Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
*Author for correspondence (e-mail: akiyama{at}niehs.nih.gov)
Accepted June 1, 2001
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SUMMARY |
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Key words: Integrin activation, Cell-cell adhesion, cAMP, PKA, F-actin, Signal transduction
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INTRODUCTION |
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Integrins can be activated by divalent cations, synthetic peptides and certain antibodies (Humphries, 1996). Recently, we described the effects of an activating anti-ß1 mAb, 12G10, that can specifically and rapidly induce both cell-substrate and cell-cell adhesion of a number of different cell types (Mould et al., 1995; Whittard and Akiyama, 2001). Binding of mAb 12G10 also induced clustering of cell-surface integrins, F-actin polymerization and the preferential localization of ß1 integrins expressing the 12G10 epitope at sites of cell-cell adhesion. Integrin activation-induced HT-1080 cell-cell adhesion minimally requires the interaction of activated 2ß1 with nonactivated
3ß1. MAb 12G10 is thought to recognize an activation-dependent epitope located close to the ligand-binding pocket of the ß1 integrin (Mould et al., 1998). This epitope can be expressed by ß1 integrins in the absence of ligand (Whittard and Akiyama, 2001) and may represent a naturally existing conformer of the receptor. The signaling pathways associated with activation of integrin receptors are not well characterized.
Protein phosphorylation is a pivotal mechanism in the transduction of signaling events, and the relative activities of protein kinases and protein phosphatases are highly regulated. The cAMP-dependent protein kinase (PKA) is activated as a result of interaction with cAMP, a well-studied second messenger signal classically generated through G-protein-coupled receptor stimulation of adenylyl cyclase (Gilman, 1987). The PKA holoenzyme is a tetramer consisting of two catalytic subunits and a regulatory (R) subunit dimer (Potter et al., 1978). The cAMP-PKA pathway can regulate many cellular processes such as cell metabolism (Edelman et al., 1987), proliferation (Boynton and Whitfield, 1983) and gene transcription (Montminy, 1997).
Here, we show that PKA is essential for mAb 12G10-induced HT-1080 cell-cell and cell-substrate adhesion. Binding of mAb 12G10 to ß1 integrins stimulates an increase in intracellular cAMP levels and PKA activity. We also show that two processes required for HT-1080 cell-cell adhesion, that is, integrin clustering and F-actin polymerization, are both dependent on PKA. Taken together, our data suggest that PKA is a key part of a signaling pathway that is stimulated by activation of integrins and may be required for enhanced levels of cell-substrate and cell-cell adhesion.
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MATERIALS AND METHODS |
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Cell culture and reagents
HT-1080 human fibrosarcoma cells were obtained from American Type Culture Collection (Manassas, VA) and cultured as described (Whittard and Akiyama, 2001). Staurosporine, calphostin C, genistein, H89, myristoylated heat-stable protein kinase A inhibitor (MPKI, 14-22) peptide, myristoylated protein kinase C (PKC) and ß inhibitor (20-28) peptide, forskolin, Sp-adenosine-3',5'-cyclic monophosphorothioate (Sp-cAMPS), 8-Bromo-cAMP, Sp-5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole-3'5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS) and Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS) were obtained from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). Poly-D-lysine and collagen type IV were obtained from Becton Dickinson Labware (Franklin Lakes, NJ). SignaTECT® cAMP-dependent protein kinase assay system was obtained from Promega (Madison, WI). [
32P]ATP (4000 Ci/mmol) was obtained from ICN (Costa Mesa, CA). cAMP enzyme immunoassay kit was obtained from Biomedical Technologies Incorporated (Stoughton, MA). ProLongTM antifade and AlexaTM 488 phalloidin were obtained from Molecular Probes.
Cell attachment assay
Cell-substrate attachment assays were performed as described (Whittard and Akiyama, 2001), using a 30 minute pretreatment with inhibitors when indicated. Treatment with each of the inhibitors had no effect on HT-1080 cell viability, as judged by using trypan blue exclusion.
Cell-cell adhesion assay
Cell-cell adhesion assays were performed as described (Whittard and Akiyama, 2001). HT-1080 cells were treated with inhibitors or activators for either 30 minutes (staurosporine, calphostin C, H89, MPKI peptide, myristoylated PKC and ß inhibitor peptide, forskolin, Rp-cAMPS, 8-Bromo-cAMP, Sp-5,6-DCl-cBiMPS and Sp-cAMPS) or 4 hours (genistein). ß1 integrins were clustered by pretreating cells with 10 µg/ml K20 for 30 minutes at 37°C followed by the addition of 10 µg/ml goat mAb directed against the Fc region of mouse IgG for 20 minutes at 37°C. Cell-cell adhesion was calculated using the following formula: Cell-cell adhesion index (CCAI)=(Total number of cells-total number of aggregates)/total number of cells, in which an aggregate is defined as one cell, or multiple cells that form a single cluster. Treatment with each of the inhibitors had no effect on HT-1080 cell viability as judged by trypan blue exclusion. Photographs were taken using a SPOT 2 digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI) on a Zeiss microscope equipped with a 30x objective (Axiovert 35; Carl Zeiss, Jena, Germany).
