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Originally published In Press as doi:10.1074/jbc.M404599200 on August 9, 2004

J. Biol. Chem., Vol. 279, Issue 43, 44889-44896, October 22, 2004
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The cAMP-Epac-Rap1 Pathway Regulates Cell Spreading and Cell Adhesion to Laminin-5 through the {alpha}3{beta}1 Integrin but Not the {alpha}6{beta}4 Integrin*

Jorrit M. Enserink{ddagger}§, Leo S. Price||, Trond Methi{ddagger}, Milada Mahic{ddagger}, Arnoud Sonnenberg**, Johannes L. Bos||, and Kjetil Taskén{ddagger}{ddagger}{ddagger}

From the {ddagger}Biotechnology Centre of Oslo, University of Oslo, Blindern, N-0317 Oslo, Norway, the ||Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Centre Utrecht, Universiteitsweg 100, 3584CG Utrecht, The Netherlands, and the **Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

Received for publication, April 26, 2004 , and in revised form, August 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Laminin-5 is an important constituent of the basal lamina. The receptors for laminin-5, the integrins {alpha}3{beta}1 and {alpha}6{beta}4, have been associated with epithelial wound migration and carcinoma invasion. The signal transduction mechanisms that regulate these integrins are not well understood. We report here that the small GTPase Rap1 regulates the adhesion of a number of cell lines to various extracellular matrix proteins including laminin-5. cAMP also mediates cell adhesion and spreading on laminin-5, a process that is independent of protein kinase A but rather dependent on Epac1, a cAMP-dependent exchange factor for Rap. Interestingly, although both {alpha}3{beta}1 and {alpha}6{beta}4 mediate adhesion to laminin-5, only {alpha}3{beta}1-dependent adhesion is dependent on Rap1. These results provide evidence for a function of the cAMP-Epac-Rap1 pathway in cell adhesion and spreading on different extracellular matrix proteins. They also define different roles for the laminin-binding integrins in regulated cell adhesion and subsequent cell spreading.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Laminins are extracellular matrix proteins important for epithelial cell migration and adhesion. Laminin-5 (Ln-5),1 a dynamic component of the basement membrane, is a ligand for integrins {alpha}3{beta}1 and {alpha}6{beta}4 (1). {alpha}3{beta}1 couples to the actin cytoskeleton, and its function in epithelial cell adhesion and migration is established (2). Recently, {alpha}6{beta}4 has received much attention, because it was found to function as a signaling adaptor for growth factor-induced invasive growth (3–5). In contrast to all other integrins, {alpha}6{beta}4 couples to the intermediate filament cytoskeleton, and its regulation is likely to be different from other integrins. {alpha}6{beta}4 is a major constituent of hemidesmosomes (6–8), but in aggressive, late stage tumors, {alpha}6{beta}4 relocalizes from hemidesmosomes to membrane protrusions associated with cell migration (9, 10). The upstream factors that regulate adhesion and migration to Ln-5 are not well understood, although cAMP has been shown to regulate the small GTPases RhoA and Rac in a protein kinase A (PKA)-dependent manner, thereby affecting cell migration on Ln-5 (for a review see Ref. 11).

cAMP is a common second messenger that regulates many cellular processes. Until recently, PKA was thought to be the main target of cAMP in eukaryotic cells. However, Epac (exchange factor directly activated by cAMP), a widely expressed exchange factor for the small GTPases Rap1 and Rap2, has been shown to be a receptor for cAMP as well (12, 13). Importantly, Epac controls a number of cellular processes previously attributed to PKA (for a review see Ref. 14). Rap1 has recently attracted much attention, because it was shown to be involved in the regulation of cell adhesion in a variety of cell types (for reviews see Refs. 15 and 16). cAMP also controls cell adhesion in many cell types, and recently a link between cAMP, Epac-Rap1, and regulation of cell adhesion has been established (17). Furthermore, cAMP is known to control cell spreading, which is thus far thought to be mediated by PKA (18).

Although cAMP has previously been shown to regulate specific integrin functions on Ln-5 in a PKA-dependent manner, the involvement of the Epac-Rap1 pathway has not been appreciated in these studies. Therefore, the goal of this research was to test the role of the cAMP-Epac-Rap1 pathway in the regulation of adhesion to Ln-5. Furthermore, because {alpha}3{beta}1 and {alpha}6{beta}4 are regulated by two separate mechanisms, close investigation of Rap1-mediated adhesion to Ln-5 provides an excellent opportunity to gain more insight into how Rap1 may regulate integrins in general. Here we present evidence that cAMP acts as an upstream regulator of cell adhesion and cell spreading to Ln-5 through an Epac-Rap1-dependent pathway. Importantly, we found that although both {alpha}3{beta}1 and {alpha}6{beta}4 mediate adhesion to Ln-5, only {alpha}3{beta}1-dependent adhesion is dependent on Rap1, showing that Rap1 specifically regulates {alpha}3{beta}1 and not {alpha}6{beta}4. This suggests that Rap1 exclusively regulates factors that couple to the actin cytoskeleton, without affecting the intermediate filament cytoskeleton. In addition, these results define different roles for the laminin-binding integrins in cAMP-regulated cell adhesion and subsequent cell spreading.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Constructs, and Transfections—804G and HaCaT cells were maintained in DMEM (4.5 g/liter glucose), and K562 cells, NIH3T3:Ovcar-3 cells and HEK293T cells were maintained in RPMI medium. All cell media were supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 units/ml streptomycin. The cells were transfected with cDNAs for CMV-luciferase (17) or HA-tagged cDNAs in the pMT2-HA expression vector for Rap1V12 (19), Epac1 (13), PDZ-GEF1 (20), or Rap1GAPI (21), using the FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's procedures, whereas K562 cells were transfected by electroporation.

