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• W1985264276 abstract "Understanding the mechanisms that regulate cell migration is important for devising novel therapies to control metastasis or enhance wound healing. Previously, we demonstrated that β2-adrenergic receptor (β2-AR) activation in keratinocytes inhibited their migration by decreasing the phosphorylation of a critical promigratory signaling component, the extracellular signal-related kinase (ERK). Here we demonstrate that β2-AR-induced inhibition of migration is mediated by the activation of the serine/threonine phosphatase PP2A. Pretreating human keratinocytes with the PP2A inhibitor, okadaic acid, prevented the β2-AR-induced inhibition of migration, either as isolated cells or as a confluent sheet of cells repairing an in vitro “wound” and also prevented the β2-AR-induced reduction in ERK phosphorylation. Similar results were obtained with human corneal epithelial cells. In keratinocytes, immunoprecipitation studies revealed that β2-AR activation resulted in the rapid association of β2-AR with PP2A as well as a 37% increase in association of PP2A with ERK2. Finally, β2-AR activation resulted in a rapid and transient 2-fold increase in PP2A activity. Thus, we provide the first evidence that β2-AR activation in keratinocytes modulates migration via a novel pathway utilizing PP2A to alter the promigratory signaling cascade. Exploiting this pathway may result in novel therapeutic approaches for control of epithelial cell migration. Understanding the mechanisms that regulate cell migration is important for devising novel therapies to control metastasis or enhance wound healing. Previously, we demonstrated that β2-adrenergic receptor (β2-AR) activation in keratinocytes inhibited their migration by decreasing the phosphorylation of a critical promigratory signaling component, the extracellular signal-related kinase (ERK). Here we demonstrate that β2-AR-induced inhibition of migration is mediated by the activation of the serine/threonine phosphatase PP2A. Pretreating human keratinocytes with the PP2A inhibitor, okadaic acid, prevented the β2-AR-induced inhibition of migration, either as isolated cells or as a confluent sheet of cells repairing an in vitro “wound” and also prevented the β2-AR-induced reduction in ERK phosphorylation. Similar results were obtained with human corneal epithelial cells. In keratinocytes, immunoprecipitation studies revealed that β2-AR activation resulted in the rapid association of β2-AR with PP2A as well as a 37% increase in association of PP2A with ERK2. Finally, β2-AR activation resulted in a rapid and transient 2-fold increase in PP2A activity. Thus, we provide the first evidence that β2-AR activation in keratinocytes modulates migration via a novel pathway utilizing PP2A to alter the promigratory signaling cascade. Exploiting this pathway may result in novel therapeutic approaches for control of epithelial cell migration. When skin is wounded, keratinocytes within the epidermis must migrate from the wound edges to re-epithelialize the wound surface (1Woodley D. O'Toole E. Nadelman C.M. Li W. Dermatol. Found. 1999; 33: 1-12Google Scholar). One of the best characterized mediators of keratinocyte migration is the epidermal growth factor receptor (EGFR) 1The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; MAP, mitogen-activated kinase; ERK, extracellular signal-regulated kinase; β2-AR, β2-adrenergic receptor; ISO, isoproterenol; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; OA, okadaic acid; KGM, keratinocyte growth medium; AHCE, adult human corneal epithelial. (2Ando Y. Jensen P.J. J. Invest. Dermatol. 1993; 100: 633-639Abstract Full Text PDF PubMed Google Scholar, 3Fang K.S. Farboud B. Nuccitelli R. Isseroff R.R. J. Invest. Dermatol. 