Vol. 12, 641– 647, December 2001
Cell Growth & Differentiation
Mitogen-activated Protein Kinase Is Required for Bryostatin 1-induced Differentiation of the Human Acute Lymphoblastic Leukemia Cell Line Reh1 Nathan R. Wall, Ramzi M. Mohammad, and Ayad M. Al-Katib2 Division of Hematology and Oncology, Department of Internal Medicine, Karmanos Cancer Institute at Wayne State University School of Medicine, Detroit, Michigan 48201
oligonucleotides blocked bryo 1-induced expression of CD11c. Our analysis also shows that CD11c’s gene promoter activity is augmented by bryo 1. Therefore, we conclude that activation of the MEK/ERK signaling pathway is necessary for bryo 1-induced differentiation of the pre-B Acute Lymphoblastic Leukemia cell line Reh.
Abstract Bryostatin 1 (bryo 1) is known to induce the differentiation and cell cycle arrest of human lymphoid leukemia cells in vitro. The extracellular signal-regulated kinase (ERK), originally identified as a participant in mitogenic signaling, has recently been implicated in the signaling of cellular differentiation. To examine the role of the ERK/mitogen-activated protein (MAP) kinase pathway in B-lymphoid cell differentiation of the Reh Acute Lymphoblastic Leukemia cell line, the effects of bryo 1 on ERK activation were determined. On bryo 1 treatment, the activity of ERK2 (p42) rapidly increased, with ERK1 (p44) protein levels remaining constant. p44/42 immunoprecipitates from lysates of bryo 1-treated cells had increased their ability to phosphorylate the transcription factor Elk-1. Constitutive AP-1 activity was shown to be potentiated after bryo 1 treatment using electrophoretic mobility shift assays. The protein composition of the AP-1 transcription factor complex activated by bryo 1 was analyzed using supershift analysis with specific antibodies against c-Fos, Fos B, c-Jun, Jun B, and Jun D proteins. Supershift analysis revealed that the bryo 1-induced AP-1 complex was composed predominantly of Fos B and Jun D. Therefore, we evaluated the effects of inhibiting MAP/ERK kinase (MEK) on both DNA binding and cellular differentiation. Treatment of Reh cells with 20 M PD98059, a specific inhibitor of MEK, inhibited bryo 1-induced ERK activity and DNA binding. Furthermore, PD98059 blocked the bryo 1-induced differentiation of Reh cells, as assessed by a number of features associated with lymphoid differentiation, including changes in morphology, cell growth arrest, attachment, and increased expression of the leukocyte integrin CD11c. Moreover, transient transfection of Reh cells with antisense MAP kinase
Received 5/7/01; revised 10/2/01; accepted 10/9/01. 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 in part by NIH Grant P30 CA22453-20. 2 To whom requests for reprints should be addressed, at Division of Hematology and Oncology, Wayne State University School of Medicine, P. O. Box 02143, Detroit, MI 48201. Phone: (313) 745-8217; Fax: (313) 993-0307; E-mail: [email protected]
Introduction It is well recognized that the ERK3/MAPK signal transduction pathway is important in relaying signals from receptors on a cell surface to the nucleus to stimulate cellular responses, including proliferation, differentiation, and regulation of specific metabolic pathways (1, 2). In particular, signaling via receptor tyrosine kinases leads to a characterized chain of biochemical events, among which include the sequential activation of p21ras, Raf, MEK, ERK/MAPK, and the transcription factor Elk1 (1, 3–5). The role of ERK/MAPK in cellular differentiation has been well documented in some systems. Nerve growth factor induction of neural differentiation in PC12 pheochromocytoma cells requires sustained activation and associated nuclear translocation of ERK/MAPK (6, 7). Microinjection of constitutively active mutants of MEK induces neurite extension in PC12 cells, and dominantnegative mutants of MEK block nerve growth factor induction of neurite extension (8). Development of thymocytes from immature CD4-, CD8- cells to intermediate CD4⫹, CD8⫹ cells also requires activation of ERK/MAPK (9), and positive thymic selection but not negative selection or T-cell receptor-induced proliferation requires activation of the ERK/ MAPK pathway (10). Many types of cellular differentiation, including myogenesis and adipogenesis, appear to proceed in a MAPK-independent fashion (11). However, in hematopoietic differentiation, the role of the ERK/MAPK pathway has not yet been fully characterized. Previous studies in our laboratory and in others have shown that modulation of PKC by two PKC activators/deactivators, the phorbol ester TPA, and the marine natural agent bryo 1 induce leukocyte differentiation of the early pre-B ALL cell line Reh and the CLL cell line WSU-CLL (12–15). Differentiation in these cell lines was evaluated by the development of filopodia and numerous vacuoles indicating phagocytic activity. Cells also increase in size with many adhering
3 The abbreviations used are: ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; bryo 1, bryostatin 1; ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; EMSA, electrophoretic mobility shift assay; AP-1, activator protein-1; FACS, fluorescence-activated cell sorter; MoAb, monoclonal antibody; DOTAP, N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate.
