Activation of p38 Has Opposing Effects on the Proliferation and Migration of Endothelial Cells

Pathological conditions such as hypertension and hyperglycemia as well as abrasions following balloon angioplasty all lead to endothelial dysfunction that impacts disease morbidity. These conditions are associated with the elaboration of a variety of cytokines and increases in p38 activity in endothelial cells. However, the relationship between enhanced p38 activity and endothelial cell function remains poorly understood. To investigate the effect of enhanced p38 MAPK activity on endothelial cell function, we expressed an activated mutant of MEK6 (MEK6E), an upstream regulator of p38. Expression of MEK6E activated p38 and resulted in phosphorylation of its downstream substrate, heat shock protein 27 (Hsp27). Activation of p38 was not sufficient to induce apoptosis; however, it did induce p38-dependent cell cycle arrest. MEK6E expression was sufficient to inhibit ERK phosphorylation triggered by growth factors and integrin engagement. MAPK phosphatase-1 (MKP-1) expression was increased upon p38 activation, and expression of a “substrate-trapping” MKP-1 was sufficient to restore ERK activity. Activation of p38 was sufficient to induce cell migration, which was accompanied by alterations in actin architecture characterized by enhanced lamellipodia. Co-expression of a mutant form of Hsp27, lacking all three phosphorylation sites, reversed MEK6E-induced cell migration and altered the cytoskeletal changes induced by p38 activation. Collectively, these results suggest that cellular decisions regarding migration and proliferation are influenced by p38 activity and that prolonged activation of p38 may result in an anti-angiogenic phenotype that contributes to endothelial dysfunction. Pathological conditions such as hypertension and hyperglycemia as well as abrasions following balloon angioplasty all lead to endothelial dysfunction that impacts disease morbidity. These conditions are associated with the elaboration of a variety of cytokines and increases in p38 activity in endothelial cells. However, the relationship between enhanced p38 activity and endothelial cell function remains poorly understood. To investigate the effect of enhanced p38 MAPK activity on endothelial cell function, we expressed an activated mutant of MEK6 (MEK6E), an upstream regulator of p38. Expression of MEK6E activated p38 and resulted in phosphorylation of its downstream substrate, heat shock protein 27 (Hsp27). Activation of p38 was not sufficient to induce apoptosis; however, it did induce p38-dependent cell cycle arrest. MEK6E expression was sufficient to inhibit ERK phosphorylation triggered by growth factors and integrin engagement. MAPK phosphatase-1 (MKP-1) expression was increased upon p38 activation, and expression of a “substrate-trapping” MKP-1 was sufficient to restore ERK activity. Activation of p38 was sufficient to induce cell migration, which was accompanied by alterations in actin architecture characterized by enhanced lamellipodia. Co-expression of a mutant form of Hsp27, lacking all three phosphorylation sites, reversed MEK6E-induced cell migration and altered the cytoskeletal changes induced by p38 activation. Collectively, these results suggest that cellular decisions regarding migration and proliferation are influenced by p38 activity and that prolonged activation of p38 may result in an anti-angiogenic phenotype that contributes to endothelial dysfunction. Under normal conditions, vascular injury triggers the proliferation and migration of normally quiescent endothelial cells, leading to repair of the injured vessel. However, several pathological conditions such as hypertension and hyperglycemia are accompanied by a dysfunctional endothelium characterized by impaired re-endothelialization. These conditions are associated with elevated levels of cytokines such as tumor necrosis factor and transforming growth factor-β as well as enhanced activation of p38 (1Nakagami H. Morishita R. Yamamoto K. Yoshimura S.I. Taniyama Y. Aoki M. Matsubara H. Kim S. Kaneda Y. Ogihara T. Diabetes. 