1998; 111: 751-756Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 4Fang K.S. Ionides E. Oster G. Nuccitelli R. Isseroff R.R. J. Cell Sci. 1999; 112: 1967-1978PubMed Google Scholar). The mitogen-activated protein (MAP) kinases, extracellular signal-related kinase 1 (ERK1) and ERK2, are activated upon EGF binding to its cognate receptor (reviewed in Ref. 5Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1663) Google Scholar). They are activated by phosphorylation on both conserved threonine and tyrosine residues and inactivated upon dephosphorylation by dual specificity phosphatases, tyrosine phosphatases, and serine/threonine-specific phosphatases (5Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1663) Google Scholar, 6Gomez N. Cohen P. Nature. 1991; 353: 170-173Crossref PubMed Scopus (419) Google Scholar, 7Alessi D.R. Gomez N. Moorhead G. Lewis T. Keyse S.M. Cohen P. Curr. Biol. 1995; 5: 283-295Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 8Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (712) Google Scholar, 9Zhou B. Wang Z.X. Zhao Y. Brautigan D.L. Zhang Z.Y. J. Biol. Chem. 2002; 277: 31818-31825Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). The activation of ERK is tightly controlled. The MAP kinase pathway is usually only transiently activated, and its constitutive activation is sufficient to cause the oncogenic transformation of some cell types (10Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar). Inhibiting ERK phosphorylation and activation prevents growth factor-induced keratinocyte migration (11Zeigler M.E. Chi Y. Schmidt T. Varani J. J. Cell. Physiol. 1999; 180: 271-284Crossref PubMed Scopus (206) Google Scholar), demonstrating the pivotal role of ERK in the keratinocyte promigratory signaling pathways (11Zeigler M.E. Chi Y. Schmidt T. Varani J. J. Cell. Physiol. 1999; 180: 271-284Crossref PubMed Scopus (206) Google Scholar, 12Leng J. Klemke R.L. Reddy A.C. Cheresh D.A. J. Biol. Chem. 1999; 274: 37855-37861Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 13Glading A. Chang P. Lauffenburger D.A. Wells A. J. Biol. Chem. 2000; 275: 2390-2398Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). In addition to the EGFR, keratinocytes also express high levels of the G-protein-coupled receptor, the β2-adrenergic receptor, (β2-AR) (14Schallreuter K.U. Wood J.M. Pittelkow M.R. Swanson N.N. Steinkraus V. Arch. Dermatol. Res. 1993; 285: 216-220Crossref PubMed Scopus (52) Google Scholar, 15Steinkraus V. Steinfath M. Korner C. Mensing H. J. Invest. Dermatol. 1992; 98: 475-480Abstract Full Text PDF PubMed Scopus (62) Google Scholar), whose functional role in the epidermis has not yet been elucidated. Keratinocytes do not express the β1-AR (16Steinkraus V. Mak J.C. Pichlmeier U. Mensing H. Ring J. Barnes P.J. Arch. Dermatol. Res. 1996; 288: 549-553Crossref PubMed Scopus (51) Google Scholar). Earlier studies have investigated the role of β-ARs in epithelial wound healing and migration, but the results have been paradoxical. β-Antagonists have been reported to either delay (17Haruta Y. Ohashi Y. Matsuda S. Eur. J. Ophthalmol. 1997; 7: 334-339Crossref PubMed Scopus (18) Google Scholar, 18Liu G.S. Trope G.E. Basu P.K. J. Ocul. Pharmacol. 1990; 6: 101-112Crossref PubMed Scopus (16) Google Scholar) or enhance (19Reidy J.J. Zarzour J. Thompson H.W. Beuerman R.W. Br. J. Ophthalmol. 1994; 78: 377-380Crossref PubMed Scopus (28) Google Scholar) corneal epithelial wound healing, and β-agonists can stimulate proliferation and migration of transformed corneal epithelial cells (20Murphy C.J. Campbell S. Araki-Sasaki K. Marfurt C.F. Cornea. 1998; 17: 529-536Crossref PubMed Scopus (29) Google Scholar), SW 480 colon carcinoma cells (21Masur K. Niggemann B. Zanker K.S. Entschladen F. Cancer Res. 2001; 61: 2866-2869PubMed Google Scholar), and bovine bronchial epithelial cells (22Spurzem J.R. Gupta J. Veys T. Kneifl K.R. Rennard S.I. Wyatt T.A. Am. J. Physiol. 2002; 282: L1108-L1116Crossref PubMed Scopus (43) Google Scholar). Conversely, β-AR agonists delay wound healing in the hind limbs of adult newts (23Donaldson D.J. Mahan J.T. Comp. Biochem. Physiol. C. 1984; 78: 267-270Crossref PubMed Scopus (34) Google Scholar) and inhibit neutrophil chemotaxis to the lungs (24Silvestri M. Oddera S. Lantero S. Rossi G.A. Respir. Med. 1999; 93: 416-423Abstract Full Text PDF PubMed Scopus (24) Google Scholar). Since keratinocytes have the capacity to synthesize β2-AR agonists and express a high level of the receptor (25Schallreuter K.U. J. Invest. Dermatol. Symp. Proc. 1997; 2: 37-40Abstract Full Text PDF PubMed Scopus (81) Google Scholar), we initiated studies to examine the role of β2-ARs in keratinocyte migration. Surprisingly, we found that unlike other cell types studied, where β2-AR agonist binding activates ERK (26Zou Y. Yao A. Zhu W. Kudoh S. Hiroi Y. Shimoyama M. Uozumi H. Kohmoto O. Takahashi T. Shibasaki F. Nagai R. Yazaki Y. Komuro I. Circulation. 