MAPK Is Required for Bryo 1-induced Differentiation
Fig. 1. A, effect of MEK inhibitors PD 98059 (20 M) and U0126 (15 M) on bryo 1-induced cell growth inhibition. B, Reh cell differentiation. Reh cells were treated with bryo 1, the MEK inhibitor PD 98059, or the combination of bryo 1 and PD 98059 for 24 h. Bryo 1-induced cellular differentiation was reduced significantly when cells were pretreated for 30 min with PD 98059 as shown by cellular adhesion and surface expression of CD11c.
to the surface of the culture flask. There was induction in the cell surface antigens CD11c and CD22 with the concomitant down-regulation of CD10 and CD19. Enzymatically, bryo 1 and TPA induced tartrate-sensitive acid phosphatase expression but failed to induce periodic acid Schiff and nonspecific esterase. The ERK/MAPK pathway, known to be activated by PKC and to be involved in some models of cellular differentiation, represents an interesting target in the study of bryo 1induced cellular differentiation. To address the question of whether the ERK/MAPK signaling pathway is involved in bryostatin-induced B-cell differentiation, we examined the effects of bryo 1 on ERK/MAPK activation. Our results show that on bryo 1 treatment of the Reh ALL cell line, ERK2 (p42) activity increases as does the phosphorylation of the ERK/ MAPK target transcription factor Elk-1 by kinase assay. Furthermore, bryo 1 potentiated the binding kinetics of AP-1 as determined by EMSA. The phosphorylation of Elk-1, AP-1binding, cellular adherence and expression of CD11c were all down-regulated or abolished when the Reh cells were pretreated with the MEK inhibitor PD 98059 before bryo 1 treatment. This analysis also indicated that bryo 1 stimulates the CD11c gene promoter and identified the transcription factor AP-1 as a central target implicated in this activation.
