Activation of p38 Mitogen-activated Protein Kinase Is Required for Tumor Necrosis Factor- a -supported Proliferation of Leukemia and Lymphoma Cell Lines*

To elucidate mechanisms of tumor necrosis factor a (TNF- a )-induced proliferation of a number of human leukemia and lymphoma cell lines, we examined the role of p38 mitogen-activated protein kinase (MAPK) in TNF- a signaling in Mo7e and Hut-78 cells. TNF- a -de-pendent p38 MAPK activation was detected in both Mo7e and Hut-78 cells and was blocked by the p38 MAPK inhibitor, SB203580. Ablation of p38 MAPK activity by SB203580 abrogated TNF- a -induced Mo7e cell proliferation and TNF- a -dependent autocrine growth of Hut-78. As we have shown previously that activation of the nuclear factor k B (NF- k B) is also required for TNF- a -in-duced Mo7e cell proliferation, the involvement of p38 MAPK in NF- k B activation was assessed. SB203580 did not affect TNF- a -signaled nuclear translocation and DNA-binding activity of NF- k B, and inhibition of NF- k B function did not affect TNF- a -induced p38 MAPK activation, indicating that these events are not dependent on each other. However, SB203580 depressed the expression of NF- k B-dependent genes, as monitored by a k B-driven reporter gene. Our findings demonstrate that activation of both p38 MAPK and NF- k B plays a critical role in

TNF-␣ activates the nuclear factor-B (NF-B) in many cell lines. NF-B is a heterodimer of p65 and p50 subunits, both of which are members of the NF-B/Rel family of transcription factors, which also includes c-Rel, Rel B, and p52 (14). In response to TNF-␣ and other signals, IB␣, a protein that sequesters NF-B in the cytosol, is phosphorylated and degraded through an ubiquitin/proteasome pathway. As a result, NF-B translocates to the nucleus and promotes the expression of target genes. Increasing evidence shows that activation of NF-B is involved in cell activation and proliferation. We previously reported that NF-B activation is essential for TNF-␣induced Mo7e cell survival and proliferation (15), because inhibition of nuclear translocation of NF-B specifically blocked TNF-␣-induced cell proliferation, but had no effect on granulocyte macrophage-colony-stimulating factor-(GM-CSF) or IL-3induced Mo7e cell proliferation, in which there is no NF-B activation. On the other hand, several lines of evidence also indicate that activated NF-B may participate in apoptosis (16,17).
TNF-␣ is also a potent activator of p38 MAPK. To date, there are at least three different known subtypes of MAP kinases: the p42 and p44 MAPKs, which are also termed as extracellular signal-regulated kinases (ERK); the c-Jun N-terminal kinase/ stress-activated protein kinases; and p38 MAPK (18 -20). Mammalian p38 MAPK was originally identified in murine pre-B cells transfected with the lipopolysaccharide complex receptor CD14 and in macrophages in which p38 MAPK was activated in response to a lipopolysaccharide. Like ERKs and c-Jun N-terminal kinases, p38 MAPK requires phosphorylation of a closely spaced tyrosine and threonine for activation. However, p38 MAPK is distinguished by the sequence TGY in its activation domain, which differs from the TEY sequence found in ERKs, and the TPY sequence in c-Jun N-terminal kinase and MAP kinase homologues. p38 MAPK is activated by a wide spectrum of stimuli, such as physical and chemical stresses, lipopolysaccharide, and cytokines. Despite rapid progress in the elucidation of the structural elements of the p38 MAPK pathway, the physiological consequences of its stimulation by stress agents largely remain to be defined. A variety of evidence suggests that p38 MAPK plays a key role in regulating anti-apoptotic and inflammatory responses (21)(22)(23). In addition, inhibition of p38 MAPK has been found to prevent interleukin-6 and GM-CSF mRNA synthesis, indicating that p38 MAPK may regulate transcriptional events (24 -26). Some other studies indicated that p38 has been implicated in the activation of transcription factors, such as ATF-2, ELK-1, c-Fos, c-Jun, CHOP, MAX, and NF-B. Activa-tion of p38 MAPK was reported to have anti-apoptotic effects in some cell lines and to play a role in cell proliferation in other systems (21)(22)(23). However, several studies have shown that activation of p38 MAPK plays a role in apoptosis in the PC12 neuronal cell line and mouse CD8ϩ T cells (27,28).
Although our previous studies have established the necessity of NF-B activity for the TNF-␣-mediated growth and survival of human leukemic cell lines (15), TNF-␣ initiated proliferative and anti-apoptotic signaling pathways upstream or independent of NF-B activation have yet to be described. In this study, we investigated the activation and role of the p38 MAPK signaling pathway in TNF-␣-treated Mo7e and other human leukemia or lymphoma cell lines. Our data show that TNF-␣ activates p38 MAPK and that this activity is required for TNF-␣-supported survival and proliferation of human leukemia and lymphoma cells. In addition, we found that abrogation of p38 activity markedly repressed TNF-␣-induced expression of a B-driven reporter gene without affecting the nuclear translocation or DNA-binding activity of NF-B. Our findings demonstrate that p38 MAPK plays an essential role in the TNF-␣-mediated proliferation and survival of human leukemic cells and acts at least in part by promoting the expression of the B-driven genes.
