p38 Mitogen-activated protein kinase regulation of JB6 Cl41 cell transformation promoted by epidermal growth factor.

The relationship between cell transformation and p38 MAP kinase, a major mitogen-activated protein (MAP) kinase pathway converting signals of various extracellular stimuli into expression of specific target genes through activation of transcription factors, still remains unclear. The aim of the present study was to investigate the role of the p38 MAP kinase pathway in epidermal growth factor (EGF)-induced cell transformation in JB6 cells. Our data show that a dominant negative mutant of p38 MAP (DN-p38) kinase inhibits EGF-promoted JB6 Cl41 cell transformation and that SB202190, an inhibitor of p38 MAP kinase, also inhibits JB6 Cl41 cell transformation in a dose-dependent manner. Moreover, our results show that DN-p38 MAP kinase inhibits the phosphorylation of EGF-stimulated activating transcription factor-2 (ATF-2) and signal transducer and activator of transcription 1 (STAT1). Additionally, DN-p38 MAP kinase inhibits EGF-induced phosphorylation of c-Myc (Thr58/Ser62). Gel shift assays indicate that DN-p38 MAP kinase inhibits EGF-induced activator protein-1 (AP-1) DNA binding in a dose-dependent manner. These results show that p38 MAP kinase plays a key role in the regulation of EGF-induced cell transformation in JB6 cells through regulation of phosphorylation of p38 MAP kinase and activation of its target genes in phosphorylation, c-Myc cell transformation-related genes, and AP-1 binding ability.

Myc complex, and myocyte enhancer factor 2 (MEF2) (1, 4 -6). On the other hand, ERKs are predominantly activated by mitogenic stimuli, including mitogens and growth factors, and activated ERKs are involved in cell differentiation and development (2,3). Although one major function of the p38 kinase and JNKs pathways is regulation of inflammation and apoptosis, in many cases the biological consequences of p38 kinase and JNK activation overlap with those of ERKs in mediation of cell growth and differentiation (2,3,6,7).
Previously, we studied ERKs regulation of epidermal growth factor (EGF)-induced cell transformation in promotion sensitive (P ϩ ) derivatives of the mouse epidermal JB6 cell line (8).
Results showed that inhibition of ERKs appeared to be an important contributor to the tumor promotion-resistant phenotype in JB6 cells. A recent study showed that the p38 kinase and JNKs pathways cooperate to transactivate the vitamin D receptor through the c-Jun/activator protein-1 (AP-1) (9). This result indicates that a connection exists between p38 MAP kinase and AP-1. But AP-1 activation is involved in JB6 cell transformation promoted by EGF and the phorbol ester 12-Otetradecanoyl-phorbol-13-acetate (TPA) (10,11). AP-1 is a dimeric complex consisting of proteins encoded by the jun and fos gene families, and the AP-1 binding region in these genes is referred to as the TPA response element (TRE). AP-1 induces the transcription of genes that are related to cell proliferation, metastasis, and metabolism (12)(13)(14). Increased AP-1 activity is associated with malignant transformation, and repression of AP-1 activity has been shown to lead to suppression of cell transformation and tumor promotion (15).
Activation of MAP kinase cascades is known to play a considerable role in malignant transformation (3,8). However, whether p38 MAP kinase is involved in the regulation of cell transformation is not known. A recent study showed that only p38 MAP kinase, a MAP kinase usually associated with stress responses, growth arrest, and apoptosis, was consistently increased in human non-small cell lung cancer samples compared with the normal tissues examined (16). More interesting are the results showing that ERKs and JNKs, the MAP kinase pathways traditionally associated with cell growth and perhaps malignant transformation, were not activated in the human non-small cell lung tumor samples. These results indicate that p38 MAP kinase is probably involved in malignant cell transformation. The purpose of this research project was to explore the mechanism of p38-mediated JB6 cell transformation using CMV-neo plasmid-transfected JB6 cells and dominant negative mutant p38 (DN-p38) plasmid-transfected JB6 cells following EGF stimulation. The results obtained show that p38 MAP kinase plays a critical role in regulation of JB6 Cl41 cell transformation promoted by EGF through the inhibition of the phosphorylation of ATF-2, STAT1, and c-Myc and the repression of AP-1 binding ability.