2001; 50: 1472-1481Crossref PubMed Scopus (155) Google Scholar, 2McGinn S. Saad S. Poronnik P. Pollock C.A. Am. J. Physiol. 2003; 285: E708-E717Crossref PubMed Scopus (65) Google Scholar, 3Ju H. Behm D.J. Nerurkar S. Eybye M.E. Haimbach R.E. Olzinski A.R. Douglas S.A. Willette R.N. J. Pharmacol. Exp. Ther. 2003; 307: 932-938Crossref PubMed Scopus (59) Google Scholar, 4Kishore R. Luedemann C. Bord E. Goukassian D. Losordo D.W. Circ. Res. 2003; 93: 932-940Crossref PubMed Scopus (25) Google Scholar, 5Sheetz M.J. King G.L. J. Am. Med. Assoc. 2002; 288: 2579-2588Crossref PubMed Scopus (825) Google Scholar). Interestingly, high glucose levels present in diabetic patients have also been shown to induce the activation of p38 (1Nakagami H. Morishita R. Yamamoto K. Yoshimura S.I. Taniyama Y. Aoki M. Matsubara H. Kim S. Kaneda Y. Ogihara T. Diabetes. 2001; 50: 1472-1481Crossref PubMed Scopus (155) Google Scholar). These data suggest that p38 activity may play a direct role in endothelial dysfunction. The direct effect of chronic p38 activity on the proliferation and migration of endothelial cells has not been tested. p38 is a member of the mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; DNp38, dominant negative p38; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FGF, fibroblast growth factor; GFP, green fluorescent protein; Hsp, heat shock protein; HUVEC, human umbilical vein endothelial cell; MEK, MAPK/ERK kinase; MKP-1, MAPK phosphatase-1; mutHsp27, mutant form of Hsp27; VEGF, vascular endothelial growth factor. family, which includes extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase. These serine/threonine protein kinases transmit signals from the membrane to the nucleus (6Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1402) Google Scholar). Phosphorylation of both the threonine and tyrosine residues of the conserved Thr-X-Tyr motif of MAPKs is required for their activation. A variety of dual specificity protein phosphatases dampen activity by dephosphorylating these residues (7Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (720) Google Scholar). The MAPK family has classically been viewed as separate signaling cascades activated by distinct stimuli and upstream kinases (8Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2292) Google Scholar). ERK is activated by MEK1/2 kinases, whereas p38 is typically activated following phosphorylation of its upstream kinases MEK3 and MEK6 (9Yashima R. Abe M. Tanaka K. Ueno H. Shitara K. Takenoshita S. Sato Y. J. Cell. Physiol. 2001; 188: 201-210Crossref PubMed Scopus (40) Google Scholar). Functionally, these kinases are also thought of as distinct. ERK is involved in cell proliferation and survival responses in a variety of cell types, including endothelial cells (8Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2292) Google Scholar, 10Meadows K.N. Bryant P. Pumiglia K. J. Biol. Chem. 2001; 276: 49289-49298Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Activation of p38 is implicated in inflammation, cell growth control, cell differentiation, cell migration, and apoptosis (6Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1402) Google Scholar). The p38 MAPK family consists of four different isoforms including α, β, δ, and γ. The α and β isoforms are ubiquitously expressed, whereas γ expression is found predominantly in skeletal muscle, and δ expression is enriched in the lung, kidney, testis, pancreas, and small intestine (6Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1402) Google Scholar). A growing body of evidence indicates that these isoforms can be activated differentially and may control different downstream cellular processes, depending on cell type (11Uddin S. Ah-Kang J. Ulaszek J. Mahmud D. Wickrema A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 147-152Crossref PubMed Scopus (136) Google Scholar, 12Pramanik R. Qi X. Borowicz S. Choubey D. Schultz R.M. Han J. Chen G. J. Biol. Chem. 2003; 278: 4831-4839Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Efimova T. Broome A.M. Eckert R.L. J. Biol. Chem. 2003; 278: 34277-35285Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 14Harnois C. Demers M.J. Bouchard V. Vallee K. Gagne D. Fujita N. Tsuruo T. Vezina A. Beaulieu J.F. Cote A. Vachon P.H. J. Cell. Physiol. 