Cell Lines-The human Mo7e megakaryoblastic leukemic cell line, originally described by Avanzi et al. (30), was obtained from Genetics Institute (Boston, MA) and was maintained in Iscove's modified Dulbecco's medium (Life Technologies, Inc.) containing 10% fetal bovine serum, 1% glutamine, and 5 ng/ml recombinant human GM-CSF. The human lymphoma Hut-78 cell line, originally described by Gazdar et al. (31), and leukemic HEL cell line (3,32,33) were purchased from ATCC and were maintained in Iscove's modified Dulbecco's medium with 10% fetal bovine serum. In experiments to detect effects of cytokines, Mo7e cells were prepared by washing three times with serum-free medium and were starved for 18 h in medium without cytokine (3). HEL cells were cultured in serum-free medium with 1 ϫ Nutridoma HU (Roche Molecular Biochemicals) for 18 h before cytokine treatments.
Effects of TNF-␣ on Cell Growth-To determine the effect of TNF-␣ on cell proliferation, DNA synthesis was measured by [ 3 H]TdR incorporation in freshly prepared cells. The assays were performed in triplicate, using a total of 4 ϫ 10 5 Mo7e or Hut-78 cells or 2 ϫ 10 5 HEL cells (fewer HEL cells were used because of their shorter doubling time). Mo7e cells in Iscove's modified Dulbecco's medium with 10% fetal calf serum and HEL cells in medium with 1x Nutridoma HU were cultured for 72 h in the presence or absence of TNF-␣ or other cytokines. Cells were then labeled with 4 Ci/ml of [ 3 H]TdR for an additional 4 h. The radioactivity incorporated into DNA (cpm) was determined by a liquid scintillation counter according to a previously described protocol (3). All assays for [ 3 H]TdR incorporation were repeated at least three times. Some of our data from [ 3 H]TdR incorporation assays also were compared with those obtained from the nonradioactive Cell Proliferation Wst-1 kit (Roche Molecular Biochemicals). Both methods yielded consistent results. To determine cell growth, cell numbers were counted manually in a hemacytometer, and cell viability was assessed by trypan blue staining.

Effect of Inhibitors of MAPK and NF-B Activation on TNF-␣-induced Mo7e
Cell Proliferation-SN50, a peptide that blocks the nuclear translocation of activated NF-B (15,29), contains membrane-permeable signal sequences of Kaposi's fibroblast growth factor and the nuclear translocation motif (VQRKRQKLMP) of human NF-B p50. A mutant SN50 (SN50mt) contains membrane-permeable signal sequences of Kaposi's fibroblast growth factor and a nonfunctional mutant nuclear translocation motif of human NF-B p50 (29). Both SN50 and SN50mt were synthesized commercially (Genemed Synthesis, South San Francisco, CA). The peptides were purified by reverse-phase high pressure liquid chromatography, and the molecular weight of the purified peptides was verified by mass spectrometry analysis. To track the intracellular localization of the membrane-permeable peptides in Mo7e cells, SN50 was labeled with FITC. The synthesized peptides were dissolved in Me 2 SO to a final concentration of 100 g/l and mixed directly with culture medium (1:500 -1000) before use. To investigate the effect of inhibition of p38 MAPK or ERK on cell proliferation and on other signaling molecules including NF-B and JAK/STAT5, SB203580 or PD098059 was used to incubate with cells in the absence or presence of cytokines.
Western Blotting Analysis-For the detection of unphosphorylated or phosphorylated p38 MAPK, p44/p42 MAPK, or IB␣, cells treated with or without cytokines were boiled in SDS buffer. Total cellular proteins (30 g) were loaded into each lane and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene difluoride membranes and probed with the anti-total (unphosphorylated and phosphorylated) or antiphospho-p44/p42, -p38, or -IB␣ antibodies (New England Biolabs, Beverly, MA). Anti-phospho-p38 MAPK reacts only with p38 MAPK that is activated by dual phosphorylation at Thr 180 and Tyr 182 .
Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays-Preparation of nuclear extracts and electrophoretic mobility shift assays were performed according to methods described previously (15,34). The sequence of the NF-B-binding oligonucleotide used as a radioactive DNA probe was 5Ј-CGACAGAGGGGACTTTCCGAGAGGC-3Ј. Equal amounts of nuclear proteins (5-10 g) for each sample were incubated with 1 ng of 32 P-labeled probe. The DNA binding reaction was performed at room temperature in a volume of 25 l, which contained binding buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 20 g/ml bovine serum albumin, and 0.2% Nonidet P-40, 1.8 g/ml salmon sperm DNA), 1 ng of 3Ј-labeled probe, and 5-10 g of nuclear proteins. After incubation for 15 min, the DNA-protein complexes and the unbound probe were separated electrophoretically on 6% native polyacrylamide gels in 0.25ϫ buffer (44.5 mM Tris, pH 8.0, 1 mM EDTA, and 44.5 mM boric acid). The gels were fixed and dried, and the DNA-protein complexes were visualized by autoradiography at Ϫ70°C with Kodak X-OMAT film and a DuPont Cranex lightning-plus intensifying screen.
Flow Cytometry Analysis-A FACScan flow cytometer in the H. Lee Moffitt Cancer Center Flow Cytometry Core Facility was used to examine Mo7e cell cycle status, intracellular localization of SN50 peptides, and apoptosis. Mo7e cells were incubated with 50 g/ml FITC-labeled SN50 peptide for various periods or with various amounts of FITC-SN50 for 30 min to investigate the intercellular incorporation of the peptides. The status of apoptosis was analyzed by incubating cells with FITC-labeled Annexin V and propidium iodide, following the manufacturer's suggested procedure.
Effects of p38 MAPK Phosphorylation on Expression of a B-driven Alkaline Phosphatase Reporter Gene-To examine the role of p38 MAPK phosphorylation in the expression of B-driven genes, a reporting system pNF-B (SEAP, the secreted alkaline phosphatase) (CLON-TECH, Palo Alto, CA), which carries a secreted alkaline phosphatase reporter gene driven by a basic promoter element (TATA box) and tandem repeats of the B site, was transfected into Mo7e cells. Mo7e cells (5 ϫ 10 6 ) were plated in 60-mm dishes at a density of 1 ϫ 10 6 cells/ml. The following day, the cells were transfected with 8 g of B/phosphatase plasmid by the liposome method (GenPORTER, Gene Therapy System, San Diego, CA). The transfected cells were cultured in Iscove's modified Dulbecco's medium containing 10% fetal bovine serum, 1% glutamine, and 5 ng/ml recombinant human GM-CSF. After 24 h, the cells were washed three times with serum-free medium and were starved for 18 h in medium without cytokine. On the next day, the transfectants were divided into three groups and treated without TNF-␣, with 5 ng/ml TNF-␣ for 1-6 h, or with 50 M SB203580 for 2 h, followed by 5 ng/ml TNF-␣ for 1-6 h. At each time point, 250 l of supernatant was collected and used for determination of SEAP activity. The activity of SEAP was determined with a PNPP Phosphatase Sub-strate Kit (Pierce) following the manufacturer's suggested protocol. The relative SEAP activity was calculated by dividing the reading at 405 nM from each treated group by that of the untreated cells. The activity of SEAP in supernatant also was determined by dot-blotting proteins onto nitrocellulose, and the reaction of SEAP and its substrate was visualized with enhanced chemiluminescence (Lumi-Phos WB, Pierce) following the manufacturer's suggested procedure.

TNF-␣, but Not GM-CSF or IL-3, Induced Phosphorylation of p38 MAPK in Leukemic Cell
Lines-We have shown previously that p44/p42 MAPK is not activated by TNF-␣ in Mo7e cells (3).
In the experiments described here, we examined the effects of TNF-␣ on the activation of p38, using an antibody that specifically recognizes phosphorylated and thus activated p38. Phosphorylation of p38 MAPK was not detected in Mo7e or HEL human leukemic cell lines in the absence of TNF-␣ but was induced significantly upon exposure of the cells to TNF-␣ (Fig.  1a). In Mo7e cells treated with 5 ng/ml TNF-␣, phosphorylation of p38 MAPK was maximal at 30 -60 min and declined thereafter (Fig. 1a, Mo7e). In Mo7e cells (1 ϫ 10 6 cells/ml) treated with various doses of TNF-␣, the maximal level of p38 MAPK phosphorylation was reached by treating cells with 1-5 ng/ml TNF-␣ for 30 min, and higher TNF-␣ concentrations failed to further increase levels of p38 MAPK phosphorylation (Fig. 1b). The level of total (phosphorylated and unphosphorylated) p38 MAPK was unaffected by TNF-␣ (Fig. 1). We reported previ-ously that GM-CSF and IL-3 induced significant p44/p42 MAPK phosphorylation but did not activate NF-B in Mo7e cells (15). Incubation of Mo7e cells with 10 ng/ml GM-CSF ( Fig.  1c) or IL-3 (data not shown) for 5-60 min also failed to induce p38 MAPK phosphorylation.