Antibodies and Reagents—Monoclonal antibodies against HA were from Babco, phospho-specific polyclonal antibodies against CREB were from Cell Signaling, function-blocking {alpha}3 antibodies (P1B5, 10 µg/ml) were from Chemicon International, function-blocking antibodies against {alpha}6 integrin (GoH3, 10 µg/ml) were from BD Biosciences, function-blocking antibodies against {beta}1 integrin (AIIB2) were kindly obtained from Dr. C. H. Damsky (UCSF, San Franscisco, CA), and Rap1 antibodies were from Santa Cruz Biotechnology, Inc. H-89 (10 µM) and forskolin (10 µM) were from Calbiochem, isoproterenol (10 µM) was from Sigma, 8-(4-chloro-phenylthio)-2-O-methyladenosine-3,5 cyclic monophosphate (8CPT-2Me-cAMP) (50 µM) was from Biolog Life Science Institute, and phalloidin-TRITC (50 µg/ml) was obtained from Sigma-Aldrich.

Extracellular Matrix Preparation—All cell adhesion assays, except K562 cells, were carried out using the laminin-5-rich matrix of 804G cells, which was prepared as described previously (22). Briefly, 804G cells were plated on 6-well plates or 24-well plates (Costar) and allowed to adhere overnight. After removal of the culture medium, the cells were washed in PBS and removed from their matrix by treatment with 20 mM NH4OH for 10 min at room temperature. The plates were then washed four times with PBS, and complete removal of any residual cells was confirmed by microscopy. Alternatively, the plates were coated overnight at 4 °C with conditioned medium from 804G cells, yielding identical results. The plates were blocked with serum-free DMEM containing 20 mM Hepes, pH 7.4, and 1% fatty acid-free bovine serum albumin (BSA) for 1 h at 4 °C. K562 cell adhesion assays were performed using the laminin-5 matrix of Rac-11P/SD cells as described previously (23). Briefly, the cells were grown in 96-well tissue culture plates for 2 days until confluent. The cells were then detached with 10 mM EDTA overnight at 4 °C. The resulting laminin-5-coated wells were washed three times with PBS and checked under the microscope to ensure that all of the cells had been removed. The coated wells were then blocked with 1% heat-denatured BSA for 1 h at 37 °C. Fibronectin matrix (5 µg/ml) was prepared as previously described (17).

Adhesion Assays—Adhesion assays were performed in triplicate as described previously (17, 24), with minor modifications. Briefly, as indicated, transfected or untransfected adherent cells were trypsinized and recovered in serum-free DMEM containing 20 mM Hepes, pH 7.4, and 1% fatty acid-free BSA for 1.5 h at 37 °C while rocking gently, to allow re-expression of cell surface markers. Serum-starved K562 cells, which grow in suspension, were not trypsinized but centrifuged and resuspended at a concentration of 1 x 106 cells ml-1 and rolled gently at 37 °C for 1 h. In studies with H-89 (10 µM), the cells were preincubated with the inhibitor during the final 40 min of the recovery period, whereas stimuli like forskolin, 8CPT-2Me-cAMP, isoproterenol, and PMA were added during the final 10 min of the recovery period. After the recovery period, the cells (2.5 x 104/well for 24-well plates and 10 x 104/well for 6-well plates in case of adherent cells; 5 x 105 cells/well of 96-well plates (Nunc Maxisorp) in the case of K562 cells) were directly transferred to plates. The cells were allowed to adhere at 37 °C for either 10 min or as indicated otherwise. Subsequently, the nonadherent cells were removed by washing gently but extensively with wash buffer (PBS with 1.8 mM CaCl2 and 2 mM MgCl2) five times. The cells were then lysed in luciferase lysis buffer (15% glycerol, 25 mM Tris-phosphate, pH 7.8, Triton X-100 1%, 8 mM MgCl2, 1 mM dithiothreitol), and luciferase activity (light units) was quantified with the addition of an equal volume of luciferase assay buffer (25 mM Tris-phosphate, pH 7.8, 8 mM MgCl2, 1 mM dithiothreitol, 1 mM ATP, 1 mM luciferin) using a luminometer. Unseeded cells were also measured to determine luciferase activity in the total input of cells. Expression of transfected cDNAs was confirmed by Western blotting. Specific adhesion (%) was determined (light units in cells bound/light units in total input x 100) and plotted relative to the basal adhesion of untreated, HA vector-transfected cells. Alternatively, cell adhesion was measured as follows. After washing the tissue culture plates with PBS, the cells were fixed in 4% formaldehyde in PBS for 30 min at 4 °C. The cells were then permeabilized and blocked in ice-cold TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 100 mM glycine, and 1% fatty acid-free BSA) for 30 min. Subsequently, the cells were incubated with phalloidin-TRITC overnight at 4 °C, washed with TBST, mounted in fluorescence mounting medium (DAKO), and photographed at 100x magnification using a CCD camera mounted on an inverted fluorescence microscope (Olympus IX 81, fitted for tissue culture dishes), after which the cells were counted directly.