2004; 198: 209-222Crossref PubMed Scopus (57) Google Scholar). The role of p38 in regulating endothelial cell function is currently not clear, and which isoforms are present in endothelial cells has not been defined. P38 can be activated by vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), two well known angiogenic factors, and p38 activation has been shown to be critical for endothelial cell migration in response to VEGF (15McMullen M. Keller R. Sussman M. Pumiglia K. Oncogene. 2004; 23: 1275-1282Crossref PubMed Scopus (62) Google Scholar, 16Rousseau S. Houle F. Landry J. Huot J. Oncogene. 1997; 15: 2169-2177Crossref PubMed Scopus (732) Google Scholar). Studies utilizing pharmacological inhibitors of p38 have suggested that p38 activation may promote endothelial cell apoptosis (17Matsumoto T. Turesson I. Book M. Gerwins P. Claesson-Welsh L. J. Cell Biol. 2002; 156: 149-160Crossref PubMed Scopus (177) Google Scholar, 18Gratton J.P. Morales-Ruiz M. Kureishi Y. Fulton D. Walsh K. Sessa W.C. J. Biol. Chem. 2001; 276: 30359-30365Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) and vascular permeability (19Issbrucker K. Marti H.H. Hippenstiel S. Springmann G. Voswinckel R. Gaumann A. Breier G. Drexler H.C. Suttorp N. Clauss M. FASEB J. 2003; 17: 262-264Crossref PubMed Scopus (146) Google Scholar, 20Goldberg P.L. MacNaughton D.E. Clements R.T. Minnear F.L. Vincent P.A. Am. J. Physiol. 2002; 282: L146-L154PubMed Google Scholar) while negatively regulating the tubular morphogenesis associated with angiogenesis (17Matsumoto T. Turesson I. Book M. Gerwins P. Claesson-Welsh L. J. Cell Biol. 2002; 156: 149-160Crossref PubMed Scopus (177) Google Scholar, 19Issbrucker K. Marti H.H. Hippenstiel S. Springmann G. Voswinckel R. Gaumann A. Breier G. Drexler H.C. Suttorp N. Clauss M. FASEB J. 2003; 17: 262-264Crossref PubMed Scopus (146) Google Scholar). Other studies have shown that p38 is not required for neovessel outgrowths (21Zhu W.H. MacIntyre A. Nicosia R.F. Am. J. Pathol. 2002; 161: 823-830Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Mice lacking p38α demonstrate severe defects in placental development and vascularization (22Mudgett J.S. Ding J. Guh-Siesel L. Chartrain N.A. Yang L. Gopal S. Shen M.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10454-10459Crossref PubMed Scopus (329) Google Scholar), suggesting a role in endothelial cell function. Collectively, these divergent pieces of data suggest that p38 activity is a tightly regulated component of the neovascularization response. In the present study, we have utilized a constitutively active form of MEK6 to investigate directly the effects of sustained p38 activity in vascular endothelial cells. Our results indicate that activation of p38 is sufficient to inhibit endothelial cell proliferation while promoting migration. Our data suggest a model whereby a shift in the balance of p38 and ERK signaling, mediated in part by induction of MKP-1, alters the ability of endothelial cells to respond normally to angiogenic factors. As a result, chronic activation of p38 may contribute directly to pathological endothelial cell dysfunction. Materials—VEGF was obtained from the NCI, National Institutes of Health developmental therapeutics program. Polyclonal anti-phosphop38, anti-pan-p38 antibodies, anti-p38α, anti-p38δ, anti-phospho-Hsp27, anti-Hsp27, anti-phospho-MEK1/2, and anti-MEK1/2 antibodies were obtained from Cell Signaling Technology (Beverly, MA). Monoclonal anti-phospho-ERK and polyclonal anti-ERK1, anti-ERK2, anti-FAK, anti-p38β, and anti-MKP-1 antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Polyclonal anti-phospho-FAK-Tyr 397 antibody and anti-MKP-1 antibody used to show expression of MKP-1 C/S (retained activity against non-human protein) were obtained from Upstate Biotechnology (Lake Placid, NY). Monoclonal anti-FLAG antibody was obtained from Sigma. Ro-31-8220, was purchased from Calbiochem. The cDNA for dominant negative p38α (DNp38α) was from Roger Davis (University of Massachusetts Medical School). Adenoviruses were all created using the Adeasy system, essentially as described previously (10Meadows K.N. Bryant P. Pumiglia K. J. Biol. Chem. 2001; 276: 49289-49298Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 15McMullen M. Keller R. Sussman M. Pumiglia K. Oncogene. 