Cell Culture and Establishment of Stably Transfected JB6 Cells-Using the CMV-neo plasmid and DN-p38 kinase plasmid, we established stable transfectants according to the protocol from Invitrogen. All of these cells were selected in media containing 400 g/ml of G-418 for 2 weeks, and then the G418 concentration was decreased to 200 g/ml and maintained. Empty vector CMV-neo plasmid-transfected JB6 cells (Cl41 CMV-neo) and DN-p38 plasmid-transfected JB6 cells (Cl41 DN-p38) were cultured as adherent monolayers in MEM supplemented with 5% (v/v) heat-inactivated FBS and glutamine (2 mM) at 37°C in a humidified atmosphere of 5% CO 2 .
Immunoblotting-Cl41 CMV-neo and Cl41 DN-p38 cells were cultured as described above, and immunoblotting was carried out as described previously (17). In brief, cells were cultured to 80% confluence and then starved in 0.1% FBS/MEM for 24 h at 37°C in a 5% CO 2 incubator. The media were changed to fresh 0.1% FBS/MEM, the cells were incubated for another 2-4 h at 37°C, and the cells were then treated with EGF for different time periods at the concentrations indicated. Cells were then disrupted with lysis buffer (62.5 mM Tris-HCl, pH 6.8, 50 mM dithiothreitol, 2% (w/v) SDS, 10% (v/v) glycerol, and 0.1% bromphenol blue). The lysed samples were transferred into fresh 1.5-ml tubes and sonicated for 5-10 s. Samples containing an equal amount of protein were loaded into each lane of an SDS-polyacrylamide gel for electrophoresis and subsequently transferred onto a polyvinylidene difluoride membrane. Phosphorylation of ERKs, JNKs, ATF-2, STAT1, and c-Myc were selectively detected by Western immunoblotting using a chemiluminescent detection system and a phospho-specific antibody against phosphorylation of p38 kinase (Thr 180 /Tyr 182 ), ERKs (Thr 202 / Tyr 204 ), JNKs (Thr 183 /Tyr 185 ), ATF-2 (Thr 71 ), STAT1 (Ser 727 ), and c-Myc (Thr 58 /Ser 62 ). Antibodies against non-phosphorylated levels of p38 kinase, ERKs, JNKs, ATF-2, STAT1, and c-Myc were used as internal controls to determine loading efficiency.
p38 MAP Kinase Activity Assay-A p38 MAP kinase activity assay was carried out following the instructions from Cell Signaling Technology Inc. Cl41 CMV-neo cells and Cl41 DN-p38 cells were starved in 0.1% FBS/MEM at 37°C in a 5% CO 2 incubator for 24 h and then treated with EGF (10 ng/ml) for various times as indicated. Cells were harvested with lysis buffer (20 mM Tris, pH 7.5, 5 mM ␤-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM Na 3 VO 4 , and 10 mM MgCl 2 ), and protein concentration was determined by the Modified-Lowry protein assay. Total cell lysates were centrifuged at 10,000 rpm for 10 min at 4°C. The cell extracts were incubated overnight at 4°C with 20 l of an immobilized dual phospho-specific p38 MAPK (Thr 180 /Tyr 182 ) monoclonal antibody. After extensive washing, the kinase reaction was performed at 37°C for 10 min in the presence of 100 M ATP and 2 g of an ATF-2 fusion protein. Phosphorylation of ATF-2 at Thr 71 was measured by Western blot using a specific antibody against phosphorylation of ATF-2 (Thr 71 ).
AP-1 DNA Binding Study-Nuclear protein extracts were prepared from cells by a modification of the method of Ye et al. (18). Briefly, Cl41 CMV-neo cells and Cl41 DN-p38 cells were cultured in 10-cm dishes and starved in 0.1% FBS/MEM at 37°C in a 5% CO 2 incubator. After 24 h of starvation, the cells were exposed to different concentrations of EGF for another 12 h. The cells were then harvested and disrupted in 500 l of lysis buffer A (50 mM KCl, 0.5% Nonidet P-40, 100 M dithiothreitol, 25 mM HEPES, pH 7.8, 10 g/ml leupeptin, 25 g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). After a 1-min centrifugation (16,000 ϫ g at 4°C), the pellets containing the nuclei were washed once with 500 l of Buffer B (Buffer A without Nonidet P-40). The pellets were then resuspended in 100 l of extraction buffer (Buffer B, but with 500 mM KCl and 10% glycerol) and strongly shaken at 4°C for 30 min. After a 10-min centrifugation (16,000 ϫ g at 4°C), the supernatant solutions were moved into fresh tubes and stored at Ϫ70°C until analysis. The DNA binding reaction was incubated at room temperature for 30 min in a mixture containing 5 g of nuclear protein, 1 g of poly(dI⅐dC), and 15,000 cpm of a ␣-32 P-labeled double-stranded AP-1 oligonucleotide (5Ј-CGCTTGATGAGTCAGCCGGAA-3Ј). The samples were separated on a 5% polyacrylamide gel, and the gels were analyzed using the Storm 840 phosphorimaging system (Amersham Biosciences).