Constitutive Activation of p38 MAPK Was Detected in Hut-78 Cells-We then investigated activation of p38 MAPK in the human lymphoma Hut-78 cell line. The Hut-78 cell line was chosen because it constitutively expresses activated NF-B as a result of TNF-␣ autocrine stimulation (9, 10). When we utilized antiphosphorylated p38 MAPK antibody to determine p38 phosphorylation status in Hut-78 cells, the results from Western blotting show that p38 MAPK was constitutively activated in Hut-78 cells without any cytokine exposure (Fig. 2, lane 1).
Treating cells with 1-20 ng/ml TNF-␣ for 30 min did not have any further effect on constitutively activated p38 MAPK (Fig.  2, lanes 2-4). GM-CSF, IL-6, or TPO also did not significantly enhance or inhibit the constitutive p38 MAPK phosphorylation (data not shown). However, incubation of Hut-78 cells with anti-TNF-␣ neutralizing antibody reduced the level of constitutively phosphorylated p38 MAPK in a dose-dependent manner (Fig. 2, lanes 5-6), indicating that the activation of p38 MAPK in Hut-78 cells is via TNF-␣ autocrine stimulation.
Inhibition of Nuclear Translocation of Activated NF-B Had No Effect on TNF-␣-induced p38 MAPK Phosphorylation-Because our previous and present studies indicated that treating cells with TNF-␣ not only activated NF-B (15), but also induced p38 MAPK phosphorylation (Figs. 1 and 2), we designed experiments to examine the relationship of these events. NF-B is sequestered in the cytoplasm as an inactive complex with IB␣ in the basal state and becomes active after exposure of cells to TNF-␣. To investigate whether TNF-␣-induced NF-B activation was required for activation of p38 MAPK, SN50 was used to investigate the effect of blockage of NF-B nuclear translocation on p38 MAPK phosphorylation. The results showed that preincubation of Mo7e cells with various amounts of SN50 for 2 h inhibited TNF-␣-induced nuclear translocation of NF-B in a dose-dependent manner (Fig. 3a, lanes 3-5 and also see our previous report (15)) but had no effect on TNF-␣-induced p38 MAPK phosphorylation (Fig. 3b,  lanes 3-5) or on the level of total p38 MAPK (data not shown).  5) and from HEL cells treated without or with 5 ng/ml TNF-a for 30 min (lanes 6 and 7). Equal amounts of cellular proteins were immunoblotted with anti-phospho-p38 (p-p38) or antitotal p38 (t-p38) MAPK antibodies. B, whole cell lysates prepared from Mo7e cells treated with the indicated amounts of TNF-␣ for 30 min were immunoblotted with anti-phospho-p38 or anti-total p38 MAPK antibodies. C, whole cell lysates were prepared from untreated Mo7e cells (lane 2) or Mo7e cells treated with 5 ng/ml TNF-␣ for 30 min as a positive control (lane 1) or with 5 ng/ml GM-CSF for 5-120 min (lanes 3-7). Equal amounts of cellular proteins were immunoblotted with antiphospho-p38 or anti-total p38 MAPK. Similar results were obtained in three separate experiments, and one of each is displayed here.  5 and 6). Equal amounts of cellular proteins were immunoblotted with anti-phospho-p38 (p-p38) or anti-total p38 (t-p38) MAPK antibodies. Similar results were obtained in three separate experiments, one of which is displayed here.
p38 MAPK in TNF-␣-induced Cell Proliferation ( Fig. 4a) or the constitutively activated p38 MAPK in Hut-78 cells (data not shown). In experiments to investigate the effect of SB203580-induced blockage of p38 phosphorylation on TNF-␣-induced nuclear translocation and DNA-binding activity of NF-B, antibody to the NF-B p65 subunit was used to assess the subcellular location of NF-B. p65 was not detectable in the nuclei of untreated Mo7e cells (Fig. 4b, lane 1), whereas marked accumulation of nuclear p65 was apparent in cells treated with 10 ng/ml TNF-␣ for 30 min (Fig. 4b, lane 2). Preincubation of Mo7e cells with 1-50 M SB203580 for 2 h did not affect the TNF-␣-induced nuclear accumulation of p65 (Fig.  4b, lanes 3-5).
As assessed by electrophoretic mobility shift assay, DNAbinding activity of NF-B was observed in the nuclear extracts of TNF-␣-treated, but not untreated Mo7e cells (Fig. 4c, lanes 1  and 2), and the induction of this activity by TNF-␣ was unaffected by pretreatment of cells with SB203580 (Fig. 4c, lanes  3-5). In a separate experiment, we found that treating Mo7e cells with the same amounts of SB203580 as used above had no effect on TNF-␣-induced phosphorylation and degradation of IB␣ (data not shown).