2004; 23: 1275-1282Crossref PubMed Scopus (62) Google Scholar, 23He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3256) Google Scholar). The mutant form of Hsp27 (mutHsp27) adenovirus was a generous gift of William Gerthoffer (University of Nevada School of Medicine). The MKP-1 C/S adenovirus was a generous gift of Andrey Sorokin (Medical College of Wisconsin). Cell Culture—Human umbilical vein endothelial cells (HUVECs) from pooled donors were supplied by VEC Technologies (Troy, NY) through the NCI, National Institutes of Health angiogenesis resource center and cultured as we have described previously (10Meadows K.N. Bryant P. Pumiglia K. J. Biol. Chem. 2001; 276: 49289-49298Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). For experiments requiring serum deprivation, MCDB-131 supplemented with 1% penicillin/streptomycin and 2 mm l-glutamine was used as indicated. Western Blotting—When appropriate, HUVECs were infected with an adenovirus overnight as indicated at an multiplicity of infection of 5–10. For experiments requiring growth factor stimulation, nearly confluent cells were serum-starved for 16 h prior to stimulation with 50 ng/ml VEGF or 100 ng/ml FGF for the indicated time points. Experiments conducted with subconfluent cells gave identical results. For experiments requiring integrin-induced activation, cells were trypsinized and placed into suspension for 30 min prior to replating at 50% confluency. Subsequently, whole cell lysates were collected in Laemmli sample buffer. Blotting was performed according to procedures we have described previously (10Meadows K.N. Bryant P. Pumiglia K. J. Biol. Chem. 2001; 276: 49289-49298Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). All figures are representative of at least three independent experiments. Apoptosis Assay—Caspase 3/7 activities were measured by an Apo-ONE™ homogeneous caspase 3/7 assay (Promega). A 96-well plate coated with 0.2% gelatin was seeded with 2 × 104 cells/well. The following day, experimental manipulations were initiated. After 48 h, an equal volume of lysis buffer containing the caspase substrate benzyloxycarbonyl-DEVD-R100 was added and incubated at room temperature for 1 h. The cell lysates at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a PerkinElmer Life Sciences HTS 7000 plus BioAssay Reader. Cell Proliferation and Cell Migration—These experiments were conducted essentially as described previously by us (10Meadows K.N. Bryant P. Pumiglia K. J. Biol. Chem. 2001; 276: 49289-49298Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 15McMullen M. Keller R. Sussman M. Pumiglia K. Oncogene. 2004; 23: 1275-1282Crossref PubMed Scopus (62) Google Scholar, 24Meadows K.N. Bryant P. Vincent P.A. Pumiglia K.M. Oncogene. 2004; 23: 192-200Crossref PubMed Scopus (80) Google Scholar). Labeling of Actin Cytoskeleton—HUVECs seeded onto coverslips were infected with GFP or the MEK6E adenovirus and serum-starved overnight. Subsequently, the cells were treated with VEGF (50 ng/ml) for 15 min to observe the changes in actin cytoskeleton in adherent cells. To examine changes in cells actively remodeling the actin cytoskeleton, cells infected with GFP or the MEK6E adenovirus were trypsinized, and 2.5 × 104 cells were plated onto gelatin-coated coverslips. After 4 h, cells were fixed with 3.7% formaldehyde, and actin was visualized using Texas Red phalloidin (Molecular Probes) (10Meadows K.N. Bryant P. Pumiglia K. J. Biol. Chem. 2001; 276: 49289-49298Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Statistical Analysis—All quantitative data were pooled from multiple independent experiments. One- or two-way analysis of variance was conducted as appropriate using Statistica software (Tulsa, OK). A Newman-Keuls post hoc test was performed to determine statistically significant differences (p < 0.05). As several studies have shown chronically