2001; 104: 102-108Crossref PubMed Scopus (99) Google Scholar, 27Jordan B.A. Trapaidze N. Gomes I. Nivarthi R. Devi L.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 343-348PubMed Google Scholar, 28Crespo P. Cachero T.G. Xu N. Gutkind J.S. J. Biol. Chem. 1995; 270: 25259-25265Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 29Ahn S. Maudsley S. Luttrell L.M. Lefkowitz R.J. Daaka Y. J. Biol. Chem. 1999; 274: 1185-1188Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 30Bogoyevitch M.A. Andersson M.B. Gillespie-Brown J. Clerk A. Glennon P.E. Fuller S.J. Sugden P.H. Biochem. J. 1996; 314: 115-121Crossref PubMed Scopus (158) Google Scholar, 31Maudsley S. Pierce K.L. Zamah A.M. Miller W.E. Ahn S. Daaka Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2000; 275: 9572-9580Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 32Schmitt J.M. Stork P.J. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), in keratinocytes the β2-AR agonist isoproterenol (ISO) reduced ERK phosphorylation. Keratinocyte migration was also reduced in a dose-dependent and cAMP-independent manner (33Chen J. Hoffman B.B. Isseroff R.R. J. Invest. Dermatol. 2002; 119: 1261-1268Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The mechanisms by which a β2-AR agonist reduced MAP kinase signaling and keratinocyte migration, however, remained enigmatic. We reasoned that the serine/threonine phosphatase, PP2A, known to play a role in a multitude of cellular functions (reviewed in Ref. 34Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1549) Google Scholar), could be a good candidate for the mediation of the observed β2-AR agonist effects. A number of studies have linked β2-AR activation to either PP2A function, ERK activation, or migration. ISO can increase the activation of PP2A in the rat heart (35Boknik P. Fockenbrock M. Herzig S. Knapp J. Linck B. Luss H. Muller F.U. Muller T. Schmitz W. Schroder F. Neumann J. Naunyn-Schmiedeberg's Arch. Pharmacol. 2000; 362: 222-231Crossref PubMed Scopus (72) Google Scholar), PP2A can dephosphorylate mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1) and ERK family kinases in vitro (6Gomez N. Cohen P. Nature. 1991; 353: 170-173Crossref PubMed Scopus (419) Google Scholar, 7Alessi D.R. Gomez N. Moorhead G. Lewis T. Keyse S.M. Cohen P. Curr. Biol. 1995; 5: 283-295Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 9Zhou B. Wang Z.X. Zhao Y. Brautigan D.L. Zhang Z.Y. J. Biol. Chem. 2002; 277: 31818-31825Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 36Anderson N.G. Maller J.L. Tonks N.K. Sturgill T.W. Nature. 1990; 343: 651-653Crossref PubMed Scopus (797) Google Scholar, 37Garcia L. Garcia F. Llorens F. Unzeta M. Itarte E. Gomez N. FEBS Lett. 2002; 523: 90-94Crossref PubMed Scopus (45) Google Scholar), and decreased PP2A activity promotes migration in a number of transformed cells and cancer cell lines (38Benefield J. Meisinger J. Petruzzelli G.J. Young M.R. Invasion Metastasis. 1997; 17: 210-220PubMed Google Scholar, 39Young M.R. Adv. Exp. Med. Biol. 1997; 407: 311-318Crossref PubMed Google Scholar, 40Jackson J. Meisinger J. Patel S. Lim Z.C. Vellody K. Metz R. Young M.R. Invasion Metastasis. 1997; 17: 199-209PubMed Google Scholar). We therefore evaluated the possibility that the observed β2-AR-induced decrease in ERK phosphorylation and migration in human keratinocytes could be regulated by the β2-AR agonist-mediated activation of PP2A. Using okadaic acid (OA) to inhibit intracellular PP2A (41Namboodiripad A.N. Jennings M.L. Am. J. Physiol. 