There Is No Cross-activation of p38 or p44/p42 MAPK Signaling Pathway-There are several members of the MAPK family, including ERK, p38, and c-Jun N-terminal kinase/ stress-activated protein kinases. It is unclear whether there is overlap and cross-talk among these signaling pathways. The pharmacological compound PD098059 has been reported to have the capacity to inhibit p44/p42 MAPK phosphorylation via specific inhibition of MEK1/2 activation. In our study, PD098059 was used to examine the effect on p38 MAPK phosphorylation. Treating Mo7e cells with PD098059 inhibited GM-CSF-induced phosphorylation of p44/p42 MAPK in a dose-dependent manner (Fig. 5a, lanes 3-6). However, treating Mo7e cells with various amounts of PD098059 had no effect on TNF-␣-induced p38 MAPK phosphorylation (Fig. 5b). On the other hand, incubation of cells with SB203580 did not show significant effects on GM-CSF-or IL-3-induced activation of p44/p42 MAPK (Fig. 5c) or JAK2/STAT5 signaling pathways (data not shown).
Inhibition of p38 MAPK Activation Inhibits TNF-␣-supported Cell Growth-Mo7e cells grow in a growth factor-dependent manner and stop growing and eventually undergo apoptotic death in medium without stimulatory cytokines.
However, treatment of cells with TNF-␣ supports continuous cell growth. SB203580 was used to examine the effect of inhibition of p38 MAPK phosphorylation on TNF-␣-dependent survival and proliferation of Mo7e cells. As shown in Fig. 6a, 2 ng/ml TNF-␣ significantly enhanced DNA synthesis in Mo7e cells, but SB203580 inhibited the TNF-␣-induced increase of DNA synthesis in a dose-dependent manner, as monitored by incorporation of [ 3 H]TdR into DNA in TNF-␣-treated Mo7e cells (lanes 3-6). 50 M SB203580 completely blocked the TNF-␣-induced increase of [ 3 H]TdR incorporation of Mo7e cells (Fig.  6a, lane 6). Similarly, when Mo7e cells were cultured in medium with 5 ng/ml TNF-␣ for 10 days in the presence of 50 M SB203580 (refeeding every three days) and cell numbers were counted in hemacytometer every other day, Fig. 6c showed that SB203580 blocked TNF-␣-supported growth of Mo7e cells (Fig.  6c, TNFϩSB). When the cultures were terminated after 10 days and cell viability was assessed by trypan blue staining, the live cells in the group treated with TNF-␣ ϩ SB203580 dropped to Ͻ10%. When Mo7e cells were co-incubated for 5 days with 5 ng/ml TNF-␣ plus various amounts of SB203580, the TNF-␣-induced protection of Mo7e cells against apoptosis was inhibited in a dose-dependent manner by SB203580, as determined by Annexin V and propidium iodide flow cytometry (data not shown). These results are virtually identical to those we reported previously, in which we showed that treating Mo7e cells with SN50 (to inhibit nuclear translocation of NF-B) prevented TNF-␣-supported Mo7e cell growth and increased apoptosis in TNF-␣-treated Mo7e cells (15).
The inhibition of p38 activation by SB293580 also showed the severe inhibitory effects on TNF-␣-supported Hut-78 cell survival and proliferation. When Hut-78 cells were incubated with various amounts of SB203580, a dose-dependent inhibition of Hut-78 cell proliferation was observed (Fig. 7a). However, incubation of cells with PD098059, an inhibitor of the MEK-p44/p42 MAPK signaling pathway, did not show significant effects on Hut-78 cell survival and proliferation (Fig. 7b).
We then investigated the effect of these inhibitors on [ 3 H]TdR incorporation in Mo7e cells treated with GM-CSF or IL-3, which stimulate Mo7e proliferation via activation of the p44/p42 MAPK and/or JAK/STAT signaling pathways. The results show that 1-50 M SB203580 had no effect on either the GM-CSF-or IL-3-induced increase of [ 3 H]TdR incorporation of Mo7e cells (Fig. 6b, lanes 3-6 for GM-CSF; IL-3, data not shown). However, as we showed previously (35), inhibition of p44/p42 MAPK activation by PD098059 significantly blocked both GM-CSF-and IL-3-induced Mo7e cell proliferation (data not shown here). In contrast, exposure of Mo7e cells to either 50 M SB203580 or PD098059 in the absence of TNF-␣ did not affect cell proliferation (data not shown).
In addition, we investigated the effect of inhibition of p38 MAPK on the growth of TNF-␣-treated HEL, Meg-01, and K562 cells. The results showed that the addition of SB203580 alone or with TNF-␣ as well as GM-CSF and IL-3 had no significant effect on survival and proliferation of these cell lines (data not shown), which grow in a growth factor-independent manner.