elevated p38 activity in disease states known to have impaired angiogenesis and endothelial dysfunction, we utilized a FLAG-tagged, constitutively active MEK6 adenovirus, MEK6E, to directly study the effects of chronic p38 activity on endothelial cells. This reagent has been described previously (25Hoover H.E. Thuerauf D.J. Martindale J.J. Glembotski C.C. J. Biol. Chem. 2000; 275: 23825-23833Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). MEK6E was expressed at ∼10-fold higher amounts than the endogenous MEK6 levels (data not shown). HUVECs showed expression of the FLAG-tagged protein and induction of p38 phosphorylation at levels similar to those induced acutely by VEGF (Fig. 1A). Although each activates p38 to similar levels, the activation of p38 by VEGF is transient, whereas the MEK6E-induced p38 activation is sustained. P38 activation by MEK6E was sufficient to induce the sustained phosphorylation of a known substrate of p38, Hsp27 (Fig. 1, B and C), as well as the transcription factor ATF-2 (Fig. 1C). MEK6 can activate all of the p38 isoforms; therefore, we tested to determine which isoforms of p38 might be present and/or activated in endothelial cells. Using commercially available isoform-specific anti-sera, we detected weak activity against p38α (data not shown). No reactivity against other isoforms was detected with the available antibodies. Because dominant negative p38 isoforms have been used to define the role of specific isoforms (14Harnois C. Demers M.J. Bouchard V. Vallee K. Gagne D. Fujita N. Tsuruo T. Vezina A. Beaulieu J.F. Cote A. Vachon P.H. J. Cell. Physiol. 2004; 198: 209-222Crossref PubMed Scopus (57) Google Scholar, 26Guo Y.L. Kang B. Han J. Williamson J.R. J. Cell. Biochem. 2001; 82: 556-565Crossref PubMed Scopus (28) Google Scholar), we also employed an adenovirus coding for DNp38α (15McMullen M. Keller R. Sussman M. Pumiglia K. Oncogene. 2004; 23: 1275-1282Crossref PubMed Scopus (62) Google Scholar). As shown in Fig. 1C, co-expression of DNp38α resulted in the inhibition of both the Hsp27 phosphorylation and ATF-2 phosphorylation induced by MEK6E, suggesting that p38α is the predominant isoform activated by MEK6E. Activation of p38 by stress signals has been shown to induce apoptosis in some systems (27Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5045) Google Scholar), and inhibitor studies have indirectly implicated p38 in the regulation of apoptosis in endothelial cells (17Matsumoto T. Turesson I. Book M. Gerwins P. Claesson-Welsh L. J. Cell Biol. 2002; 156: 149-160Crossref PubMed Scopus (177) Google Scholar, 18Gratton J.P. Morales-Ruiz M. Kureishi Y. Fulton D. Walsh K. Sessa W.C. J. Biol. Chem. 2001; 276: 30359-30365Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In contrast, other studies have indicated pro-survival effects of p38 activation (28Horowitz J.C. Lee D.Y. Waghray M. Keshamouni V.G. Thomas P.E. Zhang H. Cui Z. Thannickal V.J. J. Biol. Chem. 2004; 279: 1359-1367Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 29Zhang X. Shan P. Alam J. Davis R.J. Flavell R.A. Lee P.J. J. Biol. Chem. 2003; 278: 22061-22070Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Therefore, we tested whether chronic activation of p38 modulated apoptosis by measuring caspase 3/7 activities. Whereas serum starvation in M199 media for 48 h induced apoptosis, no increase in caspase 3/7 activity was observed in MEK6E-infected cells. (Fig. 1D). However, enhanced p38 activity did enhance stress-induced apoptosis (Fig. 1D). It should be noted that serum starvation in MCDB-131 as a basal medium did not induce apoptosis (data not shown). Therefore, MCDB-131 was used for serum starvation protocols in all other experiments. A requirement for re-endothelialization following injury, as well as for angiogenesis, is enhanced cell proliferation (30Carmeliet P. Jain R.K. Nature. 