[CANCER RESEARCH 63, 3473–3477, July 1, 2003] Advances in Brief p38 Mitogen-activated Protein Kinase Pathway Suppresses Cell Survival by Inducing Dephosphorylation of Mitogen-activated Protein/Extracellular Signal-regulated Kinase Kinase1,2 1 Song-Ping Li, Melissa R. Junttila, Jiahuai Han, Veli-Matti Ka ¨ha ¨ri, and Jukka Westermarck 2 Turku Centre for Biotechnology [S-P. L., M. R. J., V-M. K., J. W.], University of Turku and Åbo Akademi University and Departments of Medical Biochemistry and Dermatology [V-M. K.], University of Turku, FIN-20520 Turku, Finland, and Department of Immunology, Scripps Research Institute, La Jolla, California 92121 [J. H.] Abstract Raf/mitogen-activated protein/extracellular signal-regulated kinase ki- nase (MEK)1,2/extracellular signal-regulated kinase1,2 and MKK3,6/p38 mitogen-activated protein kinase pathways play an important role in cellular survival and apoptosis. The results of this study identify novel mechanisms to explain the opposing effects of these pathways in the regulation of apoptosis induction. Our results show that activation of p38 by adenoviral expression of MKK3b or arsenite treatment was followed by rapid dephosphorylation of MEK1,2 and subsequent apoptosis in human skin fibroblasts. Inhibition of p38 activity by SB203580 and ad- enoviral expression of dominant-negative forms of p38 potently inhibited MEK1,2 dephosphorylation and apoptosis. Strikingly, p38-mediated de- phosphorylation of MEK1,2, was not detected in a series of transformed human cell lines. Taken together, we provide evidence for mechanisms unidentified previously that negatively regulates survival signaling during apoptosis induction. In addition, we show that in all transformed cell lines we have studied thus far, the function of this pathway is impaired. Introduction MAPKs 3 are a family of eukaryotic serine/threonine protein kinases widely conserved among eukaryotes. MAPKs regulate many cellular processes, such as cell proliferation, migration, differentiation, and death (1). Three MAPK pathways have been characterized in detail: (a) ERK1,2; (b) JNK1,2; and (c) the p38 group of MAPKs (1). The signaling cascade Raf/MEK1,2/ERK1,2 is prototypically acti- vated by mitogenic growth factors, and it plays a crucial role in the regulation of cell proliferation and survival (1, 2). The critical role of ERK1,2 signaling pathway in cell survival is supported by findings that activated alleles of MEK1 and MEK2 promote cell survival independently of survival factors and that dominant interfering MEK1 and MEK2 alleles disrupt cell survival signaling (2). The role of ERK1,2 pathway in malignancies has been a topic of intensive re- search lately. Increased activity of this pathway has been detected in several cancers (2–5). Recently, mutations of B-Raf, which increase activity of MEK1,2-ERK1,2 pathway, were found in 66% of mel- anomas, and expression of such mutants in NIH3T3 cells led to transformation (3). These studies have indicated that inhibition of this pathway might have important implications in cancer therapy (2– 6). Chemical inhibitors of MEK1,2 are currently being evaluated for treatment of cancer, and it has been shown that they suppress tumor growth and invasion (2, 4, 5). Pro-apoptotic p38 MAPK pathway is activated by cellular stress, including UV light, arsenite, osmotic shock, and inflammatory cyto- kines (7). Recent findings indicate a requirement for a correct balance between MEK1,2-ERK1,2 and p38 signaling pathways to ensure appropriate regulation of cell survival (2, 8). However, the molecular mechanisms regulating the balance between ERK1,2 and p38 path- ways are largely unknown. We have reported previously that activa- tion of p38 MAPK by arsenite inhibits ERK1,2 signaling pathway and collagenase-1 (MMP-1) promoter activity via PP1/PP2A-dependent dephosphorylation of MEK1,2 (9). Here, we have examined whether p38-mediated inactivation of ERK1,2 signaling cascade affects cell survival. Our results show that p38-mediated dephosphorylation of MEK1,2 mediates initiation of apoptosis by arsenite or p38 activation in normal human skin fibroblasts and rat primary neurons. Impor- tantly, p38-mediated MEK1,2 dephosphorylation was not detected in several cancer cell lines, suggesting that this pathway is suppressed during malignant transformation to promote cell survival. Materials and Methods Cell Cultures. Normal human skin fibroblast cultures were established from a healthy male volunteer donor (aged 28 years). All transformed cancer cell lines were obtained from American Type Culture Collection. Cells were cultured in DMEM supplement with 10% FCS, 2 mM glutamine, 100 IU/ml penicillin G, and 100 g/ml streptomycin. Terminally differentiated rat CGNs were a generous gift from Dr. Eleanor Coffey (10). Reagents and Antibodies. TPA and sodium m-arsenite were purchased from Sigma Chemical Co. The p38 inhibitor SB203580 and MEK1,2 inhibitor PD98059 were from Calbiochem. Phospho-specific MEK1,2, ERK1,2, JNK, p38, and Akt antibodies and antibodies against total MEK1,2, and p38 were obtained from Cell Signaling Biotechnology. PARP antibody was obtained from Santa Cruz Biotechnology. Determination of MAPK Activity. The activation of MEK1,2, p38, JNK, and Akt was determined by Western blotting with antibodies specific for phosphorylated, activated forms of these kinases. Cells were maintained for 18 h in medium supplemented with 1% FCS, treated as indicated, and lyzed in 100 l of Laemmli sample buffer. Western blotting was performed as de- scribed previously (9). Determination of Cell Viability. To determine the effect of sodium ar- senite on cell viability, cells were seeded in 96-well plates and cultured in 0.5% or 1% FCS/DMEM medium for 24 h. Afterward, medium was changed, and cells were supplied with medium containing sodium arsenite, SB203580, or PD98059 for indicated time periods. The cell viability was determined by CellTiter 96 AQ ueous nonradioactive cell proliferation assay (Promega). Detection of Apoptotic Cells. Human skin fibroblasts were seeded on glass slides and cultured in medium with 1% FCS. Where indicated, cells were Received 3/5/03; accepted 5/19/03. 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. 1 Supported by grants from the Foundation of Finnish Cancer Institute, the Academy of Finland (Projects 30985, 45996, and 878179), Sigrid Juselius Foundation, Cancer Research Foundation of Finland, Turku University Hospital (EVO Grant 13336), and a research contract from the Finnish Life and Pension Insurance Companies. 2 To whom requests for reprints should be addressed, at Turku Centre for Biotechnol- ogy, University of Turku and Åbo Akademi University, FIN-20520 Turku, Finland. E-mail: jukwes@utu.fi. 3 The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extra- cellular signal-regulated kinase; MEK, mitogen-activated protein/extracellular signal- regulated kinase kinase; TPA, 12-O-tetradecanoyl-13-phorbol acetate; JNK, c-Jun NH 2 - terminal protein kinase; MKK, ; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; MOI, multiplicity of infection; PARP, poly(ADP-ribose) polymerase; CGN, cerebellar granular neuron; MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide. 3473