Inhibition of p38 MAPK Phosphorylation Reduced TNF-␣induced/supported Expression of a B-driven Phosphatase Reporter Gene-As noted above, our data show that TNF-␣ induces the activation of p38 MAPK and increases DNA-binding activity of NF-B. The co-activation of these events by TNF-␣ suggests that there may be some links between them. Thus, we investigated whether phosphorylation and activation of p38 MAPK plays a role in B-driven gene expression. To test this, Mo7e cells were transiently transfected with a B-driven phosphatase reporter gene (B-SEAP), and the effects of SB203580  1 and 2) or 5 ng/ml TNF-␣ after pretreatment of the cells with the indicated amounts of SN50 for 2 h (lanes 3-5). The extracts were subjected to SDS-polyacrylamide gel electrophoresis Western blotting, and nuclear p65 was probed with the anti-p65 NF-B antibody. Similar results were obtained in two separate experiments. b, whole cell lysates, prepared as described in a and immunoblotted with anti-phospho-p38 antibodies. Similar results were obtained in two separated experiments.

p38 MAPK in TNF-␣-induced Cell Proliferation
on activation of this gene by TNF-␣ was determined. When Mo7e cells transfected with the B/phosphatase reporter gene for 48 h were exposed to 10 ng/ml TNF-␣ for various times, phosphatase activity increased up to 7-fold over that of untreated control cells (Fig. 8a, TNF-␣). However, pretreatment of Mo7e cells with SB203580 prior to the addition of TNF-␣ severely reduced the increase in B-SEAP activity induced by TNF-␣ (Fig. 8a, TNFϩSB). For example, 50 M SB203580 almost completely blocked the TNF-␣-induced increase of B-SEAP gene expression, despite its inability to inhibit nuclear translocation or DNA-binding activity of NF-B. Preincubation of Mo7e cells with SN50 also prevented B-SEAP activation by TNF-␣ (data not shown). In addition to measurement of SEAP activity in cell extracts, we also used a dot-blot/ECL protocol to directly visualize SEAP activity. Results showed that SB203580 inhibited TNF-␣-stimulated SEAP activity in a dosedependent manner, with essentially complete inhibition occurring at 50 M SB203580 (Fig. 8b). DISCUSSION In our previous report, we demonstrated that prevention of nuclear translocation of activated NF-B blocked TNF-␣-induced Mo7e cell proliferation and increased apoptosis (15). However, TNF-␣ activates not only NF-B but also many other proliferative signaling molecules. These facts led us to investigate whether other TNF-␣-induced signaling pathways con-

FIG. 4. Effects of SB203580 on TNF-␣-induced p38 MAPK activation and on nuclear translocation and DNA-binding of NF-B. A,
Western blotting of effects of SB203580 on p38 MAPK activation. Whole cell lysates, prepared from untreated Mo7e cells or Mo7e cells preincubated with the indicated amounts of SB203580 for 2 h and then exposed to 5 ng/ml TNF-␣ for 30 min, were subjected to SDS-polyacrylamide gel electrophoresis Western blotting and probed with anti-phospho-p38 MAPK antibodies. B, Western blotting of effects of SB203580 on nuclear translocation of NF-B. Nuclear extracts were prepared as described in A and immunoblotted with anti-nuclear-p65 NF-B antibodies. C, electrophoretic mobility shift assay analysis of effects of SB203580 on DNA-binding activity of NF-B. Nuclear extracts were prepared from cells treated as described in A and incubated with the 32 P-labeled B probe. The autoradiograph shows the location of NF-B and the nonspecific bands (NS). Similar results were obtained in two separate experiments.  (lanes 3-6), were subjected to SDS-polyacrylamide gel electrophoresis Western blotting and probed with anti-phospho-p44/p42 (p-p44/p42) MAPK antibody. B, whole cell lysates, prepared from untreated cells or cells treated with 5 ng/ml TNF-␣ alone (lanes 1 and 2) or with TNF-␣ after pretreatment of the cells with the indicated amounts of PD098059 for 2 h (lanes 3-6), were immunoblotted with anti-phospho-p38 (p-p38) MAPK antibody. C, whole cell lysates, prepared from Mo7e cells pretreated as in A with the exception that they were pretreated with the indicated amounts of SB203580 instead of PD098059, were immunoblotted with anti-phospho-p44/p42 MAPK antibodies. Similar results were obtained in three separate experiments.

p38 MAPK in TNF-␣-induced Cell Proliferation
tribute to TNF-␣-mediated survival and proliferation of human leukemia and lymphoma cells. Studies presented here show that TNF-␣ transiently activates p38 MAPK in Mo7e and several other leukemia cell lines and that p38 MAPK was constitutively activated by autocrine production of TNF-␣ in the human lymphoma Hut-78 cell line. Treatment of Mo7e or Hut-78 cells with a specific p38 MAPK inhibitor SB203580 blocked TNF-␣-dependent phosphorylation of p38 MAPK but had no effect on TNF-␣-induced (in Mo7e) or constitutive (in Hut-78) NF-B activation. On the other hand, inhibition of TNF-␣-induced activation of NF-B had no effect on p38 MAPK activation. Therefore, the activation of p38 MAPK and NF-B molecules by TNF-␣ occurs in independent, parallel signaling pathways. Most significantly, we demonstrate that p38 MAPK plays a critical role in TNF-␣-mediated survival and proliferation of human leukemia and lymphoma cells and acts at least in part by facilitating the ability of NF-B to activate the transcription of the B-driven genes.