Cell Spreading Assay—As indicated, the cells were not transfected or transfected with cDNAs for Rap1GAPI or Rap1V12. GFP was co-transfected to visualize transfected cells. After 24 h, the cells were trypsinized and recovered in serum-free DMEM containing 20 mM Hepes, pH 7.4, and 1% fatty acid-free BSA for 2 h at 37 °C while rocking gently, to allow re-expression of cell surface markers. In studies with H-89 (10 µM) or function-blocking antibodies, the cells were preincubated during the final 40 min of the recovery period, whereas stimuli like forskolin, 8CPT-2Me-cAMP, and isoproterenol were added during the final 10 min of the recovery period. The cells (2.5 x 104/well in case of 24-well plates) were then directly transferred to plates and allowed to adhere at 37 °C on Ln-5 (15 min) or fibronectin (30 min), and the experiments were carried out in triplicate. After washing the tissue plates with PBS, the cells were fixed in 4% formaldehyde in PBS for 30 min at 4 °C. The cells were then permeabilized and blocked in ice-cold TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 100 mM glycine, and 1% fatty acid-free BSA) for 30 min. Subsequently, the cells were incubated with phalloidin-TRITC overnight at 4 °C, washed with TBST, mounted in fluorescence mounting medium (DAKO), and imaged at 400x magnification using a cooled CD camera mounted on an inverted fluorescence microscope (Olympus IX 81) fitted for tissue culture dishes. The total surface covered by cells (at least 33 cells/well, and a total of 3 wells/transfection) was plotted relative to the surface covered by untreated cells. The error bars represent average deviation among experiments. Alternatively, and as indicated in the figure legends, the percentage of spread cells (i.e. cells that had become flattened with their total diameter more than twice the diameter of the nucleus) per microscopic field (four different fields/treatment) was determined rather than the total cell surface.

Fluorescence-activated Cell Sorter Analysis—Ovcar-3 cells were grown to ~70% confluency, trypsinized, and recovered in serum-free DMEM containing 20 mM Hepes, pH 7.4, and 1% fatty acid-free BSA for 2 h at 37 °C while rocking gently. During the last 30 min the cells were treated with 100 µM 8CPT-2Me-cAMP or left untreated. Subsequently, the cells were washed with PBS, fixed in 4% formaldehyde in PBS for 30 min at 4 °C, washed with PBS, and incubated with 10 µl of fluorescein isothiocyanate-conjugated antibodies against {alpha}3 integrin (CD49c; Chemicon).

Rap1 Activation Assay and Phosphorylation of CREB—Rap1 activation assays were performed as described previously (25, 26). Briefly, adherent cells were stimulated as indicated and lysed in 750 µl of lysis buffer (10% glycerol, 1% Nonidet P-40, 50 mM Tris-Cl, pH 7.5, 200 mM NaCl, 2 mM MgCl2, 5 mM NaF, 1 mM NaVO3, and 1 mM phenylmethylsulfonyl fluoride). The lysates were clarified by centrifugation, and 500 µl of lysate was incubated with GST-tagged RBD of RalGDS precoupled to glutathione beads to specifically pull down the GTP-bound forms of Rap1. The samples were incubated for 1 h at 4 °C while tumbling. The beads were washed four times in lysis buffer, and the remaining fluid was removed with an insulin syringe. The proteins were eluted with Laemmli sample buffer and analyzed by SDS-PAGE and Western blotting using Rap1 antibodies. To 100 µl of clarified lysate 30 µl of 4x Laemmli sample buffer was added, and phosphorylation of CREB was analyzed by Western blotting using a phospho-specific antibody directed against phosphorylated Ser133.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Rap1 Induces Adhesion to Ln-5—To investigate whether Rap1 is able to induce adhesion to Ln-5, we used 804G rat bladder carcinoma cells, which are known to adhere to Ln-5 (27). The cells were transfected with luciferase cDNA under control of a CMV promoter in combination with either Rap1V12, an activated mutant of Rap1 that is preferentially GTP-bound, or with Rap1GAPI, an inhibitor of Rap1. After detachment with trypsin-EDTA, the cells were plated on a Ln-5 matrix, and adhesion was quantified. As shown in Fig. 1A, Rap1V12 promoted adhesion of cells to Ln-5, whereas transfection with Rap1GAPI had no stimulatory effect. Expression of Rap1V12 and RapGAPI is shown in Fig. 1B. To test whether activation of endogenous Rap1 also induces adhesion of cells to Ln-5, we transiently transfected HaCaT keratinocyte cells, which also express {alpha}3{beta}1 and {alpha}6{beta}4, and 804G cells with the exchange factor Epac1, a specific activator of the family of Rap small GTPases that does not activate any other small GTPase (12, 13). Transient transfection of cells with Epac1 cDNA has been shown previously to activate endogenous Rap1, even in the absence of stimuli that raise intracellular cAMP levels (17, 28). As shown in Fig. 1C, Epac1-expressing cells adhered better to a Ln-5 matrix than mock transfected cells. Activation of endogenous Rap1 and expression of Epac1 is shown in Fig. 1D. PDZ-GEFI is another exchange factor that does not activate any other small GTPase than the GTPases of the Rap family (20, 29). Overexpression of PDZ-GEF in 804G cells also increased adhesion to Ln-5, whereas Rap1GAPI had a slight inhibitory effect (Fig. 1E). Activation of endogenous Rap1 and expression of the transgenes is shown in Fig. 1F. Interestingly, HEK293T cells, which only express {alpha}3{beta}1 and not {alpha}6{beta}4, also adhered better to Ln-5 upon overexpression of Epac1 (Fig. 1G), which was inhibited by co-expression of the inhibitor of Rap1, Rap1GAPI. Rap1-GTP levels are shown in Fig. 1H. Taken together, these results show that activation of endogenous Rap1 results in stimulation of cell adhesion to Ln-5 in a variety of cell lines and indicate that Rap1 may specifically regulate {alpha}3{beta}1.



