and Gutkind, 2001; Neves et al., 2002). Ovcar3
cells endogenously express the
isoproterenol, a ligand for the
increased adhesion to fibronectin (Fig. 4 A). Treatment with
isoproterenol also induced both activation of Rap1 and
phosphorylation of CREB (Fig. 4 B) (EC50 for Rap1 activa-
tion and adhesion,
M). Isoproterenol-induced ad-
hesion was insensitive to short pretreatments with H-89
(Fig. 4 C, Short) but was partially inhibited when exposed
very early to H-89 (Fig. 4 C, Long). Therefore, we looked at
2-AR and stimulation with
2-AR receptor, significantly
with isoproterenol induces cell adhesion.
(A) Isoproterenol induces adhesion to fibronectin.
Ovcar3 cells transiently transfected with CMV-
luciferase plasmid were treated with increasing
concentrations of the ?2-AR agonist isoproterenol,
and cells adhering to fibronectin (2 ?g/ml) were
quantified as described in Materials and methods.
(B) Isoproterenol induces activation of Rap1 and
CREB. (Top) Ovcar3 cells were treated with
increasing concentrations of isoproterenol for 5 min.
Cells were lysed, and equal amounts of cell lysate
were analyzed for activation of Rap1 (top) and
CREB (bottom). Total levels of Rap1 in cell lysates
are shown (middle blot). (Bottom) Cells were
treated with 10 ?M of isoproterenol for the
indicated times. Cells were lysed, and equal
amounts of cell lysate were analyzed for activation
of Rap1 (top blot) and CREB (bottom blot). Total
levels of Rap1 in cell lysates are shown (middle
blot). (C) Isoproterenol-induced adhesion to
fibronectin is independent of PKA. (Top) Ovcar3
cells were pretreated with H-89 as described in the
legend to Fig. 1 C and seeded onto wells in the
absence or presence of isoproterenol (100 ?M).
Cells were allowed to adhere for 1 h, and
nonadherent cells were removed. The percentage
of adherent cells was quantified and plotted
relative to unstimulated cells (range from 2–10%).
The plot shown is representative of two (long
pretreatment) and four (short pretreatment)
Stimulation of the ?2-AR
experiments, each in triplicate. Error bars represent SD. (Bottom) Cells were pretreated with either DMSO or H-89 for 30 min before
trypsinization and during the recovery period (DMSO and long H-89 treatment, respectively) or during the last 30 min of recovery (short
H-89 treatment). Then cells were stimulated with either 50 ?M 8CPT-2Me-cAMP for 10 min or isoproterenol for 2 min, respectively. Cells
were centrifuged, cell pellets were lysed, and equal amounts of cell lysate were incubated with precoupled GST-RalGDS-RBD, and activation of
Rap1 was analyzed on Western blot using a Rap1 antibody. (D) Isoproterenol-induced adhesion to fibronectin is inhibited by Rap1GAPII.
Ovcar3 cells were transfected with either mock DNA (Vector) or HA-Rap1GapII alone or in combination with a ?2-AR expression vector
where indicated. Adhesion of cells to fibronectin in the absence or presence of isoproterenol was quantified. The percentage of adherent
cells was plotted relative to unstimulated, mock-transfected cells (range 5–15%). Summarizing data of four (for the left half of the plot) and
two (for the right half of the plot) independent experiments performed in triplicate are shown with error bars representing SD.
The Journal of Cell Biology
492 The Journal of Cell Biology
Volume 160, Number 4, 2003
activation of Rap1 under similar conditions. We observed
that after early (Fig. 4 C, Long) pretreatment with H-89,
isoproterenol-induced Rap1 activation was clearly inhibited,
whereas 8CPT-2Me-cAMP–induced Rap1 activation was
not (Fig. 4 C, bottom). Since both 8-Br-cAMP–induced
and 8CPT-2Me-cAMP–induced adhesion were not blocked
by H-89 (Fig. 1 C and Fig. 3 C), the effect of very early
treatment of H-89 on
2-AR signaling could likely be at-
tributed to slow recovery and expression of the
the cell surface. This possibility is consistent with the obser-
vation that PKA is involved in vesicle fusion (Morgan et al.,
1993). Transient transfection of Ovcar3 cells with the
AR receptor further enhanced the isoproterenol-induced ad-
hesion to fibronectin, which was sensitive to the Rap1-inac-
tivating protein, Rap1GAPII (Fig. 4 D), showing a critical
involvement of Rap1.