TNF-␣ induces growth inhibition or death of some malignant cell lines but promotes the survival and proliferation of some other cell lines (1-5, 7, 9, 10). The signaling mechanisms re-sponsible for its pleiotropic functions are not well understood. In our previous and current reports, we found that both NF-B and p38 MAPK were activated by TNF-␣. To investigate the relationship of these different signaling pathways and their role in TNF-␣-induced cell proliferation, several established inhibitors for activation of NF-B, p38 MAPK, and ERK were used to block activation of certain signaling molecules and to examine the effects on other signaling molecule(s) or on cell proliferation. In the case of p38 MAPK activation, a pyridinyl imidazole compound SB203580 (21-23, 26, 36 -38) has been demonstrated clearly to block activation of p38 MAPK in numbers of other laboratories. In our study, we confirmed that this compound specifically inhibited TNF-␣-induced p38 activation but had no effect on TNF-␣-induced NF-B activation or on IL-3-or GM-CSF-induced p44/p42 MAPK or JAK/STAT activation in several cell lines, including Mo7e, HEL, and Hut-78. For inhibition of NF-B function, we and others previously demonstrated that the SN50 peptide, which contains the p50 NF-B nuclear translocation signal, specifically inhibits nuclear translocation of NF-B (15,29). For inhibition of ERK/MEK, it has been demonstrated in numerous reports that PD098059 has a p38 MAPK in TNF-␣-induced Cell Proliferation specific inhibitory effect on the activation of MEK, which in turn prevents activation of p44/p42 MAPK (21,39,40). Thus, these molecules provide powerful tools to investigate the relationship of different TNF-␣-induced signaling pathways and the roles of these pathways in TNF-␣-induced cell proliferation.
The fact that TNF-␣ activates multiple signaling pathways raises questions whether some of these signaling molecules function by activating or working jointly with each other or with other pathways. We demonstrated that activation of p38 MAPK and NF-B signaling molecules are independent events, at least in the cell lines including Mo7e, Hut-78, HEL, and K562, based on the following evidence: 1) our data demonstrated that activation of p38 MAPK is via a different signaling pathway from that of NF-B activation, as inhibition of NF-B function had no effect on p38 phosphorylation; 2) activation of NF-B does not require activation of p38 MAPK, because inhibition of p38 MAPK activation by SB203580 had no effect on the capacity of TNF-␣ to induce the nuclear translocation of NF-B or to increase the DNA-binding activity of NF-B, thus the initial steps of NF-B activation, which require its release from its cytosolic inhibitor IB␣, and its consequent nuclear translocation and acquisition of DNA-binding activity, appear to occur independently of p38 MAPK activation; 3) given the time frames of p38 MAPK activation (within 15 min, see Fig.  1a) and of the B-driven expression of reporter gene (greater than 60 min, see Fig. 8), it is unlikely that NF-B activity plays any role in p38 MAPK activation; 4) we found that p38 MAPK can be activated by a low dose of arsenite or H 2 O 2 without the activation of NF-B, indicating that phosphorylation and activation of p38 MAPK can occur independently of NF-B activation. 2 These results are consistent with several others reports (8,36) indicating that activation of NF-B and p38 MAPK are separate events.
The p38 MAPK signaling pathway has been implicated in several biological processes, including cytokine expression, cell proliferation, and apoptosis (21,23,27,28); however, its role in TNF-␣-supported cell survival and proliferation remains undocumented. Some studies have shown that activation of the p38 MAPK pathway has a role in growth factor-supported cell proliferation. Using specific chemical inhibitors, PD098059 and SB203580, for MEK and p38 MAPK, Rausch and Marshall (21) demonstrated that both the ERK and p38 pathways are critically involved in the transduction of a proliferative signal in granulocyte-colony-stimulating factor-treated cells. Birkenkamp et al. (41) also reported that activation of p38 MAPK and ERK is involved in IL-3-induced cell proliferation. When SB203580 was used in our study to examine the effect of blockage of p38 MAPK activation on TNF-␣-supported leukemia and lymphoma cell proliferation, we demonstrated that activated p38 MAPK has an essential role in TNF-␣-supported cell proliferation, because inhibition of p38 MAPK activation markedly reduced the TNF-␣-induced increase of [ 3 H]TdR incorporation in Mo7e cells and also inhibited autocrine TNF-␣supported Hut-78 cell survival and proliferation. However, TNF-␣-supported cell proliferation appears to be via a distinct signaling mechanism from that of other growth factors such as IL-3, granulocyte-colony-stimulating factor, and GM-CSF. In TNF-␣-induced cell proliferation, both p38 MAPK and NF-B are required, but the ERK-MEK (p44/p42) signaling pathway does not play any role, because TNF-␣ failed to activate this signaling pathway in the cell lines we examined. On the other hand, we and others reported that the NF-B signaling pathway is not involved in IL-3-, GM-CSF-, or granulocyte-colonystimulating factor-supported cell proliferation, because these cytokines are unable to induce NF-B activation and IB␣ degradation. In accord with our finding that TNF-␣ failed to activate the MEK/p44/p42 signaling pathway in human leukemia and lymphoma cells, Roulston et al. (23), in their study of TNF-␣-induced apoptosis, demonstrated that inhibition of p38 MAPK using MKK4/MKK6 (MKK, MAPK kinase) dominant negative mutants or the p38 inhibitor SB203580 increased TNF-␣-induced apoptosis, whereas expression of wild type MKK4/MKK6 enhanced survival, but the MEK inhibitor PD098059 had no stimulatory or inhibitory effect on cell survival. Thus, they demonstrated that it is activation of p38 MAPK, but not MEK/p44/p42 MAPK, that protects cells from TNF-␣-mediated apoptosis.