Results Chemical Inhibition of MAPK Reduces Bryo 1-induced Reh ALL Cell Adhesion and CD11c Protein Expression. Fig. 1A shows that MEK inhibitors PD98059 and U0126 have no
significant growth inhibition compared with control. Additionally, there was no toxicity associated with adding bryo 1 to them. We examined the effect of one chemical inhibitor of ERK/MAPK, PD 98059 (16), on bryo 1-induced Reh cellular adherence and CD11c expression. Bryo 1 treatment of the Reh cells for 24 h induced ⬃75 and 50% cellular adherence and CD11c expression, respectively (Fig. 1). Reh cells treated with 20 M PD 98059 for 30 min before bryo 1 diminished adherence and CD11c induction to ⬃12 and 30%, respectively. Bryo 1 Induces ERK/MAPK Activity in the Reh ALL Cell Line. To study bryo 1’s effects on the ERK/MAPK pathway, Reh cells were analyzed by two independent techniques: immunoblotting with an antibody specific for activated ERK kinases and immunoprecipitation-kinase assays with an antiERK antibody. Fig. 2A shows a representative immunoblot of Reh cellular extracts pre and postbryo 1 using an antibody that detects the dual phosphorylated forms of p42 ERK2 and p44 ERK1. Bryo 1 induced the activity of p42 ERK2 in Reh cells while not affecting the level of p44 ERK1 activity. The immunoprecipitation-kinase assay shown in Fig. 2B confirmed the immunoblot data. Treatment of Reh ALL cells for 3 h with bryo 1 caused a 4-fold increase in ERK/MAPK activity as shown by phosphorylation of the p42 ERK2 nuclear target, Elk-1 (Fig. 2B). PD 98059 treatment of the Reh cells 30 min before bryo 1 treatment for 3 h significantly reduced but did not completely inhibit ERK/MAPK activity. A second chemical inhibitor of ERK/MAPK, UO126 (18), also showed the ability to significantly inhibit bryo 1’s ability to
Cell Growth & Differentiation
Fig. 2. Bryo 1 induced ERK/MAPK activity in the Reh cell line. In A, Western blots were run using total cell lysates from Reh cells treated with bryo 1 and probed with MoAbs specific for the phosphorylated p44/p42 MAPK. Bryo 1 is shown inducing the phosphorylated form of p42. In B, a kinase assay was run using phospho-p44/p42 MAPK immunoprecipitates, which was then incubated with Elk-1 substrate. Western blots were run and probed with antibodies specific for phopho-Elk-1. Each experiment was completed in duplicate with a representative experiment shown.
induce ERK/MAPK activity in Reh cells when it was used at 15 M (Fig. 2B). Bryo 1 Induces the Formation of AP-1/DNA Complexes in Reh ALL Cells. Fos and Jun proteins dimerize to form the AP-1 transcription factor complex. Thus, we investigated bryo 1’s ability to induce the formation of AP-1 complexes in Reh cells. EMSAs revealed that control cells contain very modest levels of preformed AP-1 complexes. However, bryo 1 induces a time-dependent increase in AP-1-DNA binding activity (Fig. 3A). The formation of AP-1 complexes was completely inhibited by the addition of excess cold AP-1 Consensus DNA sequence, indicating that the DNA-protein interactions were sequence specific. The protein composition of the AP-1 transcription factor complex activated by bryo 1 was analyzed using supershift analysis with specific antibodies against c-Fos, Fos B, c-Jun, Jun B, and Jun D proteins. Supershift analysis revealed that the bryo 1induced AP-1 complex was composed predominantly of Fos B and Jun D (Fig. 3A). When Reh cells were again pretreated for 30 min with chemical inhibitors of the ERK/MAPK signal transduction pathway, AP-1 complexes failed to assemble on the DNA (Fig. 3B). Role of AP-1 on CD11c Gene Promoter Activation by Bryo 1. To investigate the effect of bryo 1 on CD11c gene transcription, we transfected Reh cells with reporter plasmids containing the region of the CD11c promoter spanning from ⫺61 to ⫹43 (pCD11c61-Luc). This construct displayed basal promoter activity that was clearly increased in bryo 1-treated cells (Fig. 4). The transfection of a longer fragment of the CD11c gene promoter spanning from ⫺160 to ⫹43 (pCD11c160-Luc) yielded an increased level of promoter
Fig. 3. Characterization of AP-1 binding by EMSA. In A, nuclear extracts from bryo 1-differentiated Reh cells were tested for their capacity to recognize the AP-1 consensus sequence. Specific mobility shift and supershift of AP-1-radiolabeled, double-stranded oligonucleotides were used. Complex formation can be seen in Lanes 1 and 3–13. Lane 1, nuclear extract derived from 3T3 SR cells that is used as a positive control. For competition experiments, a 100-fold molar excess of unlabeled, double-stranded, wild-type, or mutant oligonucleotide was included in the DNA binding reaction. Bryo 1 is shown to potentiate AP-1 complex formation in a time-dependent fashion. Polyclonal antibodies against c-Fos, c-Jun, Fos B, Jun B, and Jun D were included in the DNA binding reaction for supershift analysis. B, nuclear lysates taken from Reh cells treated with the MEK-specific inhibitors PD 98059 at a concentration of 20 M and U0126 at a concentration of 15 M before bryo 1 showed an inhibition of AP-1/DNA complex formation.