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  		FIG. 1.
The small GTPase Rap1 stimulates adhesion to Ln-5. A, overexpression of a constitutively active mutant of Rap1 induces adhesion to Ln-5. 804G cells were transfected with CMV-luciferase in combination with mock DNA, cDNA for Rap1V12, or cDNA for Rap1GAPI, respectively, and plated on a Ln-5 matrix for the indicated times. Nonadherent cells were washed away, adherent cells were lysed, and luciferase units were measured. B, expression of Rap1GAPI and Rap1V12 was confirmed by Western blotting using HA antibodies. C, overexpression of Epac1 increases cell adhesion to Ln-5. HaCaT and 804G cells were transfected with CMV-luciferase in combination with either mock DNA or Epac1, and an adhesion assay was performed as described for A. D, overexpression of Epac1 results in activation of endogenous Rap1. HaCaT and 804G cells were transfected with Epac cDNA as indicated, and the cell lysates were incubated with immobilized GST-RalGDS-RBD to specifically pull down the GTP-bound form of Rap1 (top panel). To confirm equal loading, total Rap1 levels in whole cell lysates were determined by Western blotting with Rap1 antibodies (middle panel), and expression of Epac is shown using HA antibodies (bottom panel). E, expression of PDZ-GEF increases adhesion of 804G cells to Ln-5. The cells were transfected with CMV-luciferase in combination with either mock DNA or PDZ-GEF, and an adhesion assay was carried as described for A. F, regulation of Rap1 by PDZ-GEF and Rap1GAPI. 804G cells were transfected as indicated, and the cell lysates were incubated with immobilized GST-RalGDS-RBD to specifically pull down the GTP-bound form of Rap1 (top panel). Total Rap1 in whole cell lysates is shown (middle panel), and expression of transfected cDNAs was confirmed by blotting with HA antibodies (bottom panel) G, expression of Rap1GAPI inhibits Epac-induced cell adhesion. HEK293T cells were transfected with CMV-luciferase in combination with indicated cDNAs, and an adhesion assay was performed as described for A. H, activation of endogenous Rap1 in transfected HEK293 cells. HEK293 cells were transfected as indicated, and the cell lysates were incubated with immobilized GST-RalGDS-RBD to specifically pull down the GTP-bound form of Rap1 (top panel). Total Rap1 in whole cell lysates is shown (middle panel), and expression of transfected cDNAs was confirmed by blotting with HA antibodies (bottom panel).

 
cAMP Promotes Cell Adhesion to Ln-5 through Epac—Cyclic AMP is a versatile second messenger implicated in numerous cellular processes, many of which are believed to be carried out by PKA. However, some of the effects of cAMP have been shown to be mediated by the Epac1 signaling pathway rather than the PKA pathway, like calcium release, insulin secretion, and secretion of soluble amyloid precursor protein {alpha} (sAPP{alpha}), the major component of senile plaques in patient brains inflicted with Alzheimer's disease (30–32). In addition, cAMP has recently been reported to induce adhesion of Ovcar-3 cells to fibronectin (FN) through the Epac-Rap1 pathway (17). Ovcar-3 cells originate from a human ovarian carcinoma and have been reported to express both {alpha}3{beta}1 and {alpha}6{beta}4 and to adhere to laminins (33). All this makes the Ovcar-3 cell line an ideal cell line to study the role of cAMP in adhesion of cells to Ln-5. As shown in Fig. 2A, treatment of Ovcar-3 cells with the cAMP-elevating agent forskolin, a direct activator of adenylate cyclase, resulted in increased adhesion to Ln-5, which was resistant to the PKA inhibitor H-89. Importantly, treatment with isoproterenol, an agonist of the G{alpha}s-coupled {beta}2-adrenergic receptor, also resulted in increased cell adhesion (Fig. 2A), indicating that physiologically relevant stimuli also regulate adhesion to Ln-5. The finding that cAMP-induced cell adhesion was resistant to H-89 indicates that the effect of cAMP on cell adhesion is mediated by the Epac pathway rather than the PKA pathway. Therefore, we made use of a recently described Epac-specific cAMP analog, 8CPT-2Me-cAMP, which does not activate PKA even at high concentrations or long incubation times (21). 8CPT-2Me-cAMP has previously been used to rapidly and very specifically "switch on" the endogenous Epac-Rap1 pathway and has been established as a tool for studying Epac signaling in a wide range of cellular processes (17, 30, 34–36). As shown in Fig. 2B (top panel), 8CPT-2Me-cAMP rapidly activates Rap1 in Ovcar-3 cells, which is not affected by H-89, confirming that cAMP-induced Rap1 activation is mediated by the Epac pathway. The same results were obtained with forskolin and isoproterenol (Fig. 2B, top panel). As a control, phosphorylation of CREB, a direct target of PKA (37), was not affected by 8CPT-2Me-cAMP, whereas forskolin and isoproterenol clearly induced PKA-dependent phosphorylation of CREB (Fig. 2B, bottom panel). Equal loading was confirmed by probing total cell lysates with Rap1 antibodies (Fig. 2B, middle panel). Importantly, as observed for forskolin and isoproterenol, stimulation of Ovcar-3 cells with 8CPT-2Me-cAMP resulted in increased adhesion to Ln-5 (Fig. 2C), indicating the involvement of the Epac-Rap1 pathway. To confirm whether cAMP-induced cell adhesion to Ln-5 is indeed mediated by Rap1, we transfected cells with Rap1GAPI and performed an adhesion assay. As shown in Fig. 2D, overexpression of Rap1GAPI completely blocks forskolin-induced cell adhesion. Finally, we observed that Ovcar-3 cells undergo extensive cytoskeletal rearrangements upon treatment with forskolin, resulting in increased cell spreading (see Fig. 4 below). To test whether the increase in cell spreading is actually required for augmented cell adhesion, we performed an adhesion assay in which cells were only allowed to adhere for 5 min to an Ln-5 matrix. At this short time interval, forskolin-treated Ovcar-3 cells already adhered better to a Ln-5 matrix than untreated cells (Fig. 2E, left panel). The forskolin-treated cells were still round at this time point, without having undergone the large cytoskeletal changes that result in cell spreading (Fig. 2E, right panel). The fact that the rapid forskolin-induced increase in cell adhesion precedes the increase in cell spreading suggests that extensive cytoskeletal rearrangements are not required for Rap1-induced augmented cell adhesion. This view is in line with previous reports that Rap1 can regulate integrin affinity and avidity (16). Taken together, these results show that cAMP induces rapid, Epac-Rap1-dependent cell adhesion of Ovcar-3 cells to Ln-5.