1996; 270: C449-C456Crossref PubMed Google Scholar, 42Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar, 43McMahon K.A. Wilson N.J. Marks D.C. Beecroft T.L. Whitty G.A. Hamilton J.A. Csar X.F. Biochem. J. 2001; 358: 431-436Crossref PubMed Scopus (18) Google Scholar, 44Fernandez J.J. Candenas M.L. Souto M.L. Trujillo M.M. Norte M. Curr. Med. Chem. 2002; 9: 229-262Crossref PubMed Scopus (134) Google Scholar), we demonstrate that OA pretreatment completely prevented the β2-AR agonist-induced decrease in keratinocyte migration in two different in vitro migration assays. Likewise, OA pretreatment also prevented the β2-AR agonist-induced reduction in ERK phosphorylation. Similar results were obtained in human corneal epithelial cells; OA pretreatment completely prevented the β2-AR agonist-induced decrease in wound healing and also prevented the β2-AR agonist-induced reduction in ERK phosphorylation. Further, treating keratinocytes with ISO increased the intracellular association of PP2A with ERK2 and initiated the association of β2-AR with PP2A. Finally, the β2-AR agonist rapidly and transiently increased intracellular PP2A activity. Here we provide the first evidence that PP2A plays a crucial role in the β2-AR agonist-mediated decrease in ERK phosphorylation and provides a novel regulatory mechanism of epithelial cell migration. Keratinocyte Growth—Human keratinocytes were isolated from primary keratinocyte cultures derived from neonatal and adult foreskins as we have reported previously (45Isseroff R.R. Ziboh V.A. Chapkin R.S. Martinez D.T. J. Lipid Res. 1987; 28: 1342-1349Abstract Full Text PDF PubMed Google Scholar), using a modification of the method of Rheinwald and Green (46Rheinwald J.G. Green H. Cell. 1975; 6: 331-343Abstract Full Text PDF PubMed Scopus (3936) Google Scholar). Cells were grown in keratinocyte growth medium (KGM) (Medium 154; 0.2 mm Ca2+), supplemented with human keratinocyte growth supplement (0.2 ng/ml EGF, 5 μg/ml insulin, 5 μg/ml transferrin, 0.18 μg/ml hydrocortisone, and 0.2% bovine pituitary extract) (Cascade Biologics, Inc., Portland, OR) and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin) (Gemini Bio-Products, Inc., Calabasas, CA) at 37 °C in a humidified atmosphere of 5% CO2. Since keratinocyte migration is optimal on a collagen matrix (1Woodley D. O'Toole E. Nadelman C.M. Li W. Dermatol. Found. 1999; 33: 1-12Google Scholar, 47Sheridan D.M. Isseroff R.R. Nuccitelli R. J. Invest. Dermatol. 1996; 106: 642-646Abstract Full Text PDF PubMed Scopus (100) Google Scholar), all experiments were performed on cells plated for 2 h at 37 °C on glass coverslips (Eppendorf, Hamburg, Germany) or plastic tissue culture dishes (Corning Glass) that had been coated for 1 h at 37 °C with 60 μg/ml collagen I (Celtrix Pharmaceuticals, Santa Clara, CA). Cell strains isolated from at least two different foreskins were used, and experiments were performed with subcultured cells between passages 4 and 7. Neonatal keratinocytes were used in all experiments except where noted. Corneal Epithelial Cell Growth—Human adult corneas that had been donated for research were obtained from the Sierra Eye and Tissue Donor Services (Sacramento, CA; a regional center of Dialysis Clinics Inc., Donor Services, Nashville, TN) within 2–10 days of collection. Corneas were stored in Optisol-GS corneal storage medium (Chiron Ophthalmics, Irvine, CA) at 2–8 °C and were transported to the laboratory on ice. The research followed the tenets of the Declaration of Helsinki, tissue was obtained with appropriate consents from either donor or next of kin, and the research was approved by the University institutional review board. Corneal epithelial cells were isolated as we have described (48Han B. Schwab I.R. Madsen T.K. Isseroff R.R. Cornea. 2002; 21: 505-510Crossref PubMed Scopus (111) Google Scholar) and maintained in a 37 °C incubator with 5% CO2 in EpiLife medium (Cascade Biologics) supplemented with 0.18 μg/ml hydrocortisone, 5 μg/ml transferrin, 0.2% bovine pituitary extract, and 1 ng/ml mouse EGF, calcium (final concentration 0.06 mm), and antibiotics/antimycotic (100 units of penicillin G/ml, 100 μg of streptomycin/ml, and 0.25 μg of amphotericin B/ml). Passage 3–5 cells were used for all experiments. Scratch Assay—Cells were grown to confluence on collagen-coated plastic tissue culture dishes. A sterile pipette tip was used to scratch a 1-mm-wide wound along the center of the dish, and a demarcated area of the wound was photographed on an inverted Nikon Diaphot microscope at the time of wounding (time 0) up to wound healing (49Haas A.F. Isseroff R.R. Wheeland R.G. Rood P.A. Graves P.J. J. Invest. Dermatol. 