Anchorage-independent Transformation Assay-The role of p38 MAP kinase in EGF-promoted cell transformation was investigated in Cl41 CMV-neo cells and Cl41 DN-p38 cells. The effect of the inhibitor SB202190 on EGF-promoted cell transformation was explored in JB6 Cl41 cells. In brief, 8 ϫ 10 3 /ml cells were exposed to EGF (5-10 ng/ml) with or without SB202190 (0.1-0.5 M) in 1 ml of 0.3% basal medium Eagle (BME) agar containing 10% FBS. The cultures were maintained at 37°C in a 5% CO 2 incubator for 10 days, and the cell colonies were scored as described by Colburn et al. (19). The effect of DN-p38 on JB6 Cl41 cell transformation is presented as colony number per 10,000 seeded Cl41 CMV-neo cells or Cl41 DN-p38 cells in soft agar, and the effect of SB202190 on JB6 cell transformation is also presented as the number of colonies per 10,000 cells.
Amplification, Mutagenesis, and Expression Vector Construction of the STAT1-To further explore the role of transcriptional factor STAT1, one of the targets of p38 MAP kinase in JB6 Cl41 cell transformation, we gained stable expression of the point mutation at S727A (STAT1-S727A) and expression of only plasmid pcDNA 3.1. The JB6 Cl41 cell lines used included JB6 Cl41-STAT1-mt (S727A) and JB6 Cl41-pcDNA3.1 cells. All of these cells were employed to detect cell transformation ability promoted by EGF. In brief, the cDNA fragment of STAT1, including a 2253-base pair open reading frame (ORF), was reverse-transcribed with an oligo(dT) primer and SuperScript II RNase H Ϫ reverse transcriptase (Invitrogen), amplified by PCR with primers 5Ј-GCA GGA TCC TGT CTC AGT GGT ACG AAC T-3Ј (BamHI site underlined) and 5Ј-TCG ACG CGT TGC TCT ATA CTG TGT TCA TC-3Ј (MluI site underlined), and then cloned into the pACT vector (Promega, Madison, WI) via the BamHI and MluI restriction sites. The STAT1 coding fragment digested with BamHI/EcoRV from the pACT was cloned into the BamHI/SmaI sites of the pUC19 vector. To introduce the point mutation in the position of the Ser 727 residue, a pair of sense and antisense primers for STAT1 S727A, 5Ј-C CTG CTC CCC ATG GCT CCT GAG GAG-3Ј and 5Ј-CTC CTC AGG AGC CAT GGG GAG CAG G-3Ј (underlined nucleotides indicate the codon coding a mutated amino acid) were synthesized (Sigma). Then, the nucleotide substitutions were accomplished using the QuikChange site-directed mutagenesis kit (Stratagene). To construct the mammalian expression vector, the PCR products of wild type STAT1 and STAT1-S727A were amplified using primers 5Ј-CGG GAT CCA TGT CTC AGT GGT ACG AAC TTC A-3Ј (BamHI site underlined) and 5Ј-GGG ATA TCC TAT ACT GTG TTC ATC ATA CTG-3Ј (EcoRV site underlined) and then introduced into same restriction sites of pcDNA3.1-neo (Invitrogen). All of products, including cloning, mutagenesis, or expression vectors, were confirmed by DNA sequence analysis (Sigma).
Statistical Analysis-Significant differences between groups were determined by the Student's and Welch's t tests.

EGF Induces Phosphorylation of p38 Kinase and ERKs but Not JNKs-MAPKs play very important roles in the transformation of mouse JB6 Cl41 cells stimulated by EGF or TPA (8).
Our results showed that EGF induced phosphorylation of ERKs in both time-and dose-dependent manners (Fig. 1, A and G). We further investigated whether EGF also contributed to phosphorylation of other members of MAPKs such as p38 kinase and JNKs. Our results showed that EGF also induced phosphorylation of p38 kinase in both time-and dose-dependent manners (Fig. 1, C and I), but not JNKs (Fig. 1, E and K). These data correspond with other findings that show that EGF mediates cell growth through both MEK/ERK and p38 kinase signal transduction pathways (20,21).