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  		FIG. 2.
cAMP promotes cell adhesion to Ln-5 through Epac. A, cAMP-induced cell adhesion is independent of PKA. As indicated, Ovcar-3 cells were pretreated with H-89 for 30 min and incubated with either forskolin (Forsk) or isoproterenol (Iso) for 15 min before being plated on Ln-5 for 10 min. Nonadherent cells were washed away, and adherent cells were fixed with formaldehyde, stained with phalloidin-TRITC, and imaged. Subsequently, the cells in three different microscopic fields were counted. The experiment was performed in triplicate. B, cAMP activates Rap1 through Epac. Ovcar-3 cells were pretreated with H-89 or left untreated before incubation with 8CPT-2Me-cAMP, forskolin, or isoproterenol, respectively. Subsequently, the cells were lysed and incubated with immobilized GST-RalGDS-RBD, to specifically pull down the GTP-bound form of Rap1 (top panel). Total lysates were probed with Rap1 antibodies to show equal input (middle panel) or with phospho-specific CREB antibodies to monitor PKA activity (bottom panel). C, 8CPT-2Me-cAMP increases cell adhesion to Ln-5. Ovcar-3 cells were treated with 8CPT-2Me-cAMP and plated on Ln-5, and an adhesion assay was carried out as in A. D, overexpression of Rap1GAPI blocks cAMP-induced cell adhesion. Ovcar-3 cells were transfected with mock cDNA or with Rap1GAPI, treated with forskolin, and plated on Ln-5, and an adhesion assay was performed as in Fig. 1A. The inset shows expression of Rap1GAPI. E, short term forskolin-induced adhesion of Ovcar-3 cells occurs without extensive cytoskeletal rearrangements. Ovcar-3 cells were treated with forskolin and plated on Ln-5 for 5 min, and an adhesion assay was performed as in Fig. 2A (left panel). The forskolin-treated cells were photographed at 100x magnification using phase contrast microscopy to show morphology (right panel). Bar, 20 µm.

 



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  		FIG. 4.
cAMP promotes cell spreading through Epac and Rap1. A, 804G cells were transiently transfected with mock DNA, Rap1V12, or Rap1GAPI. GFP was co-transfected to visualize transfected cells. The cells were detached, allowed to recover, and plated on Ln-5 for 15 min at 37 °C. Subsequently, the cells were fixed, and cells positive for GFP were photographed at 400x magnification. Bar, 15 µm. B, quantification of cell spreading. The data are shown as fold induction of total cell surface relative to mock transfected cells. The error bars represent standard error of three independent experiments, with n > 33. C, cAMP promotes Epac-Rap1-mediated cell spreading on fibronectin. Ovcar-3 cells were detached, allowed to recover, and plated on fibronectin (1 µg/ml) for 30 min at 37 °C. Subsequently, the cells were fixed, permeabilized, and stained with phalloidin-TRITC to visualize the actin cytoskeleton. The cells were photographed at 400x magnification. Representative images are shown. Bar, 20 µm. D, cAMP induces Ovcar-3 cell spreading on Ln-5. Ovcar-3 cells were detached and allowed to recover. During the last 40 min of recovery either Me2SO or H-89 was added, and the cells were treated with forskolin (Forsk) or isoproterenol (Iso) for the final 10 min. The cells were then plated on Ln-5 for 15 min at 37 °C, fixed, and photographed at 400x. The percentage of spread cells (i.e. cells that had become flattened with their total diameter more than twice the diameter of the nucleus) in four microscopic fields was determined. Representative data of one experiment are shown, and the experiment was performed four times with similar results. E, 8CPT-2Me-cAMP promotes cell spreading on Ln-5. Ovcar-3 cells were detached and allowed to recover, and 8CPT-2Me-cAMP was added during the final 10 min. Subsequently, a cell spreading assay was carried out as described for D. F, Ovcar-3 cell spreading is blocked by antibodies against {alpha}3 integrins. Ovcar-3 cells were detached and allowed to recover. During the last 40 min the cells were incubated with goat anti-mouse IgG or antibodies against {alpha}3, {alpha}6, or a combination of {alpha}3 and {alpha}6 integrins. Forskolin was added during the final 15 min of recovery, and a cell spreading assay was carried out as described for D.