1990; 94: 822-826Abstract Full Text PDF PubMed Google Scholar). The area of the wound was determined using NIH Image 1.6 (public domain image processing and analysis program for the Macintosh developed at the National Institutes of health and available on the Internet at rsb.info.nih.gov/nih-image/), and the percentage of wound healing was calculated by dividing the area of the wound at time X by the area of the wound at time 0 and multiplying by 100. In some experiments, to prevent cell proliferation, which could confound the analysis of cell migration into the wound, cells were preincubated with mitomycin C (10 μg/ml) for 1 h at 37 °C (50You Y.S. Lim S.J. Chung H.S. Lee Y.G. Kim C.Y. Hong Y.J. Yonsei Med. J. 2000; 41: 185-189Crossref PubMed Scopus (11) Google Scholar). To confirm that the observed reduction in keratinocyte migration induced by ISO was due to β2-AR activation, in some experiments, human keratinocytes were preincubated for 15 min at 37 °C in the presence or absence of timolol (20 μm), a β2-AR antagonist. Significance was determined using Student's t test. Single Cell Migration Assay—Cells were plated onto collagen-coated glass coverslips at a density of 50 cells/mm2 for 2 h at 37 °C. The coverslips were then placed into a migration chamber to monitor individual cell migration over a 1-h period at 37 °C, as described previously (4Fang K.S. Ionides E. Oster G. Nuccitelli R. Isseroff R.R. J. Cell Sci. 1999; 112: 1967-1978PubMed Google Scholar). The migration chamber was placed on an inverted Nikon Diaphot microscope. Time lapse images of the cell migratory paths were digitally captured to a video frame grabber on a Power Macintosh 8500/120 every 10 min for a 1-h period and imported to a recompiled version of NIH Image 1.60. After each cell's center of mass was tracked, the data were automatically exported to FileMaker Pro 3.0, where they were analyzed and stored. Cell migration was quantitatively analyzed, and the speed and distance traveled by each cell were calculated using a cell tracking program compiled in this laboratory (3Fang K.S. Farboud B. Nuccitelli R. Isseroff R.R. J. Invest. Dermatol. 1998; 111: 751-756Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Significance was determined using Student's t test. Cell Treatments—1–2 × 106 cells were preincubated with either KGM alone or KGM containing 10 nm OA for 45 min at 37 °C. OA inhibits purified PP2A with a half-maximal concentration on the order of 1 nm; it also inhibits PP1, but at 100-fold higher concentrations in vitro (51Bialojan C. Takai A. Biochem. J. 1988; 256: 283-290Crossref PubMed Scopus (1518) Google Scholar, 52Cohen P. Klumpp S. Schelling D.L. FEBS Lett. 1989; 250: 596-600Crossref PubMed Scopus (425) Google Scholar). OA is membrane-permeant (41Namboodiripad A.N. Jennings M.L. Am. J. Physiol. 1996; 270: C449-C456Crossref PubMed Google Scholar), and it can be added to cells up to concentrations of 1 μm without any detectable intracellular inhibitory effect on PP1, PP2B, or PP2C (42Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). Cells were then stimulated with KGM containing either 10 ng/ml EGF (Biomedical Technologies Inc., Stoughton, MA), 10 nm ISO (Calbiochem), 10 nm OA (Calbiochem), 10 nm ISO plus 10 nm OA, or KGM alone (control) for 15 min at 37 °C unless otherwise noted. Cells were placed immediately on ice, washed twice with ice-cold phosphate-buffered saline containing phosphatase inhibitors (50 mm NaF and 1 mm Na3VO4), and scraped in 50 μl to 1 ml of lysis buffer (phosphate-buffered saline containing 0.5% Triton X-100, 50 mm NaF, 1 mm Na3VO4, 10 μg/ml leupeptin, 30 μg/ml aprotinin, 200 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin A). The lysates were transferred into 1.5-ml tubes, incubated on ice for 20 min, and then centrifuged at 14,000 × g for 10 min at 4 °C (53Pullar C.E. Repetto B. Gilfillan A.M. J. Immunol. 