The p38 Kinase Inhibitor SB202190 Inhibits the EGF-induced Phosphorylation of p38 Kinase but Not ERKs-To explore the role of p38 kinase in the EGF-stimulated signal transduction pathway, SB202190, a p38 kinase inhibitor (22)(23)(24)(25), was used to pre-treat JB6 cells at different doses for 1 h. Our results showed that SB202190 inhibited the EGF-induced phosphorylation of p38 kinase in a dose-dependent manner ( Fig. 2B) but had no effect on EGF-induced ERK phosphorylation ( Fig. 2A). These results indicate that p38 MAP Kinase Regulates Cell Transformation Induced by EGF SB202190 can specifically inhibit EGF-induced p38 kinase phosphorylation.
The p38 Kinase Inhibitor SB202190 Suppresses JB6 Cl41 Cell Anchorage-independent Transformation Promoted by EGF-To confirm the role of p38 MAP kinase in JB6 cell transformation, we explored EGF-promoted cell transformation in JB6 Cl41 cells using the p38 kinase inhibitor SB202190. JB6 cells were treated with EGF (10 ng/ml) alone or with SB202190 (0.1-0.5 M) in a soft agar matrix at 37°C in a 5% CO 2 incubator for 10 days. The colony number was determined as described previously, and experiments were repeated three times. Compared with JB6 Cl41 cells stimulated only by EGF, the colony numbers were greatly decreased after EGF and SB202190 treatment, and this inhibition occurred in a dose-dependent manner (Fig. 3, A-E). These data provide more evidence that p38 MAP kinase contributes to JB6 cell transformation promoted by EGF, and this study is the first to report that SB202190 inhibits EGF-induced JB6 Cl41 cell transformation.
DN-p38 MAP Kinase Inhibits Phosphorylation of p38 Kinase but Not ERKs or JNKs-Because SB202190 may affect other molecular targets besides p38 kinase, we used stable transfec-tants of Cl41 CMV-neo cells and Cl41 DN-p38 cells to further study the biological activity of p38 kinase (24,25). Cells were treated with EGF at various concentrations for different times, Cells were then incubated in fresh 0.1% FBS/MEM for another 2 h before being treated with EGF (10 ng/ml) for 5-60 min or EGF (5-20 ng/ml) for 30 min. Phosphorylation of p38 kinase (C and I), ERKs (A and G), and JNKs (E and K) were determined by Western blot analysis as described under "Experimental Procedures" using specific antibodies against phosphorylation of p38 kinase (Thr 180 /Tyr 182 ), ERKs and JNKs. Total protein levels of p38 kinase (D and J), ERKs (B and H), and JNKs (F and L) were determined as described under "Experimental Procedures" using corresponding antibodies against non-phosphorylated proteins.
FIG. 2. SB202190 inhibits phosphorylation of p38 kinase but not ERKs. JB6 cells were cultured and starved as described above. SB202190 (0.25-1.0 M) was used to pretreat JB6 cells for 1 h, and then JB6 cells were treated with EGF (10 ng/ml) for 30 min. Phosphorylation of ERKs (A) and p38 kinase (B) were determined by Western blot analysis as described under "Experimental Procedures" using specific antibodies against phosphorylation of p38 kinase and ERKs. Total protein levels of p38 kinase and ERKs were determined as described above.
FIG. 3. SB202190 inhibits JB6 Cl41 anchorage-independent transformation promoted by EGF. JB6 Cl41 anchorage-independent transformation promoted by EGF was performed as described previously. Final concentrations of 0.1 or 0.5 M SB202190 were added into soft agar, and the colonies were counted automatically after 10 days of incubation at 37°C in a 5% CO 2 atmosphere. Almost no colony formation was observed in JB6 cells without EGF stimulation (A), and many colonies were observed in JB6 Cl41 cells treated only with EGF (B). SB202190 suppressed EGF-stimulated JB6 Cl41 cell colony formation in a dose-dependent manner (C and D). The average number of colonies was determined from three separate experiments; ctrl, control (E).