 
cAMP-induced Cell Adhesion to Ln-5 Is Mediated by {alpha}3{beta}1—Because overexpression of Epac1 resulted in increased adhesion to Ln-5 of HEK293T cells, which lack {alpha}6{beta}4, we hypothesized that Rap1 may regulate the {alpha}3{beta}1 integrin rather than {alpha}6{beta}4. Therefore, we performed an adhesion assay with Ovcar-3 cells (which, as mentioned above, express both {alpha}3{beta}1 and {alpha}6{beta}4) in the presence of function-blocking antibodies against {alpha}3 or {alpha}6 integrins. As shown in Fig. 3A, anti-{alpha}6 antibodies failed to influence forskolin-induced adhesion of Ovcar-3 cells to Ln-5, indicating that {alpha}6{beta}4 is not required. In contrast, anti-{alpha}3 antibodies greatly impaired adhesion of Ovcar-3 cells to Ln-5 (Fig. 3A), whereas as a control adhesion to FN was not affected by antibodies against either {alpha}3 or {alpha}6 (data not shown). These results show that although both {alpha}3{beta}1 and {alpha}6{beta}4 integrins may mediate adhesion of Ovcar-3 cells to Ln-5, cAMP-induced cell adhesion to Ln-5 is specifically mediated by the {alpha}3{beta}1 integrin. To further substantiate the finding that Rap1 regulates {beta}1 integrins and not {beta}4 integrins, we made use of human erythroleukemic K562 cells stably transfected with {alpha}6{beta}4 (38). Importantly, these cells do not express {alpha}3{beta}1, and therefore adhesion to Ln-5 is strictly mediated by {alpha}6{beta}4 (23). First, we tested whether activation of Rap1 results in increased adhesion to FN, which is mediated by {beta}1 integrins. Indeed, upon stimulation with the phorbol ester PMA, which is a strong stimulus for Rap1 (39), these cells rapidly adhered to FN. This was dependent on Rap1, because overexpression of Rap1GAPI strongly inhibited adhesion (Fig. 3B). As expected, this PMA-induced, Rap1-mediated induction of cell adhesion was almost completely blocked by function-blocking antibodies against {beta}1 integrins, whereas as a control antibodies against {alpha}6 had no effect. In contrast, PMA did not enhance cell adhesion to Ln-5, and overexpression of Rap1GAPI had no effect on cell adhesion, showing that Rap1 does not regulate {alpha}6{beta}4 (Fig. 3C). Rap1 has previously been shown to regulate the avidity and affinity, but not cell surface expression of various integrins. As shown in Fig. 3D, Rap1 also does not increase the cell surface expression of {alpha}3{beta}1 integrins, indicating that the cAMP-induced increase in cell adhesion to Ln-5 is mediated by increased {alpha}3{beta}1 avidity or affinity or both, but not the number of integrins on the cell surface. Altogether, we conclude that Rap1-dependent cell adhesion to Ln-5 is mediated by {alpha}3{beta}1 and is independent of the number of {alpha}3{beta}1 integrins on the cell surface. Importantly, these data indicate that Rap1 specifically regulates {beta}1 integrins and not {beta}4 integrins.



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  		FIG. 3.
Rap1 specifically regulates {alpha}3{beta}1 and not {alpha}6{beta}4. A, cAMP-induced cell adhesion to Ln-5 is mediated by {alpha}3{beta}1 integrins. Ovcar-3 cells were incubated with goat anti-mouse IgG or antibodies against {alpha}3, {alpha}6, or a combination of {alpha}3 and {alpha}6 integrins before receiving treatment with forskolin for 15 min. The cells were then plated on Ln-5 for 10 min, and nonadherent cells were washed away. Adherent cells were fixed with formaldehyde, stained with phalloidin-TRITC, and imaged, and the cells in three different microscopic fields were counted. The experiment was performed in triplicate. B, Rap1 regulates {beta}1 integrins. K562 cells stably transfected with {alpha}6{beta}4 were transfected with mock DNA or with Rap1GAPI. The cells were treated with function-blocking antibodies against {alpha}6 or {beta}1 before stimulation with PMA. The cells were then plated on FN, and an adhesion assay was performed as described in Fig. 1A. C, Rap1 does not regulate {alpha}6{beta}4. {alpha}6{beta}4-expressing K562 cells were transfected with mock DNA or with Rap1GAPI as indicated. The cells were pretreated with function-blocking antibodies against {alpha}6 or {beta}1, stimulated with PMA, and plated on Ln-5, and an adhesion assay was performed as described for Fig. 1A. D, Rap1 does not change cell surface expression of {alpha}3{beta}1 integrins. Ovcar-3 cells were trypsinized, recovered, and left untreated or treated with 100 µM 8CPT-2Me-cAMP for 30 min. Subsequently, the cells were fixed, incubated with fluorescein isothiocyanate-conjugated antibodies against {alpha}3 (CD49c), and analyzed by flow cytometry. The fluorescence-activated cell sorter profile is representative for three independent experiments.