Our results demonstrate a clear connection between cell
surface receptors that induce cAMP, cAMP signaling, and in-
tegrin-mediated cell adhesion and show that this pathway is
independent of PKA but mediated by the cAMP target Epac
and the small GTPase Rap1. This conclusion is based on the
observations that isoproterenol, which raises cAMP levels
through activation of endogenous
integrin-mediated cell adhesion to fibronectin in a Rap1-
dependent, PKA-independent manner. Furthermore, impor-
tantly, a cAMP analogue that specifically activates Epac but
not PKA is also able to induce cell adhesion. However, our
results do not entirely exclude a role for PKA in this process.
Both Rap1 and Rap1GAP are substrates for PKA (Bos et al.,
2001), and thus PKA may modulate the effect of the Epac-
Rap1 signaling pathway on the adhesion process.
This novel function of cAMP was found in Ovcar3, an
ovarian carcinoma cell line that expresses the fibronectin-
1 and in NIH3T3 cells stably trans-
fected with Epac1 (unpublished data). However, the effect
may be more general and may include different cell types ex-
pressing Epac. Epac is particularly highly expressed in ovary,
thyroid, kidney, adrenal gland, and brain (de Rooij et al.,
1998; Kawasaki et al., 1998), and it is expected that the
cAMP-Epac pathway leading to integrin activation may op-
erate particularly in these tissues. In addition, Rap1 is impli-
cated in the activation of a variety of integrins, including
3, and thus the effect may not be restricted
?1 integrins. The regulation of integrin-mediated ad-
hesion plays an important role in many cellular processes in-
cluding cell migration, cell division, and reactions to me-
chanical stress, and cAMP may impinge on these processes
by activation of the Epac-Rap1 pathway. In ovarian cancer
cells, for instance, functional integrins and molecular events
that regulate them are important for invasion into the sub-
mesothelial ECM (Cannistra et al., 1995; Buczek-Thomas
et al., 1998; Strobel and Cannistra, 1999; for review see
Brakebusch et al., 2002).
How Rap1 regulates integrin-mediated cell adhesion re-
mains elusive. Integrin activity is regulated through various
mechanisms, including cell surface expression (change of
number), redistribution at the cell surface (change of avid-
ity), and conformational changes (change of affinity) (Stew-
art and Hogg, 1996; Porter and Hogg, 1998; van Kooyk
and Figdor, 2000; Brakebusch et al., 2002; Sebzda et al.,
2-AR, is able to induce
2002). Studies using activation-specific antibodies show that
Rap1 regulates both avidity and affinity but not cell surface
expression. For instance, in Jurkat cells Rap1 inhibits both
the clustering and the increased affinity of ?L?2 (Katagiri et
al., 2000; Reedquist et al., 2000; Sebzda et al., 2002),
whereas in megakaryocytes Rap1 increases the affinity of
?IIb?3 (Bertoni et al., 2002). In addition, Rap1 is required
for the direct activation of integrins by integrin-activating
antibodies or manganese ions (de Bruyn et al., 2002). Ap-
parently, Rap1 modulates a process before integrin activa-
tion, for instance, the recruitment of an essential cofactor.
Interestingly, it has been reported recently that Rap1 is es-
sential in the formation of adherens junctions, though it is
less clear whether the process involves integrin-mediated sig-
naling (Knox and Brown, 2002).
Materials and methods
Cells, plasmids, and transfections
NIH-OVCAR3 (Ovcar3) cells were maintained at 37?C in RPMI 1640 sup-
plemented with 10% heat-inactivated (30 min at 56?C) FBS and 0.05%
glutamine in the presence of penicillin and streptomycin. Hemagglutinin
(HA)-tagged constructs of Epac1 and Rap1GapII in the PMT2HA expres-
sion vector have been described previously (de Rooij et al., 1998; de
Bruyn et al., 2002). Transient transfection of Ovcar3 cells was performed
using the FuGENE 6 transfection reagent (Roche Diagnostics Corporation)
according to the manufacturer’s procedures using 6 ?g total DNA includ-
ing either a TK-luciferase plasmid (1 ?g) or CMV-luciferase plasmid (0.2
?g) as indicated. Cells were serum starved at least 16 h before stimulation.
Western blotting of protein samples was performed using polyvinylidene
difluoride membranes. Antibodies against dually phosphorylated p42/
44MAPK and phosphorylated CREB (directed against phosphorylated Ser133)
were obtained from Cell Signaling, and antibodies against K-Rev/Rap1 and
polyclonal anti-HA were obtained from Santa Cruz Biotechnology, Inc.
The following inhibitor and stimuli were used at the indicated concentra-
tions: RGD peptide (100 ?M) and H-89 (10 ?M), obtained from Biomol
Research Laboratories Inc. (Plymouth Meeting). Isoproterenol (10 ?M, un-
less indicated otherwise) was obtained from Sigma-Aldrich and 8-Br-cAMP
(1 mM, unless indicated otherwise) and 8CPT-2Me-cAMP (100 ?M, unless
indicated otherwise) was obtained from Biolog Life Science Institute.