2000; 407: 249-257Crossref PubMed Scopus (7531) Google Scholar, 31Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7235) Google Scholar). We investigated the effect of activating p38 on growth factor-induced DNA synthesis of endothelial cells. Analysis of [3H]thymidine incorporation revealed that the increase in the number of cells entering S phase upon VEGF treatment was completely inhibited following expression of MEK6E (Fig. 2A). Similarly, MEK6E was sufficient to inhibit FGF-induced cell cycle progression (Fig. 2B), demonstrating that this effect was not specific to VEGF responses. Expression of DNp38α was sufficient to rescue the inhibition of cell proliferation induced by MEK6E (Fig. 2B). We also measured bromodeoxyuridine incorporation, a method for measuring DNA synthesis independent of changes in total cell number (survival), and found similar results (Fig. 2C). These results clearly demonstrate that the activation of p38α by MEK6E inhibits S phase entry in response to several angiogenic factors. ERK activity is known to be essential for proliferation in many cell types (8Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2292) Google Scholar, 32Schwartz M.A. Assoian R.K. J. Cell Sci. 2001; 114: 2553-2560Crossref PubMed Google Scholar, 33Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3218) Google Scholar), including endothelial cells (10Meadows K.N. Bryant P. Pumiglia K. J. Biol. Chem. 2001; 276: 49289-49298Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Therefore, we investigated whether activation of p38 by MEK6E affected growth factor-mediated induction of ERK activity. Time course experiments of VEGF-mediated activation of ERK in control GFP-infected cells revealed peak activity at 5–10 min that remained elevated for ∼4 h (Fig. 3A). In contrast, ERK phosphorylation levels in MEK6E-infected cells were reduced significantly at all time points (Fig. 3B), although VEGF still elevated levels above basal. Similar results were observed in FGF-stimulated cells (data not shown). These data indicate that activation of p38 reduces the basal level of ERK activity and the total level of ERK activity following growth factor stimulation. ERK can also be activated by integrin engagement (34Wary K.K. Mainiero F. Isakoff S.J. Marcantonio E.E. Giancotti F.G. Cell. 1996; 87: 733-743Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar), and the integrin-mediated activation of ERK is critical for cell cycle progression (32Schwartz M.A. Assoian R.K. J. Cell Sci. 2001; 114: 2553-2560Crossref PubMed Google Scholar). We postulated that the reduced level of ERK activity might be due to inhibition of an integrin-stimulated component and investigated the effect of MEK6E on integrin-induced ERK activity. Infected cells were placed in suspension and then seeded onto fibronectin-coated dishes for the indicated times (Fig. 4A) to synchronize the integrin-mediated activation of ERK. The cells expressing GFP showed a rapid induction of ERK phosphorylation within 15 min of replating. In contrast, expression of MEK6E substantially inhibited the integrin-induced ERK activity (Fig. 4A). Similar results were observed when experiments were performed with collagen and vitronectin (Fig. 4B). This finding argues that the inhibition of ERK is likely the result of inhibition of a shared adhesion-dependent activation mechanism rather than down-regulation of a specific integrin subunit or changes in matrix-integrin affinity. Integrin signaling to FAK has been reported as being an essential component of integrin-mediated ERK activation (35Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1448) Google Scholar) and would represent a common or shared signaling protein through which p38 activation might be exerting its effects. Therefore we measured the phosphorylation of the FAK autophosphorylation site, Tyr-397, as an indicator of FAK activation. When endothelial cells were replated onto fibronectin, FAK was phosphorylated at Tyr-397, and MEK6E had no affect on this response (Fig. 4C). Taken collectively, these data indicate that MEK6E inhibits attachment-induced ERK activation. This inhibition is independent of the extracellular matrix ligand and occurs subsequent to FAK activation. To gain more insight into where the down-regulation of ERK might be occurring, we examined the effect of MEK6E expression on MEK1/2 phosphorylation levels. Time course experiments of VEGF-induced MEK1/2 activity