activity induction, suggesting that a cis-acting element, such as Sp1, located within the ⫺160 to ⫹43 proximal region of the CD11c promoter, is assisting in the bryo 1-induced CD11c gene promoter activity. To further determine the functional contribution of the AP-1 site in the activation of CD11c gene promoter by bryo 1, we used the plasmids pCD11c160(-60mut)-Luc and pCD11c160(70mut)-Luc, which contained bp substitutions within the AP-1 and Sp1 binding sites, respectively. These bp substitutions were shown to affect AP-1 and Sp1 binding activity by EMSA. The activities of the AP-1 mutant construct, pCD11c160(60mut)-Luc, in response to bryo 1 were significantly lower than that recorded from the wild-type (pCD11c61-Luc and pCD11c160-Luc) constructs. However, the activity of the Sp1 mutant construct, pCD11c160(-70mut)-Luc, did not decrease significantly from that of its wild-type (pCD11c160-Luc) construct (Fig. 4). This suggests that bryo 1-induced CD11c gene expression is regulated at the transcriptional level involving the functional activation of AP-1. However, cis-acting elements other than Sp1, responsible for transcriptional activation mediated by bryo 1, are located within the ⫺160 to ⫹43 proximal region of the CD11c promoter. Effect of MAPK Antisense Oligonucleotides on CD11c Expression and Cellular Adhesion in Reh Cells. The utilization of drugs as inhibitors for specific molecules in a signal transduction pathway is commonly taken advantage of. However, nonspecific effects of these drugs or their metabolites on other signaling molecules cannot be entirely controlled for. To confirm the role of MAPK in the regulation of
MAPK Is Required for Bryo 1-induced Differentiation
Fig. 4. Contribution of the AP-1 transcription factor to regulated activity of the CD11c promoter. Reh cells were transfected with the indicated reporter constructs and treated with bryo 1. For each construct, fold induction represents the ratio between the luciferase and Renilla activity produced in the presence of bryo 1. Each construct was assayed three times, and a representative experiment is shown. The wild-type and mutant AP-1 and Sp1 sites are depicted as E and F (AP-1) or 䡺 (Sp1), respectively. The pCD11c160(-70mut)Luc reporter construct is mutated at the Sp1binding site at ⫺70; the pCD11c160(-60mut)Luc reporter construct is mutated at the AP-1-binding site at ⫺60. The pCD11c160-Luc reporter construct contains the ⫺160/⫹43 region of the promoter, whereas the pCD11c61Luc reporter construct contains the region ⫺61/ ⫹43 but lacks the Sp1-binding site at ⫺70; the pXP2 construct is the promotorless control.
CD11c and cellular adhesion, MAPK antisense 17-mer oligodeoxynucleotides with all phosphorothioate linkages were used. This oligonucleotide is directed against a sequence that is identical in the p42 and p44 MAPK isoforms (ERK2 and ERK1), which are conserved in humans, mice, and rats. MAPK oligonucleotides have no effect on expression of the MAPK homologues, p38 and JNK, or activation of MEK (26). In Reh cells, Bryo 1-induced cellular adhesion (Fig. 5A) and CD11c expression by FACS analysis (Fig. 5B) was significantly inhibited in the presence of antisense MAPK oligonucleotides. In contrast, scrambled MAPK oligonucleotides had no effect on Bryo 1-induced cellular adhesion or CD11c expression.