Protein kinase A assay
Tissue culture dishes (60 mm; Becton Dickinson Labware) were coated with 2.5 ml of 10 µg/ml poly-D-lysine overnight at 4°C. Wells were blocked with 10 mg/ml heat-denatured BSA (Calbiochem-Novabiochem Corp., La Jolla, CA) and near-confluent HT-1080 cells were harvested with 1% (w/v) trypsin (GIBCO-BRL). Cells were resuspended to a density of 2.0x106 cells/ml in 37°C DMEM with 0.5 mg/ml soybean trypsin inhibitor and 25 mM Hepes, and allowed to recover for 15 minutes at 37°C. Cells (2.5 ml) were added to dishes with or without antibodies for 20 minutes at 37°C, washed with PBS, and then suspended in 0.5 ml of ice-cold extraction buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM ß-mercaptoethanol, 1 µg/ml leupeptin and 1 µg/ml aprotinin). Samples were homogenized with a Pellet PestleTM homogenizer (Kimble-Kontes, Vineland, NJ) at 4°C, and nuclei were removed by centrifugation at 14,000 g for 5 minutes at 4°C. The protein concentrations of the supernatants were determined by using the BCA protein assay (Pierce Chemical Company). PKA activity in the supernatants was measured using the Promega SignaTECTTM assay system as described (Goueli et al., 1995).
cAMP assay
Tissue culture dishes (35 mm; Becton Dickinson Labware) were coated with 1.0 ml aliquots of 10 µg/ml poly-D-lysine overnight at 4°C. Dishes were blocked and near-confluent cells were harvested as described for PKA assays. Cells (1.0 ml) were added to dishes with or without antibodies for 20 minutes at 37°C. Cells were washed three times with ice-cold PBS and extracted with 0.5 ml 5% (w/v) trichloroacetic acid for 5 minutes. Supernatant was combined with 9 ml 5% trichloroacetic acid and homogenized with a Pellet PestleTM homogenizer (Kimble-Kontes). cAMP in the supernatant was quantitated using the Biomedical Technologies Inc. enzyme immunoassay system kit according to the manufacturers instructions.
Immunofluorescence microscopy
Immunofluorescence localization was performed as described (Whittard and Akiyama, 2001). F-actin was stained with 0.5 units/ml AlexaTM 488 phalloidin, the PKA RII subunits were localized using 5 µg/ml polyclonal antibody and ß1 integrins were localized using 10 µg/ml mAb 12G10 directly labeled with Alexa 568TM, all for 30 minutes at room temperature. The antibody directed against the PKA RII subunits was detected using AlexaTM 488-coupled donkey anti-goat IgG for 30 minutes at room temperature.
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RESULTS |
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Inhibiting PKA blocks F-actin polymerization and integrin clustering induced as a result of integrin activation
Previously, we showed that mAb 12G10-induced cell-cell adhesion may require both integrin clustering and polymerization of F-actin (Whittard and Akiyama, 2001). We therefore decided to explore whether inhibiting PKA would affect either of these processes. On nonactivated HT-1080 cells, integrins expressing the 12G10 epitope were uniformly distributed on the surface of cells (Fig. 6, top panels). There was also a small amount of F-actin around the periphery of HT-1080 cells. By contrast, cells treated with mAb 12G10 exhibited clustering of integrins expressing the 12G10 epitope and also increased levels of F-actin polymerization in single cells (Fig. 6, middle panels). Locations of integrin clustering coincided with sites of increased F-actin. MAb 12G10-induced integrin clustering and F-actin polymerization were both inhibited when cells were treated with 10 µM MPKI peptide prior to the addition of mAb 12G10 (Fig. 6, bottom panels). These results suggest the hypothesis that the mAb 12G10-induced increase of PKA activity was required for integrin clustering and F-actin polymerization, and thus was likely to be upstream of both processes.
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DISCUSSION |
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There is increasing evidence suggesting that integrins can regulate the activation of a number of signaling pathways inside adherent cells, including the MAP kinase cascade, Rho family of guanosine triphosphatases, tyrosine kinases, phosphatidylinositol 3-kinase and Jun amino-terminal kinase pathways (Giancotti and Ruoslahti, 1999). Results presented here demonstrate that the cAMP-PKA pathway can be stimulated in response to activating ß1 integrins with mAb 12G10. Recent work has shown that mechanically stressed ligand-bound ß1 integrins can activate the cAMP-PKA pathway in a G-protein-dependent manner (Meyer et al., 2000). The cAMP-PKA pathway can also be stimulated upon adhesion of endothelial cells to extracellular matrix proteins (Fong and Ingber, 1996) and transiently activated following detachment of 3T3 fibroblasts (Howe and Juliano, 2000).