revealed peak activation at 10 min, similar to the profile of ERK activity. Surprisingly, MEK6E-infected cells had high levels of MEK1/2 phosphorylation prior to VEGF stimulation that remained elevated at all time points (Fig. 5A). Similar results were observed in FGF-stimulated cells (Fig. 5B) and in cells replated onto fibronectin (data not shown). The control experiments confirmed that MEK6E was running at a different molecular weight than the phospho-MEK1/2 and that no antibody cross-reactivity was occurring (data not shown). These data indicate that the cross-talk between p38 and ERK is occurring at the level of ERK in our system. Furthermore, these data suggest ERK activation may normally provide negative feedback that results in the down-regulation of MEK1/2 phosphorylation. Induction of phosphatase expression can regulate MAPK cross-talk in other cell types (36Westermarck J. Li S.P. Kallunki T. Han J. Kahari V.M. Mol. Cell. Biol. 2001; 21: 2373-2383Crossref PubMed Scopus (177) Google Scholar). The dual specificity phosphatase MKP-3 has been shown previously to be an important and relatively selective MAPK phosphatase for ERK (7Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (720) Google Scholar). To determine whether this phosphatase was up-regulated by MEK6E expression, we measured levels of MKP-3 and observed no change in its expression levels (data not shown). Another dual specificity MAPK phosphatase, MKP-1, has also been shown to dephosphorylate ERK (37Pervin S. Singh R. Freije W.A. Chaudhuri G. Cancer Res. 2003; 63: 8853-8860PubMed Google Scholar, 38Sandberg E.M. Ma X. VonDerLinden D. Godeny M.D. Sayeski P.P. J. Biol. Chem. 2004; 279: 1956-1967Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 39Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1028) Google Scholar), and p38 has been linked to the induction of MKP-1 expression (40Li J. Gorospe M. Hutter D. Barnes J. Keyse S.M. Liu Y. Mol. Cell. Biol. 2001; 21: 8213-8224Crossref PubMed Scopus (170) Google Scholar). Therefore, we investigated the potential role of MKP-1 in the p38-dependent down-regulation of ERK activity. Expression of MEK6E induced MKP-1 expression (Fig. 6A), whereas pretreatment with the p38 inhibitor, SB203580, inhibited the MEK6E-induced MKP-1 expression (Fig. 6A). These data suggest that MKP-1 could play a role in the MEK6E-induced down-regulation of ERK activity. The compound Ro-31-8220 has been shown previously to inhibit MKP-1 expression by an indeterminate mechanism (41Beltman J. McCormick F. Cook S.J. J. Biol. Chem. 1996; 271: 27018-27024Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). We utilized this compound to determine whether the inhibition of MKP-1 could modulate ERK phosphorylation. Pretreatment with Ro-31-8220 inhibited MEK6E-induced MKP-1 expression (Fig. 6B) and produced an increase in the basal ERK activity in MEK6E-expressing cells; however, it was not sufficient to completely restore integrin-induced ERK activity (Fig. 6C). These data suggest that MKP-1 is playing a role in the modulation of ERK activity. However, studies have indicated that Ro-31-8220 can also modulate the activity of other signaling molecules (41Beltman J. McCormick F. Cook S.J. J. Biol. Chem. 1996; 271: 27018-27024Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Therefore, we sought to confirm these results with a more specific strategy of expressing a catalytically inactive, “substrate-trapping” mutant of MKP-1 (MKP-1 C/S) (42Pratt P.F. Bokemeyer D. Foschi M. Sorokin A. Dunn M.J. J. Biol. Chem. 2003; 278: 51928-51936Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Infecting endothelial cells with an adenovirus coding for this mutant produced an increase in immunoreactive MKP-1, documenting expression of the mutant protein (Fig. 6D). MKP-1 C/S was co-infected with MEK6E prior to replating onto fibronectin. Results show that MKP-1 C/S was also able to elevate basal levels and attenuate the inhibition of integrin-induced ERK activity (Fig. 6E). Because MKP-1 can also inhibit p38 activation, we examined the effect of expressing MKP-1 on the level of p38 phosphorylation induced by VEGF. Predic