Discussion To better understand the molecular mechanisms for bryo 1-induced cellular differentiation in the Reh ALL model, a first step is to identify the signal transduction pathways and the cellular targets involved in this process. In this study, we present evidence that the ERK/MAPK pathway is required for bryo 1-induced differentiation in the Reh ALL model. Despite recent advances in understanding signaling pathways, the role of ERK/MAPK in mediating cell differentiation in hematopoietic cells has not been well established. Previous studies in our laboratory have shown that modulation of PKC by two PKC activators/deactivators, TPA and bryo 1, induce leukocyte differentiation of the early pre-B ALL cell line Reh and the CLL cell line WSU-CLL (12–15). Differentiation in these cell lines was evaluated by the development of filopodia and numerous vacuoles indicating phagocytic activity. Cells also increase in size with many adhering to the surface
of the culture flask. There was induction in the cell surface antigens CD11c and CD22 with the concomitant downregulation of CD10 and CD19. Cell growth was inhibited with accumulation in the G0-G1 phase of the cell cycle. Enzymatically, bryo 1 and TPA induced tartrate-sensitive acid phosphatase expression but failed to induce periodic acid Schiff and nonspecific esterase. Reh cells treated with bryo 1 are phenotypically differentiated to a monocytoid B-cell lymphoma-like stage (12), whereas WSU-CLL cells treated with bryo 1 differentiate to phenotypically resemble hairy cell leukemia (15). That MAPK activation plays a direct role in Reh ALL cellular differentiation was demonstrated by using the MEK-specific inhibitors PD98059 and UO126 (16, 17). The ability of immunoprecipitated active ERK/MAPK to phosphorylate a downstream substrate, Elk-1, was significantly reduced in MEK-specific, inhibitor-treated cells (Fig. 2B). This is consistent with the role of MEK as a regulator working upstream of ERK/MAPK. PD98059 and UO126 also significantly blocked AP-1 transcription factor binding to the AP-1 consensus sequences by EMSA (Fig. 3B). Under these conditions, bryo 1-induced Reh ALL differentiation was greatly diminished by PD98059. Cellular adherence declined ⱖ60%, whereas Reh’s cellular surface expression of CD11c declined ⬃50% (Fig. 1) from that induced by bryo 1. Additional evidence supporting the role of MAPK pathway in bryo 1-induced differentiation of Reh cells was provided by molecular inhibition. Transiently transfected Reh cells with antisense MAPK oligonucleotides were not responsive to bryo 1 (Fig. 5).
Cell Growth & Differentiation
Fig. 5. Effect of MAPK antisense transfection on Reh cell adhesion (A) and CD11c expression (B). Reh cells were transiently transfected with phosphorothioated antisense or scrambled oligodeoxynucleotides. Cell adhesion was determined morphologically, whereas CD11c expression was quantitated using flow cytometry. Data are the mean ⫾ SD of at least two independent experiments.
The AP-1 transcription factor complex is comprised of a group of proteins encoded by the jun (c-Jun, Jun B, and Jun D) and fos (c-Fos, Fos B, Fra-1, and Fra-2) gene families, which can bind to the AP-1 consensus sequence either as Jun/Jun or Jun/Fos dimers (18, 19). Here we show that the AP-1 transcription factor complex is comprised primarily of Jun D and Fos B (Fig. 3A). On bryo 1 treatment, AP-1 transcription factor binding is significantly potentiated (Fig. 3A), preceding the onset of the described phenotypic changes (12). On bryo 1 treatment, the activity of ERK/MAPK p42 (ERK2) rapidly increased, with p44 (ERK1) protein levels remaining unchanged (Fig. 2A). Nuclear lysates from Reh cells treated in like manner immunoprecipitated with antibodies specific for phosphorylated ERK/MAPK, when used for kinase assays, revealed significant phosphorylation of Elk-1 (Fig. 2B). Elk-1 is phosphorylated when the p44/p42 ERK/ MAPK translocates, on activation, from the cytosol to the nucleus. The leukocyte integrin CD11c, though primarily expressed on cells of the myeloid lineage and on hairy cell leukemia cells (20), has also been found to be expressed in a subset of B-cell lymphoproliferative disorders, such as CLL (21), and our laboratory has shown CD11c’s further up-regulation by the PKC activator/deactivator bryo 1 (12). Our results indicate that bryo 1 stimulates the surface expression of CD11c by transcriptional mechanism acting on the CD11c gene