1996; 157: 1226-1232PubMed Google Scholar). The supernatants were electrophoresed immediately on 10% polyacrylamide Tris-HCl gels (Bio-Rad) or stored at –80 °C. The protein concentration of the samples was determined using the Bradford Assay (Bio-Rad). Immunoblotting—Five μg of each protein sample was added to an equal volume of 2× reducing sample loading buffer (0.0625 m Tris-HCl, pH 6.8, 3% SDS, 10% glycerol, 5% β-mercaptoethanol) and electrophoresed on 10% polyacrylamide Tris-HCl gels. Proteins were transferred to Immobilon membranes and immunoblotted with either an anti-ERK (9102), anti-MEK1 (9122), anti-phospho-ERK (9101), antiphospho-MEK (9121) (Cell Signaling Technology, Beverly, MA), anti-PP2A (C-20), anti-EGFR (1005), anti-β2-AR (H-20) (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-phosphotyrosine antibody (Ab-4) (Oncogene, Boston, MA). The immunoblots were developed by ECL according to the manufacturer's instructions (Amersham Biosciences). Densitometry was performed on scanned images using NIH Image 1.6. Immunoprecipitation—Five μg of antibody linked to 30 μl of prewashed protein A/G-Sepharose beads (Amersham Biosciences) was used to immunoprecipitate the desired proteins from 1 ml of keratinocyte cell lysate prepared from 1–2 × 107 cells. Lysates were initially precleared with 150 μl of prewashed beads for 30 min at room temperature and then incubated with the antibody-bound beads at 4 °C overnight on a rotary mixer. The beads were washed five times with lysis buffer, 1× reducing sample loading buffer was added, and the samples were boiled for 3 min and centrifuged to pellet the beads. The supernatants were loaded onto 10% polyacrylamide Tris-HCl gels (Bio-Rad), and the proteins were separated electrophoretically followed by transfer to Immobilon membrane for immunoblotting as outlined above. Phosphatase Assay—Phosphatase activity was measured using the nonradioactive serine/threonine phosphatase assay system (Promega, Madison, WI). The system determines the amount of free phosphate released from a phosphopeptide by measuring the absorbance of a molybdate-malachite green-phosphate complex. Cells were either untreated or stimulated with 10 μm ISO for 30 s at 37 °C. The plates were placed immediately on ice, washed in washing buffer (20 mm Tris, pH 8, 0.1 mm EDTA, pH 8), and lysed in a phosphate-free buffer containing no phosphatase inhibitors (20 mm Tris, pH 8, 0.1 mm EDTA, pH 8, 0.1% Triton X-100, protease inhibitor mixture (Roche Diagnostics)). The lysates were incubated on ice for 30 min and then cleared by centrifugation at 16,000 × g for 20 min at 4 °C. Control and ISO-treated keratinocyte cell lysates were centrifuged at 600 × g for 5 min at 4 °C through lysis buffer-washed Sephadex-25 spin columns to remove free phosphate. Twenty-five μg of lysate in the presence or absence of the PP2A-specific serine/threonine phosphopeptide substrate (RRApTVA, where pT represents phosphothreonine) (100 μm) was incubated for 3 min at 30 °C in PP2A-specific buffer (50 mm imidazole, pH 7.2, 200 μm EGTA, 0.02% β-mercaptoethanol, 0.1 mg/ml bovine serum albumin) in a reaction volume of 50 μl in a 96-well plate to determine PP2A activity. The reaction was terminated by the addition of an equal volume of molybdate dye additive. The plate was incubated at room temperature for 30 min to allow full color development. Absorbance was measured at 630 nm in a plate reader (Bio-Tek Instruments Inc., Winooski, VT). Significance was determined using Student's t test. OA Reversed the β2-AR Agonist-induced Inhibition of Keratinocyte Wound Healing—Our previous work demonstrated that activation of β2-AR in human keratinocytes decreased their migratory speed (33Chen J. Hoffman B.B. Isseroff R.R. J. Invest. Dermatol. 2002; 119: 1261-1268Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). We then reasoned that if β2-AR-mediated activation of PP2A was responsible for the ISO-induced decrease in migration, then preincubating cells with OA should prevent it. We chose to initially investigate the effects of ISO and OA on keratinocyte migration using a “scratch” assay that provided an in vitro model of wound healing (49Haas A.F. Isseroff R.R. Wheeland R.G. Rood P.A. Graves P.J. J. Invest. Dermatol. 