p38 MAP Kinase Regulates Cell Transformation Induced by EGF
and Western blot analysis was used to determine the level of phosphorylation of p38 kinase, ERKs, and JNKs using specific antibodies. Our data showed that EGF induced phosphorylation of p38 kinase and ERKs in a dose- (Fig. 4, A and C) and time-dependent (Fig. 4, G and I) manner but had no effect on phosphorylation of JNKs (Fig. 4, E and K). Total protein levels of these kinases did not change (Fig. 4, B, D, F, H, J, and L). These data also showed that DN-p38 inhibited the EGF-induced phosphorylation of p38 kinase in a time-and dose-dependent manner but had no effect on phosphorylation of ERKs or JNKs. Results indicate that ERKs and p38 kinase, but not JNKs, are involved in EGF-stimulated cell signal transduction.
DN-p38 Inhibits Anchorage-independent Transformation Promoted by EGF-From the results described above, we hypothesized that p38 kinase could have a role in regulating JB6 cell transformation. Cell transformation was assessed using our previously developed methods (26,27). Cl41 CMV-neo cells and Cl41 DN-p38 cells were treated separately with EGF (5-10 ng/ml) in a soft agar matrix at 37°C in a 5% CO 2 incubator for 10 days. The number of colonies formed was counted automatically by computer, and the same experiment was repeated three times. Our results showed that DN-p38 strongly inhibited the formation of EGF-induced colonies (Fig. 5D) compared with control cells (Fig. 5B). The inhibition was evident not only in colony number (Fig. 5E) but also in colony size (Fig. 5, B  versus D). However, untreated Cl41 CMV-neo cells and Cl41 DN-p38 cells showed no colony formation (Fig. 5, A and C). These data indicate that p38 MAP kinase is involved in JB6 cell transformation as a positive regulator rather than an inhibitor.
DN-p38 Inhibits EGF-induced p38 MAP Kinase Activity-To further confirm that the Cl41 DN-p38 stable transfectant cells work as expected, p38 MAPK activity toward its substrate, ATF-2, was assayed. Cells were treated with EGF (10 ng/ml) for the indicated times, and the cell extracts were incubated with an immobilized p38 kinase antibody. An ATF-2 fusion protein was used as a substrate of p38 MAP kinase, and phosphorylation of ATF-2 at Thr 71 was detected by Western blot. Our results showed that EGF-induced phosphorylation of ATF-2 at Thr 71 was decreased distinctly in Cl41 DN-p38 cells compared with Cl41 CMV-neo cells (Fig. 6). These data indicate that p38 kinase has a role in EGF-induced phosphorylation of ATF-2.

p38 MAP Kinase Regulates Cell Transformation Induced by EGF DN-p38 Inhibits EGF-induced Phosphorylation of STAT1 at Ser 727 in a Time-and Dose-dependent Manner-STAT1
is downstream of p38 MAP kinase and is a transcription factor that mediates cytokine and growth factor-induced signals that culminate in various biological responses, including proliferation and differentiation (28). Cl41 DN-p38 cells and Cl41 CMVneo cells were treated separately for various times and at various doses with EGF. We found that EGF (10 ng/ml) strongly induced phosphorylation of STAT1 at Ser 727 in Cl41 CMV-neo cells, but the phosphorylation of STAT1 at Ser 727 in Cl41 DN-p38 cells was relatively weak. However, phosphorylation of STAT1 gradually decreased within 120 min in both cell lines (Fig. 7A). Non-phosphorylated levels of STAT1 were unchanged throughout the time course and dose course (Fig. 7, B  and D). EGF-induced phosphorylation of STAT1 at Ser 727 gradually increased with increasing EGF concentrations (Fig. 7C) with no change in non-phosphorylated levels of STAT1 (Fig.  7D).
DN-p38 Inhibits Phosphorylation of c-Myc at Thr 58 / Ser 62 -To understand the mechanism of p38 MAP kinase in the regulation of JB6 cell transformation, we investigated the phosphorylation of c-Myc by Western blot using a specific antibody against phosphorylation of c-Myc at Thr 58 /Ser 62 . The c-Myc oncoprotein is associated with cell growth, development, and malignant transformation in human tumors and cell lines (29,30). The active c-Myc also contributes to cell transforming potencies in rat embryo cells (31). In this study, our results showed that phosphorylation of c-Myc at Thr 58 /Ser 62 was increased after CMV-neo JB6 cells were treated from 1-4 h with EGF (10 ng/ml), but phosphorylation of c-Myc at Thr 58 /Ser 62 in DN-p38-JB6 cells was relatively less within the same time course (Fig. 8A). EGF had no effect on total c-Myc protein levels in Cl41 CMV-neo cells or Cl41 DN-p38 cells (Fig. 8, B and D). Moreover, we studied the effects of various doses of EGF on phosphorylation of c-Myc at Thr 58 /Ser 62 . Results showed that phosphorylation of c-Myc at Thr 58 /Ser 62 increased in Cl41 CMV-neo cells but was only slightly changed in Cl41 DN-p38 cells after cells were treated with EGF (5-20 ng/ml) (Fig. 8C). These data indicate that phosphorylation of c-Myc at Thr 58 / Ser 62 is induced by EGF in a time-and dose-dependent manner in Cl41 CMV-neo cells and that DN-p38 inhibits the phosphorylation of c-Myc at Thr 58 /Ser 62 .