promoter. The AP-1 transcription factor’s importance in CD11c transactivation was evaluated using a nested deletion promoter construct (pCD11c61-Luc) containing an identified AP-1 binding site at ⫺60. Bryo 1 treatment of Reh cells transiently transfected with plasmids containing this construct showed significant promoter activity when compared with activity recorded from cells transfected with the promotorless control (pXP2; Fig. 4). Promoter activity significantly increased again when the promoter construct was lengthened to include the Sp1 binding site at ⫺70 (pCD11c160Luc). Mutation of the AP-1 binding site at ⫺60 (pCD11c160(-60mut)-Luc) significantly reduced promoter activity when compared with both previously described constructs. However, mutation of the Sp1–70 site (pCD11c160(-70mut)-Luc) within the CD11c promoter had little effect on the bryo 1 inducibility. That this mutation does not significantly alter promoter activity indicates that additional cis-acting elements, distinct from Sp1, further mediate the bryo 1 inducibility of the CD11c promoter. Sequence analysis of the ⫺160 to ⫹43 region of the CD11c promoter has shown the presence of additional putative transcription binding sites, such as AP-2, PU.1 (22), Myb, and b/HLH (23). The contribution of these additional elements to the basal and bryo 1-regulated activity of the CD11c promoter, if any, would be absolutely dependent on AP-1 because mutation of the AP-1 binding site at ⫺60 causes the greatest reduction in the CD11c promoter activity (Fig. 4). Additional evaluation of this transcriptional control is ongoing in our laboratory. The fos gene promoter has been shown to contain response elements, which when bound by the transcription factor Elk-1, leads to Fos transcription. Fos proteins dimerize with Jun proteins to form AP-1 complexes, which can transactivate specific target genes (24). Differences in the relative levels of Jun family members, which differ greatly in their trans activation capabilities, might explain the distinct bryo 1 differentiation responsiveness of the CD11c promoter in Reh cells, e.g., c-Jun/c-Fos dimers have been shown to transactivate genes, such as cyclin D1, stimulating G1 to S phase progression in the cell cycle. Jun B has been shown to inhibit the c-Jun-mediated trans activation on promoters containing a single AP-1 sequence and is a negative regulator of genes activated by c-Jun (25, 26). Therefore, it is highly plausible that the bryo 1-induced Jun D/Fos B AP-1 complex (Fig. 3A) is responsible for CD11c transactivation in the Reh cell line. Work is currently underway to more fully elucidate this association in our laboratory.
Materials and Methods Cell Culture. The human ALL cell line Reh was obtained from the American Type Culture Collection (Rockville, MD). The Reh cell line was established from a 15-year-old girl with ALL and was characterized as being at the pre-B stage (27). Reh cells were grown in RPMI 1640 supplemented with 10% heat-inactivated FCS, 1% L-glutamine, 50 units/ml penicillin, and 50 g/ml streptomycin at 37°C in an atmosphere of 5% CO2. All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), unless otherwise indicated. Culture of Reh Cells. Reh cells were seeded at 2 ⫻ 105/ml in T-75 tissue culture flasks (Falcon Labware, Oxnard,
MAPK Is Required for Bryo 1-induced Differentiation