 
cAMP Potentiates Cell Spreading on Ln-5 through Epac—Rap1 has not only been implicated in cell adhesion but also in cell spreading of a number of cell lines on FN or collagen I, including HEK293, B lymphocytes, and mouse embryonic fibroblasts (40–42). Cell spreading involves reorganization of the actin cytoskeleton, and the resulting flattening of the cell increases the contact region with the extracellular matrix, allowing stronger adhesion. To test whether Rap1 promotes spreading on a Ln-5 matrix, 804G cells were transiently transfected with either Rap1V12 or Rap1GAPI. GFP was co-transfected to visualize transfected cells. 24 h after transfection the cells were trypsinized and plated on a Ln-5 matrix for 15 min, fixed, and photographed. As shown in Fig. 4A, Rap1V12 potentiates cell spreading, whereas Rap1GAPI had no effect. Quantitative analysis showed that cells expressing Rap1V12 have a cell surface approximately twice the size of mock transfected cells (Fig. 4B). Previously, cAMP has been shown to induce cell spreading through PKA-dependent activation of Rac1 (18). We wanted to test whether cAMP might also influence cell spreading through the Epac-Rap1 pathway. Indeed, when left untreated, Ovcar-3 cells spread on a fibronectin matrix, but treatment with forskolin resulted in more rapid and more extensive cell spreading (Fig. 4C). This was not blocked by the PKA inhibitor H-89, and it was mimicked by the Epac-specific cAMP analog 8CPT-2Me-cAMP. Furthermore, treatment of Ovcar-3 cells with either forskolin of isoproterenol also promoted cell spreading on a Ln-5 matrix, which was not inhibited by H-89 (Fig. 4D), indicating that this also may be mediated by the Epac-Rap1 pathway. Indeed, treatment of cells with 8CPT-2Me-cAMP mimicked forskolin- and isoproterenol-enhanced cell spreading (Fig. 4E), showing that specific activation of the Epac pathway is sufficient to promote cell spreading. Because Rap1 mediates cell adhesion to Ln-5 specifically through {alpha}3{beta}1, we wanted to test whether cell spreading on Ln-5 is also mediated by {alpha}3{beta}1 rather than {alpha}6{beta}4. Therefore, we pretreated cells with antibodies against either {alpha}3 or {alpha}6 integrins before performing a cell spreading assay. As shown in Fig. 4F, cAMP-induced cell spreading on Ln-5 was not altered by anti-{alpha}6 antibodies, whereas anti-{alpha}3 antibodies almost completely blocked cell spreading. Together, these results show that cAMP, through Epac and Rap1, promotes cell spreading on a variety of substrates. Importantly, cell spreading on Ln-5 is {alpha}3{beta}1-dependent, indicating that Rap1 specifically regulates actin cytoskeleton-coupled {beta}1 integrins and not {beta}4 integrins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results reveal a role for the cAMP-Epac-Rap1 signaling pathway in cell adhesion on various substrates, including Ln-5, which is independent of PKA. Furthermore, we show for the first time that cAMP-induced cell spreading can also be regulated by the Epac-Rap1 pathway, which thus far was thought to be mediated by PKA, through activation of Rac (43). Finally, and in our eyes most importantly, our results indicate that Rap1 specifically regulates {beta}1 integrins and not {beta}4 integrins.

Various integrin functions on Ln-5 have previously been shown to be dependent on cAMP, PKA, and the small GTPases RhoA and Rac (44, 45). It is possible that cAMP utilizes both pathways to fine tune its effects on cell adhesion and migration. Indeed, in addition to PKA, the Epac-Rap1 pathway has recently been shown to regulate Rac (31). Furthermore, PKA and Epac have opposing roles in activation of protein kinase B in HEK293 cells (46). These findings indicate that PKA and Epac may share at least some effector proteins involved in cell adhesion and migration. It is not likely, however, that the effects of cAMP and Epac-Rap1 on cell adhesion and cell spreading described in this report are mediated by either Rac or protein kinase B, because these proteins are not activated by cAMP in Ovcar-3 cells.2

Rap1 regulates cell adhesion to a series of substrates, including fibronectin, fibrinogen, collagen, ICAM, and VCAM, through a wide variety of integrins, including {alpha}IIb{beta}3, {alpha}4{beta}1, {alpha}5{beta}1, {alpha}L{beta}2, and {alpha}M{beta}2 (16). Here we show that Rap1 also controls adhesion to Ln-5 and indicate that the {alpha}3{beta}1 integrin can be added to this still growing list. Ovcar-3 cells have previously been shown to express both {alpha}3{beta}1 and {alpha}6{beta}4 and to adhere to laminins (33). The fact that cAMP-induced adhesion of Ovcar-3 cells to Ln-5 is blocked by antibodies against {alpha}3{beta}1 integrins, whereas antibodies against {alpha}6{beta}4 have no effect, suggests that Epac and Rap1 specifically regulate the {alpha}3{beta}1 integrin and not {alpha}6{beta}4. Indeed, Rap1 fails to stimulate adhesion to Ln-5 in {alpha}6{beta}4-expressing K562 cells, which lack {alpha}3{beta}1. In contrast, {beta}1-dependent adhesion to FN was clearly potentiated by Rap1. Therefore, our results show that Rap1 specifically regulates {alpha}3{beta}1 and not {alpha}6{beta}4.