A Mutant of STAT1 (S727A) Suppresses JB6 Cl41 Cell Anchorage-independent Transformation Promoted by EGF-Stable transfectant JB6 Cl41-STAT1 (S727A) cells expressing of STAT1 (S727A) and JB6 Cl41-pcDNA3.1 cells expressing plas-mid pcDNA3.1 cells were employed to detect the cell transformation ability promoted by EGF. Cells were treated with EGF (10 ng/ml) in a soft agar matrix at 37°C in a 5% CO 2 incubator for 10 days. The colony number was determined as described previously (8). The colony numbers were greatly decreased in JB6 Cl41-STAT1 (S727A) cells promoted by EGF, but there were many more colonies in JB6 Cl41 cells and JB6 Cl41-pcDNA 3.1 stimulated by EGF under the same condition (Fig. 9, A-F). These data indicate that STAT1, one of the targets of p38 MAP kinase, also contributes to JB6 Cl41 cell transformation promoted by EGF. FIG. 6. DN-p38 MAP kinase inhibits p38 MAP kinase activity in vivo. Cl41 CMV-neo cells and Cl41 DN-p38 cells were seeded in 100-mm dishes with 5% FBS/MEM and incubated at 37°C in a 5% CO 2 atmosphere for 24 h. Cells were starved as described previously. Cells were then incubated in fresh 0.1% FBS/MEM for another 2 h before being treated with EGF (10 ng/ml) for the indicated time periods. Cells were added to lysis buffer as described under "Experimental Procedures," and the same amount of extracted proteins was immunoprecipitated with a monoclonal phospho-specific antibody against p38 MAP kinase (Thr 180 /Tyr 182 ). The resulting immunoprecipitate was incubated with an ATF-2 fusion protein in the presence of ATP and kinase buffer. Phosphorylation of ATF-2 at Thr 71 was measured by Western blotting using the phospho-ATF-2 (Thr 71 ) antibody (A), and total ATF-2 protein level was determined by Western blot using an antibody against nonphosphorylated ATF-2 protein (B) .   FIG. 7. DN-p38 MAP kinase inhibits phosphorylation of STAT1 (Ser 727 ). Culture and starvation of Cl CMV-neo cells and Cl41 DN-p38 cells were performed as described above. In the time course study (A and B), cells were treated with EGF (10 ng/ml) for the indicated time periods and then harvested with lysis buffer, and the protein concentration of each sample was determined. Equal amounts of protein were separated by 8% SDS-PAGE gel and analyzed by Western blotting. Phosphorylation of STAT1 at Ser 727 was detected using a specific phospho-STAT1 (Ser 727 ) antibody (A). Total STAT1 protein levels were detected by a non-phospho-STAT1 antibody (B). In the dose course study (C and D), Cl41 CMV-neo cells, and Cl41 DN-p38 cells were treated with EGF at the indicated concentration for 15 min. Phosphorylation of STAT1 at Ser 727 (C) and total STAT1 protein levels (D) were detected as described above.

FIG. 8. DN-p38 MAP kinase inhibits phosphorylation of c-Myc at Thr 58 /Ser 62 . Culture and starvation of Cl41 CMV-neo cells and Cl41
DN-p38 cells were performed as described above. In the time course study (A and B), cells were treated with EGF (10 ng/ml) for the indicated time periods and then harvested with lysis buffer, and the protein concentration of each sample protein was determined. Western blotting was employed to analyze the phosphorylation of c-Myc at serine 63 using a specific phospho-c-Myc (Thr 58 /Ser 62 ) antibody (A). Total c-Myc protein levels were detected by a non-phospho-c-Myc antibody (B). In the dose course study (C and D), Neo-JB6 Cl41 and DN-p38-JB6 Cl41 cells were treated with EGF at the indicated concentration for 4 h. Phosphorylation of c-Myc at Thr 58 /Ser 62 (C) and total c-Myc protein levels (D) were detected as described above.