CA). Bryo 1 (Division of Cancer Treatment and Diagnosis Center, NIH, Bethesda, MD) was dissolved in 0.05% DMSO and PBS at a concentration of 10⫺5 M and then further diluted to the final concentration in culture medium. PD 98059 at a concentration of 20 M and U0126 at a concentration of 15 M (MEK inhibitors) were purchased from Calbiochem (La Jolla, CA). Bryo 1 (1 nM) or vehicle was added to flasks containing Reh cells. Inhibitors were added 30 min before bryo 1. Cultures were incubated for ⱕ120 h at 37°C and 5% CO2. Cell viability and growth inhibition were determined daily using trypan blue (0.4%) exclusion (Life Technologies, Inc., Grand Island, NY). Flow Cytometric Analysis. Cells were stained with MoAbs using indirect immunofluorescence techniques as described previously (12). Briefly, 106 cells from appropriate aliquots were washed with PBS containing 1% BSA (Life Technologies, Inc.) and suspended in an appropriate quantity of MoAb, as suggested by the manufacturer, for 30 min at 4°C. After washing, cells were suspended in 100 l of FITC-conjugated goat antimouse antibody at 1:20 dilution for 30 min at 4°C in the dark. Appropriate normal mouse immunoglobulin isotypes were used as a control for background fluorescence. Cells were then washed with PBS and analyzed for log fluorescence intensity by flow cytometry on a Becton Dickinson FACScan (Mountain View, CA). Results were expressed as percentage-positive cells compared with background fluorescence, for which mouse IgG was used in place of MoAbs. The MoAbs anti-CD10, CD11c, CD19, and CD22 were obtained from Caltag Laboratories (Burlingame, CA). Kinase Assays for MAPK/ERK Activity. MAPK activity was measured with a p44/42 MAPK Assay Kit (New England Biolabs) according to the manufacturer’s protocol. Briefly, cells were lysed in buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium PPi, 1 mM ␤-glycerophosphate, 1 mM Na3VO4, 1 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride for 15 min at 4°C. Cell lysate (200 l, ⬃200 g protein) was mixed with monoclonal or polyclonal phospho-MAPK antibody (1:50 dilution) and incubated with gentle rocking overnight at 4°C. Immunoprecipitates were collected by protein A-Sepharose beads (10 –20 l) for 2 h at 4°C. The beads were washed twice with cold lysis buffer and twice with 500 ml of kinase buffer [20 mM Tris (pH 7.5), 5 mM ␤-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM NgCl2]. Kinase assay was performed by incubating the suspended pellet with kinase buffer containing 100 M ATP and GSTElk1 fusion protein for 30 min at 30°C. The samples were analyzed by 12% SDS-PAGE. Phospho-(Ser383)-Elk1 was detected with specific antibody using Western blot analysis. Western Blot Analysis. Nuclear extracts (20 g) or whole-cell extracts (25–50 g) were resolved by 12% SDSPAGE, transferred to Hybond C-extra membranes (Amersham Life Science, Arlington Heights, IL), and detected with antiserum specific for MAPK (p44/p42; Santa Cruz Biotechnology, Santa Cruz, CA) with the use of an enhanced chemiluminescence assay (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Briefly, cells were washed twice with cold PBS and lysed at 4°C for 30 min in lysis buffer (0.5% Triton X-100, 300 mM NaCl, 50 mM Tris.Cl, and 1 mM phenylmeth-
ylsulfonyl fluoride) with occasional vortexing. Protein concentrations were determined using the micro bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL). The Hybond C-extra membranes were blocked (5% milk, 0.05% Tween 20, and PBS) for 1 h at room temperature. The membranes were then incubated with the primary antibody (1: 1000 dilution in PBS and 0.05% Tween 20) overnight at 4°C. The membranes were washed well in PBS with 0.05% Tween 20 and then incubated with the horseradish peroxidaseconjugated antimouse secondary antibody (Santa Cruz Biotechnology; 1:5000 dilution in PBS and 0.05% Tween 20). Protein levels were visualized by peroxidase reaction using the enhanced chemiluminescence kit (Amersham Life Science). Equal sample loading was confirmed by reprobing the same blots with a rabbit polyclonal antiserum against glyceraldehyde-3-phosphate dehydrogenase (1:5000; Trevigen, Inc., Gaithersburg, MD). Blots were stripped by submerging the membranes in stripping buffer [100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7)] and incubating at 60°C for 25 min with occasional