It remains unclear how activation of the p38 MAPK signaling pathway participates in cytokine-supported cell prolifera- FIG. 8. Effects of inhibition of p38 MAPK activation on expression of a B-driven SEAP reporter gene. a, Mo7e cells were transfected with a B-driven SEAP reporter gene construct. At 48 h after transfection, cells were treated with 20 ng/ml TNF-␣ alone for 1-6 h or with 50 M SB203580 pretreatment for 2 h and then 20 ng/ml TNF-␣ for 1-6 h. At the indicated time points, supernatants were collected, and the activity of SEAP was determined by soluble PNPP phosphatase substrate kit (Pierce). The relative SEAP activity was calculated by dividing the reading at 405 nM of each treated group by that of the controls. b, Mo7e cells were transfected with a B-driven SEAP reporter gene construct. At 48 h after transfection, the cells were treated without TNF-␣, with 20 ng/ml TNF-␣ for 2, 4, or 6 h, or with pretreatment of indicated amounts of SB203580 for 2 h and then exposed to 20 ng/ml TNF-␣ for 2, 4, or 6 h in the presence of SB203580. At the indicated time points, supernatants were collected and dot-blotted onto nitrocellulose membranes. The activity of SEAP was visualized with enhanced chemiluminescence (Lumi-Phos WB, Pierce). The results shown here are the means of three separate experiments performed.
p38 MAPK in TNF-␣-induced Cell Proliferation tion. In the cases of IL-3-and granulocyte-colony-stimulating factor-induced p38-dependent cell proliferation (21,41), p38 functioned in a NF-B-independent manner and probably was involved in the activation of the STAT signaling pathway(s). However, several studies reported that activation of the p38 MAPK signaling pathway is required for NF-B-dependent gene expression. Craxton et al. (22) reported that inhibition of p38 MAPK activity in vivo with SB203580 significantly reduced expression of a reporter gene driven by a minimal promoter containing four B elements, indicating a requirement for the p38 MAPK pathway in CD40 cross-linking-induced B-driven gene activation. They also reported that cross-linking CD40-mediated NF-B binding to DNA was not affected by SB203580, suggesting that NF-B may not be a direct target for the CD40 cross-linking-induced p38 kinase. In their studies of endotoxin-induced cytokine gene transcription in monocytes and macrophages, Carter et al. (37) also reported that the p38 MAPK signaling pathway is required for NF-B-dependent gene expression. They found that inhibition of the p38 MAP kinase did not alter NF-B activation at any level, but it significantly reduced the DNA binding of the TATA-binding protein to the TATA box. The results of our studies presented here demonstrated that inhibition of p38 MAPK activation had a severe inhibitory effect on TNF-␣-induced expression of a Bdriven alkaline phosphatase reporter gene, but had no significant effect on TNF-␣-induced nuclear translocation of activated NF-B or on the capacity of activated NF-B to bind to an oligonucleotide probe containing the B element. On the other hand, our data show that treating cells with arsenite or H 2 O 2 , which activate p38 MAPK but not NF-B, failed to trigger cell proliferation or to induce the expression of a B-driven alkaline phosphatase reporter gene (data not shown). All of our data presented previously (15) and here indicate that activation of both NF-B and p38 MAPK are required for TNF-␣-induced cell proliferation, and activation of p38 MAPK has a critical role in the expression of B-driven genes, thus, providing the linkage of activation of p38 MAPK and NF-B with TNF-␣mediated cell survival and proliferation. For an optimal understanding of the mechanisms of TNF-␣-induced leukemia cell proliferation, further studies are required to determine a more detailed mechanism of how p38 MAPK activation and NF-B activation interact in stimulating the expression of B-driven genes and what specific gene products promoted by TNF-␣ exert anti-apoptotic and pro-proliferative effects in TNF-␣-induced cell survival and proliferation.