p38 MAP Kinase Regulates Cell Transformation Induced by EGF DN-p38 Inhibits EGF-induced AP-1 Binding Activity-AP-1
induces gene transcription by binding to the TPA response element site in the promoter region of its target gene and is required for cell proliferation, differentiation, and malignant transformation (32,33). Double-stranded AP-1 oligonucleotides were labeled with ␥-32 P, and electrophoretic mobility-shift assays were used to measured AP-1 DNA binding activity. Our results showed that AP-1 binding was decreased in Cl41 DN-p38 cells (Fig. 10, A and B, lanes 3, 4, and 5) compared with that in Cl41 CMV-neo cells (Fig. 10, A and B, lanes 7, 8, and 9) after cells were treated with EGF (5-20 ng/ml) for 12 h. However no significant change was observed between DN-p38-JB6 cells and Neo-JB6 cells without EGF stimulation (Fig. 10, A  and B, lanes 2 and 6). To confirm that the electrophoretic mobility-shift band was specific for AP-1 binding, a 10-fold amount of unlabeled AP-1 oligonucleotide was added, and results showed that the AP-1 binding band was completely removed (Fig. 10, A and B, lane 1). These data indicate that DN-p38 inhibits EGF-induced AP-1 binding ability. DISCUSSION Previous studies show that ERKs are involved in the JB6 cell transformation promoted by EGF or TPA (8), but an understanding of the role of p38 MAP kinase in cell transformation still remains unclear. In this study, we found that p38 MAP kinase plays a critical role in JB6 Cl41 cell transformation promoted by EGF. Both DN-p38 and the p38 MAP kinase inhibitor SB202190 suppressed JB6 Cl41 cell transformation promoted by EGF. DN-p38 inhibited EGF-stimulated phosphorylation of ATF-2, STAT1, and c-Myc, and gel shift assay results showed that DN-p38 inhibits AP-1 binding ability. These results indicate that p38 MAP kinase is involved in regulation of JB6 Cl41 cell transformation promoted by EGF through the inhibition of phosphorylation of its downstream factors, including ATF-2, STAT1, c-Myc, and also AP-1 DNA binding.
Many studies indicate that EGF induces the phosphorylation of ERKs and p38 MAP kinase (21, 34 -36). The ERK pathway is associated with activation of the EGF receptor and has been shown to play a major role in promoting several tumor phenotypes. However, the JNKs pathway has not been shown to be activated through the EGF receptor but is instead more uniformly stimulated by cellular stresses and cytokines (21). Our present data also show that EGF only induces the phosphorylation of ERKs and p38 MAP kinase but not JNKs (Fig. 1).  7-9). B, quantitation analysis of panel A from three separate electrophoretic mobility shift assays performed using nuclear extracts from different cell preparations.
SB202190 and DN-p38 only inhibit EGF-induced phosphorylation of p38 MAP kinase but not ERKs (Figs. 2 and 4). These results indicate that ERKs and p38 MAP kinase, but not JNKs, independently participate in EGF-stimulated cell signal transduction.
The p38 MAP kinase is usually associated with stress responses, growth arrest, and apoptosis (1,2,4). But a recent study (16) showed that it was activated in human lung cancer samples, suggesting an additional role for this pathway in malignant cell growth or transformation. The present study is the first to report that p38 MAP kinase is involved in JB6 Cl41 cell transformation promoted by EGF. SB202190, a p38 MAP kinase inhibitor, distinctly inhibited colony formation in EGFpromoted JB6 Cl41 cell transformation (Fig. 3). To eliminate the effects of SB202190 on other molecular targets, we successfully constructed a stable transfectant of JB6 Cl41 cells expressing a dominant negative mutant of p38 kinase and DN-p38 according to the method described previously (37,38). Our results showed that the number of colonies in EGF-treated Cl41 DN-p38 cells was significantly decreased compared with the number of colonies in Cl41 CMV-neo cells (Fig. 5). These data indicate that p38 MAP kinase plays a critical role in JB6 Cl41 cell transformation promoted by EGF.