agitation. EMSAs. EMSAs were performed using the Nushift kit (Geneka Biotechnology, Inc., Montreal, Quebec) according to the manufacturer’s protocols. Briefly, 5 g of extract and 2 l of unlabeled oligonucleotide competitor DNA (125 nM) were incubated in the same volume of buffers provided by the manufacturer for 20 min on ice. One l of 50 ng labeled probe was added, and the mixture was incubated for another 20 min on ice. Supershifts were performed by preincubating antinuclear factor B-p65, -p50 (Santa Cruz Biotechnology) or antinuclear factor B-p65, -p50 (Geneka Biotechnology) antiserum or control rabbit serum with the labeled oligonucleotide under similar assay conditions. Bound and free probes were resolved by nondenaturing PAGE. Gels (5%; acrylamide/bisacrylamide, 38:2, 1⫻ Tris glycine EDTA, 2.5% glycerol, 1.5-mm thick) were run in 1⫻ Tris glycine EDTA running buffer at constant current (60 mA) for ⬃180 min. Gels were blotted to Whatman 3 MM paper, dried under vacuum, and exposed to X-ray film from 24 to 72 h at ⫺80°C. Plasmids, Transfections, and Analysis of Luciferase Activity. The luciferase gene-derived plasmid constructs containing the nested deletion fragments of the CD11c gene promoter were the kind gift of Dr. Angel L. Corbi from the Instituto de Parasitologia y Biomedicina Lopez-Neyra, Grenada, Spain, and have been described (19, 22). For transfection experiments, a total of 3 ⫻ 106 cells were plated in 100-mm Petri plates. Cells were incubated in a mixture of DOTAP (Boehringer Mannheim, GmbH, Germany) and 5 g of plasmid DNA. Cells remained in DOTAP/DNA mixture for 24 h followed by cell harvest and resuspension in new RPMI culture medium without DOTAP/DNA. Cells were allowed to grow for 24 h. Cells were treated with bryo 1 for 6 h followed by lysis in Promega’s Dual Luciferase Passive Lysis Buffer, after which, lysates were stored at ⫺70°C overnight. Lysates were then thawed, and reagents were prepared according to the manufacturer’s protocols (Promega, Madison, WI). To determine transfection efficiency, 1 g of the plasmid Renilla Luciferase-cytomegalovirus vector containing the cytomegalovirus immediate early enhancer/promoter region, which provides strong, constitutive expression of Renilla luciferase
Cell Growth & Differentiation
gene, was included in each transfection. Renilla luciferase activity was measured following the manufacturer’s protocol. Transient Transfections of Antisense Constructs. A completely phosphorothioated antisense oligodeoxynucleotide (5⬘-GCCGCCGCCGCCGCCAT-3⬘) directed against the initiation codon and the subsequent 14 bases of the mouse p42 MAPK (ERK2) mRNA was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). This sequence is identical in rat and human p42 and p44 MAPKs (ERK2/1) and as far as is known in mouse p44 (8 of 17 bases; Ref. 28). A completely phosphorothioated oligodeoxynucleotide (5⬘-CGCGCGCTCGCGCACCC-3⬘) with the same base composition as the MAPK antisense oligo but with a scrambled sequence was used as control. Reh cells (2 ⫻ 105/ml) seeded in six-well tissue culture plates were transfected with 2 M either antisense or scrambled oligodeoxynucleotide using DOTAP (Roche Diagnostics Corp./Roche Molecular Biochemicals, Indianapolis, IN). After transfection (12 h), cells were treated with bryo 1 (10 nM) and monitored for cellular adhesion for an additional 24 h. At 24-h post bryo 1 treatment, cells were harvested and stained with antibodies directed against CD11c and analyzed using a FACScan (Becton Dickinson) as described previously (12)
Acknowledgments We thank Dr. Angel L. Corbi from the Instituto de Parasitologia y Biomedicina Lopez-Neyra, Grenada, Spain, for her kind gift of the CD11c-nested deletion constructs. For help with the flow cytometry, we also thank Eric Van Buren, Evano Piasentin, and Dr. Stephen Lerman from the Molecular and Cellular Imaging and Analytical Cytometry Core Facility of the Barbara Ann Karmanos Cancer Institute and Wayne State University School of Medicine.
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