24-well plates were coated overnight with fibronectin (Sigma-Aldrich; 1–5
?g/ml as indicated) in sodium bicarbonate buffer (Sigma-Aldrich). Poly-
L-lysine was coated for 2 h at RT (0.1% wt/vol in water), washed twice with
water, and dried overnight. Plates were washed in TSM buffer (20 mM
Tris-HCl, pH 8, 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2) and blocked for
30–45 min at 37?C with 1% BSA/TSM. Transiently transfected Ovcar3
cells, serum starved 16 h before the adhesion assay, were detached by
trypsinization. Cells were centrifuged at 1,500 rpm for 5 min and resus-
pended in serum-free RPMI containing 25 mM Hepes, 0.5% BSA, and 1 g/L
glucose to allow recovery of cell surface markers at 37?C for 1.5–2 h with
gentle rotation in suspension. Cells were centrifuged, counted, and resus-
pended at 3 ? 105 cells/ml in serum-free RPMI with 0.5% BSA. The exper-
iment was performed in triplicates, and to each well 150 ?l of cells was
added to 150 ?l of medium with or without stimulus. In studies with H-89
(10 ?M), cells were either preincubated at 37?C for 30 min with the inhib-
itor before seeding the wells (short pretreatment), or H-89 was added be-
fore trypsinization, during the recovery period, and before seeding wells
(long pretreatment). Cells were allowed to adhere for 1 h at 37?C, and non-
adherent cells were removed by gently washing plates three times with
warmed 0.5% BSA/TSM. Adherent cells were lysed in luciferase lysis
buffer (15% glycerol, 25 mM Tris-phosphate, pH 7.8, 1% Triton X-100, 8
mM MgCl2, 1 mM DTT) at 4?C for 30 min, and units of luciferase activity
were quantified with addition of equal volume of luciferase assay buffer
(25 mM Tris-phosphate, pH 7.8, 8 mM MgCl2, 1 mM DTT, 1 mM ATP, pH
7, 1 mM luciferin) using a luminometer (Lumat LB9507; Berthold Technol-
ogies). Unseeded cells (150 ?l) were lysed separately to determine lu-
The Journal of Cell Biology
A cAMP-Epac-Rap1 pathway induces cell adhesion | Rangarajan et al. 493
ciferase counts in the total input cells. Specific adhesion (%) was deter-
mined (counts in cells bound/counts in total input ? 100) and plotted
either directly or relative to the basal adhesion of HA vector–transfected
cells. Error bars represent average deviation among experiments, and
where representative experiments are depicted error bars represent aver-
age SD within each experiment. The expression of transfected constructs
was confirmed by immunoblotting of total cell lysates.
Rap1 activation assay and phosphorylation of ERK and CREB
Rap1 activation assays were performed as described previously (Franke et
al., 1997; van Triest et al., 2001). Briefly, adherent cells (unless stated oth-
erwise) were serum starved overnight, treated, and lysed in 750 ?l lysis
buffer (10% glycerol, 1% Nonidet P-40, 50 mM Tris-Cl, pH 7.5, 200 mM
NaCl, 2 mM MgCl2, 1 ?M leupeptin, 0.1 ?M aprotinin, 5 mM NaF, 1 mM
NaVO3). Lysates were clarified by centrifugation, and 500 ?l of lysate was
incubated with GST-tagged RBD of RalGDS precoupled to glutathione
beads to specifically pull down the GTP-bound forms of Rap1. Samples
were incubated for 1 h at 4?C while tumbling. Beads were washed four
times in lysis buffer, and remaining fluid was removed with an insulin sy-
ringe. Proteins were eluted with Laemmli sample buffer and analyzed by
SDS-PAGE and Western blotting using Rap1 antibodies (Santa Cruz Bio-
technology, Inc.). To 100 ?l of clarified lysate 25 ?l 5? Laemmli sample
buffer was added, and phosphorylation of ERKs was analyzed by Western
blotting using the phospho-specific antibody against p42/44MAPK. Phos-
phorylation of CREB was analyzed by Western blotting using a phospho-
specific antibody directed against phosphorylated Ser133.
We thank J. Das and M.C. Verhoeven for technical assistance and our col-
leagues for discussions and for critically reading the manuscript.
S. Rangarajan was supported by a grant from the Human Frontiers Sci-
ence Program, J.M. Enserink and H.B. Kuiperij by a grant from the Council
of Earth and Life Sciences of the Netherlands Organization for Scientific
Research, and L.S. Price by a grant from the Dutch Cancer Society.
Submitted: 23 September 2002
Revised: 20 December 2002
Accepted: 20 December 2002
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