1990; 94: 822-826Abstract Full Text PDF PubMed Google Scholar). This allowed the monitoring of keratinocyte migration at the edge of “wounds” generated in vitro by scraping a cell-free zone in a confluent sheet of cells. The contribution of cell proliferation to the in vitro wound healing was abrogated by pretreating cultures with mitomycin C. Control wounds, incubated in EGF-containing culture medium, completely healed in 24 h, whereas at that time ISO-treated cells were only 62% healed (p < 0.01) (Fig. 1A). Pretreatment of cells with OA (10 nm) prior to ISO, however, reversed the ISO-induced defects in wound repair, resulting in complete wound healing at 24 h. OA alone had no effect on the rate of wound closure. Although healing was delayed with ISO treatment, the wounds did heal by 36 h (results not shown), demonstrating that the observed decrease in migratory speed was not due to nonspecific toxicity. To demonstrate that the ISO-induced reduction in wound healing was the result of its specific receptor activation, the receptor was prebound with a β2-AR antagonist, timolol. A 15-min preincubation with timolol (20 μm) completely prevented the ISO-induced reduction in wound healing (Fig. 1B), demonstrating that the ISO-induced effects on migration were specifically through activating the β2-AR. The ISO-treated cells were only 60% healed by 24 h but did heal completely by 36 h (results not shown). OA Reversed the β2-AR Agonist-induced Reduction of Keratinocyte Migratory Speed—Since OA treatment completely prevented the β2-AR-induced reduction in wound healing, this suggested that PP2A may likewise play a role in the β2-AR agonist-induced decrease in keratinocyte migration. To more precisely measure the β2-AR agonist-induced reduction in keratinocyte speed, we quantitated the locomotory speed of individual cells (47Sheridan D.M. Isseroff R.R. Nuccitelli R. J. Invest. Dermatol. 1996; 106: 642-646Abstract Full Text PDF PubMed Scopus (100) Google Scholar). The addition of ISO to the migration chamber reduced both the mean distance traveled and the speed by half (p < 0.01) (Fig. 2, A and B). OA pretreatment completely prevented the ISO-induced reduction in both distance and speed traveled by the cells over the 1-h period. Preincubation with OA alone did not significantly affect either distance traveled or speed (Fig. 2, A and B). There was no statistically significant difference between the control, OA, and OA/ISO groups. OA pretreatment also prevented the ISO-induced reduction in migratory speed of keratinocytes isolated from adult foreskins (results not shown). These results, demonstrating OA-mediated restitution of keratinocyte migratory speed in ISO-treated cells, complement those obtained in the scratch assay, providing additional support for the hypothesis that PP2A activation mediates the decrease in migration speed observed after β2-AR agonist treatment. OA Reversed the β2-AR Agonist-induced Dephosphorylation of ERK in Human Keratinocytes—The ability of OA to restore migration in β2-AR agonist-treated cells prompted us to investigate whether it could also prevent the β2-AR-mediated decrease in ERK phosphorylation observed in our earlier work (33Chen J. Hoffman B.B. Isseroff R.R. J. Invest. Dermatol. 2002; 119: 1261-1268Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In order to determine the mechanism by which β2-AR activation resulted in decreased ERK phosphorylation, we examined critical members of the ERK/MAP kinase pathway (Fig. 7) after treating cells with ISO and or OA. First, the ability of β2-AR ligation to transactivate the EGFR, as previously reported (54Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar, 55Carpenter G. J. Cell Biol. 1999; 146: 697-702Crossref PubMed Scopus (247) Google Scholar, 56Hackel P.O. Zwick E. Prenzel N. Ullrich A. Curr. Opin. Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (546) Google Scholar, 57Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF" @default.
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• W1985264276 title "PP2A Activation by β2-Adrenergic Receptor Agonists" @default.
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