The mechanism by which activation of ß1 integrins can lead to an increase in the cAMP-PKA pathway is currently unknown. The simplest hypotheses are that an increase in PKA activity may result from the activation of adenylyl cyclase or, alternatively, activation results in inhibition of phosphodiesterases. Interestingly, elevation of intracellular free Ca2+ levels have been observed following integrin ligation (Schwartz, 1993), and these increases could activate Ca2+-sensitive adenylyl cyclases (Watson et al., 2000). However, adenylyl cyclases are more typically activated as a result of heterotrimeric Gs protein coupling to G-protein-coupled receptors at the cytoplasmic face of the plasma membrane (Gudermann et al., 1997). A recent study has shown that an
vß3 integrin-associated protein (CD47) complex could couple with, and consequently stimulate, the inhibitory trimeric G
i protein, leading to a rapid decrease in cAMP (Frazier et al., 1999). We hypothesize that an activated ß1-integrin may be involved in a protein complex that could couple with the trimeric G
s protein, and thereby stimulate an increase in the cAMP-PKA pathway.
The data reported here point to the PKA enzyme as being a crucial component of a signaling pathway(s), stimulated by activation of integrins, and that may be required for enhanced levels of cell-substrate and cell-cell adhesion. In the case of HT-1080 cells, it appears that an increase in PKA activity is required for integrin-activation-dependent cell-substrate and cell-cell adhesion. Several lines of evidence, however, suggest that integrin-mediated events can be negatively regulated by an increase in the cAMP-PKA pathway. For example, an increase in cAMP has been shown to alter cell morphology, induce the disassembly of stress fibers and focal contacts, or inhibit cell spreading and migration of certain cell types (Lamb et al., 1988; Lampugnani et al., 1990; Glass and Kreisberg, 1993; Leven, 1995). Thus, it appears that an increase in PKA activity may elicit different responses depending on how far the adhesion process has advanced, and on the cell type.
The target(s) of PKA phosphorylation that play a role in the regulation of cell-cell adhesion are unknown. PKA is a multisubstrate enzyme that can phosphorylate a broad spectrum of protein substrates (Walsh and van Patten, 1994). Conversely, PKA phosphorylation can also inhibit the function of several signaling molecules, including RhoA (Lang et al., 1996; Dong et al., 1998), actin (Ohta et al., 1987), p21-activated kinase (Howe and Juliano, 2000) and paxillin (Han and Rubin, 1996). Therefore, the involvement of PKA in the process of cell-cell adhesion may be a consequence of PKA inhibiting certain signaling molecules such as Rho, thereby leading to the upregulation of other proteins such as Rac (Sander et al., 1999). Interestingly, we observe two processes following treatment with mAb 12G10 that are typically indicative of Rac activation: F-actin polymerization and lamellipodia formation (Whittard and Akiyama, 2001).
Immunocytochemical studies suggest that activation of ß1 integrins may regulate the localization of the PKA RII subunits in HT-1080 cells. Following integrin activation, the RII subunits appear to translocate from the cytoplasm to areas where integrins expressing the 12G10 epitope reside. This result suggests that the target(s) of PKA phosphorylation may be positioned in close proximity to integrins expressing the 12G10 epitope. A change in PKA RII subunit localization may be dependent upon AKAPs, as these proteins have been shown to bind with high affinity to PKA RII subunits (Carr et al., 1991). Determining which, if any, of the AKAPs are involved in the process of cell-cell adhesion may provide valuable information in identifying downstream pathways targeted by PKA phosphorylation.
In conclusion, our data suggest that PKA plays a key role in the signaling pathway resulting from activation of integrins and that PKA may be required for enhanced levels of cell-substrate and cell-cell adhesion. To the best of our knowledge, this may be the first demonstration that an integrin-mediated increase in the activity of the cAMP-PKA pathway can positively regulate tumor-cell-adhesive interactions. There are many questions still to be answered to provide an understanding of the mechanisms by which integrin-mediated adhesive events control PKA activity, and conversely, how those events are regulated by the cAMP-PKA pathway. These answers could lead to the identification and characterization of molecular mechanisms of physiological and pathological processes that may require homotypic integrin interactions. For example, these adhesive interactions could be especially important during embryonic development and the subsequent maintenance of body organs. Furthermore, pathological processes that require both the formation and disruption of cell-cell contacts, such as tumor metastasis, could also be regulated by homotypic integrin interactions.
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ACKNOWLEDGMENTS |
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