To explore the mechanism of p38 kinase in the regulation of JB6 cell transformation, we first determined whether p38 MAP kinase activity was inhibited in Cl41 DN-p38 cells. Our results show that DN-p38 distinctly inhibits EGF-induced phosphorylation of ATF-2 at Thr 71 (Fig. 6). Thr 71 is a major ATF-2 phosphorylation site required for transcriptional activity, and a high level of phosphorylated ATF-2 protein correlates with malignant phenotypes in the multistage mouse skin carcinogenesis model (39). One recent study showed that EGF activates transcription factor ATF-2 through a two-step mechanism. The Raf-MEK-ERK pathway induces phosphorylation of ATF-2 (Thr 71 ), whereas subsequent ATF-2 Thr 69 phosphorylation requires the Ral-RalGDS-Src-p38 MAP kinase pathway (40). Moreover, cooperation between ERKs and p38 kinase was found to be essential for ATF-2 activation by EGF (41).
Some of the STAT family members have a role in the regulation of cellular transformation (42). A recent study showed that STAT3 activation is required for interleukin-6-induced transformation in tumor promotion-sensitive mouse skin epithelial cells (43). Another report showed that transformation of normal fibroblasts cells with E1A ϩ Ha-Ras oncogenes causes a constitutive activation of STAT1 and STAT3 transcription factors (44). In this study, we found that EGF-induced phosphorylation of STAT1 (Ser 727 ) also decreased in Cl41 DN-p38 cells but increased in Cl41 CMV-neo cells after the cells were separately treated with EGF (10 ng/ml) (Fig. 7). We successfully employed directed point mutation technology to obtain a mutant of STAT1 at Ser 727 (Ser 3 Ala). Stable transfectant of JB6 Cl41-STAT1 (S727A) cells expressing mutant of STAT1 at Ser 727 (Ser 727 (Ser 3 Ala) displayed its ability to inhibit cell transformation in JB6 Cl41 cells promoted by EGF (Fig. 9). These results indicate that EGF-induced phosphorylation of STAT1 at Ser 727 is at least partially dependent on p38 MAP kinase and that STAT1 is involved in JB6 cell transformation promoted by EGF.
Studies show that the c-Myc oncoprotein is associated with cancer cell proliferation and transformation (45,46). In this study, we also found that DN-p38 inhibited EGF-induced phosphorylation of c-Myc, indicating that p38 MAP kinase mediates EGF-induced c-Myc phosphorylation (Fig. 8). Thus, p38 MAP kinase may be involved in cell transformation through phosphorylation of c-Myc. On the other hand, AP-1, a heterodimer commonly comprised of the basic leucine zipper proteins Fos and Jun, plays a very important role in the induction of neoplastic transformation and the multiple genes involved in cell proliferation, differentiation, and inflammation (47,48). Blocking the tumor promoter-induced activation of AP-1 was shown to inhibit neoplastic transformation in JB6 mouse epidermal cells (49). One recent study (47) showed that p38 MAPK and ERK inhibition with SB203580 and PD98059, respectively, significantly inhibited silica-induced AP-1 activation. These findings demonstrate that AP-1 activation may be mediated through the p38 MAPK and ERKs pathways (47). In the present research, EGF-induced AP-1 binding ability decreased in Cl41 DN-p38 cells compared with Cl41 CMV-neo cells (Fig. 10). These data provide further evidence that EGF-induced AP-1 binding ability is mediated through p38 MAP kinase and that DN-p38 MAP kinase represses EGF-induced JB6 Cl41 cell transformation through inhibition of AP-1 DNA binding ability.
In summary, we report for the first time that p38 MAP FIG. 11. Proposed signal transduction pathways of EGF-promoted JB6 Cl41 cell transformation. We reported previously that ERKs are necessary factors in EGF-promoted JB6 Cl41 cell transformation. In the present study, our data show that DN-p38 MAP kinase effectively blocks EGF-promoted JB6 Cl41 cell transformation through inhibition of the phosphorylation of transcription factors, ATF-2 and STAT1, oncogenes, c-Myc, and repression of AP-1 DNA binding. Both p38 kinase and ERKs are involved in the EGF-promoted JB6 Cl41 cell transformation.

p38 MAP Kinase Regulates Cell Transformation Induced by EGF
kinase mediates EGF-promoted JB6 Cl41 cell transformation through regulation of the phosphorylation of its downstream factors, such as STAT1, ATF-2, and c-Myc, and also AP-1 DNA binding ability (Fig. 11). This study illustrates that p38 MAP kinase is another new pathway involved in JB6 Cl41 cell transformation promoted by EGF.