{alpha}3{beta}1 couples to the actin cytoskeleton, whereas {alpha}6{beta}4 couples to the intermediate filament cytoskeleton and thus recruits different proteins than {alpha}3{beta}1 (8). Regulation of {alpha}6{beta}4 may therefore differ from other integrins. {beta}1 integrins recruit proteins like talin, which in turn recruits vinculin and the Arp2/3 complex, which mediates actin polymerization. Reorganization of actin filaments in stress fibers increases integrin clustering and avidity (47). In contrast, {beta}4 integrins interact with different proteins, like bullous pemphigoid antigen-1 and -2, and plectin (48). Therefore, we speculate that Rap1 recruits or increases the activity of one of the {beta}1-binding proteins, thereby promoting actin polymerization, as has been reported previously for the {alpha}4 integrin (41), whereas it does not influence the activity of proteins recruited by the {beta}4 integrin.

Rap1 has been shown to increase integrin avidity and affinity, but not cell surface expression of integrins (16). In line with these reports, we find that the cAMP-Epac-Rap1 pathway does not increase the cell surface expression of integrin {alpha}3{beta}1, indicating that Rap1 probably influences integrin avidity or affinity or both. Furthermore, our data show that the rapid cAMP-induced increase in cell adhesion precedes the increase in cell spreading, suggesting that extensive cytoskeletal rearrangements are not required for Rap1-induced cell adhesion. Indeed, in Jurkat T cells stably transfected with Epac1 cDNA, treatment with cAMP results in increased cell adhesion, which is not blocked by inhibitors of the actin cytoskeleton.3 Therefore, although Rap1 stimulates cell spreading, the Rap1-mediated increase in cell adhesion seems to be independent of the actin cytoskeleton. Finally, Rap1 has recently been shown to regulate E-cadherins and thereby cell-cell adhesion (49). Altogether, this suggests that Rap1 may act as a global regulator of cell adhesion, functioning relatively high in the hierarchy of a number of specific signaling pathways that control several aspects of cell adhesion. Unfortunately, the direct effectors of Rap1 remain unclear (15). Rap1 has been shown to bind to and activate B-Raf, resulting in activation of ERK, although this is still a matter of debate (21). Rap1 may also regulate the cell-cell adhesion molecule AF-6 (50), which is found in adherens junctions and which may be involved in regulation of cell adhesion (51). Furthermore, in thyroid cells Rap1 regulates phosphatidylinositol 3-kinase signaling pathways (46, 52, 53), which may affect cell adhesion. Interestingly, in hematopoietic cells Rap1 has been shown to bind to RAPL, which in turn would bind directly to and activate LFA-1 integrins (54). However, it is unlikely that this protein also functions in Rap1-induced cell adhesion in fibroblastic and epidermal cells, because its expression pattern is almost exclusively restricted to hematopoietic cells and spleen (54). Furthermore, Rap1 has been shown to interact with the B4.1 domain of the cytoskeleton-associated protein Krit1 (55). Many integrin-regulating proteins contain such a B4.1 domain, including talin and FAK. Rap1 may directly interact with these proteins and thereby affect their activity. Indeed, in B cells, Rap activation results in increased phosphorylation of the FAK family member PYK2 (41). The possibility that Rap1 may directly interact with such B4.1 containing proteins is currently under investigation. Clearly, identification of the components of the Rap1 effector pathway that mediates cell adhesion remains a major challenge.

In summary, we find that cAMP regulates adhesion and cell spreading of a number of cell types to various substrates, including Ln-5. The fact that adhesion and spreading to Ln-5 is mediated by Epac and Rap1 rather than PKA provides a novel "inside-out" regulatory mechanism for {alpha}3{beta}1 adhesive properties. These results may have important consequences for our current understanding of the effect of a wide variety of cAMP-controlling hormones as for instance isoproterenol, SDF-1, serotonin, carbachol, or prostaglandin E2 on many integrin-mediated cellular functions, like wound healing, migration, axon guidance, and signaling.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Ludwig Inst. for Cancer Research, University of California, San Diego 9500 Gilman Dr., La Jolla, CA 92093-0670. Back

These authors contributed equally to this work. Back

{ddagger}{ddagger} To whom correspondence should be addressed: The Biotechnology Centre of Oslo, University of Oslo, P.O. Box 1125, Blindern, N-0317 Oslo, Norway. Tel.: 47-22840505; Fax: 47-22840506; E-mail: kjetil.tasken{at}biotek.uio.no.

1 The abbreviations used are: Ln-5, laminin-5; PKA, cAMP-dependent kinase; FN, fibronectin; 8CPT-2Me-cAMP, 8-(4-chloro-phenylthio)-2-O-methyladenosine-3,5 cyclic monophosphate; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; HA, hemagglutinin; CREB, cAMP-responsive element-binding protein; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PMA, phorbol 12-myristate 13-acetate; GFP, green fluorescent protein; GST, glutathione S-transferase; FN, fibronectin. Back

2 J. M. Enserink, unpublished observations. Back

3 L. S. Price, unpublished results. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. J. C. Jones (Feinberg School of Medicine at Northwestern University) and Dr. Anthony Montgomery (University of California, San Diego) for generously providing 804G cells, to Dr. H. Prydz (Biotechnology Centre of Oslo) for HaCaT cells, to J. H. Norum for assistance with imaging, and Dr. H. Abrahamsen for critically reading the manuscript.



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