The signal transduction networks required for phosphorylation of STAT1 at Ser727 in mouse epidermal JB6 cells in the UVB response and inhibitory mechanisms of tea polyphenols
Tatyana A. Zykova,
Yiguo Zhang1,
Feng Zhu,
Ann M. Bode and
Zigang Dong2
Hormel Institute, University of Minnesota, Austin, MN 55912, USA and 1 Biomedical Research Centre, University of Dundee, Level 5, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK
2 To whom correspondence should be addressed Email: zgdong{at}hi.umn.edu
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Abstract
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Signal transducers and activators of transcription (STATs) play a critical role in signal transduction pathways. STATs are a family of cytoplasmic proteins with roles as signal messengers and transcription factors that participate in normal cellular responses to cytokines and growth factors. Phosphorylation of STAT1 at Ser727 is essential for its activation and occurs in response to stress signals, inflammation or infection. We observed that UVB induced phosphorylation of STAT1 (Ser727) in mouse epidermal JB6 Cl41 cells. This stimulation was inhibited by PD98059 and UO126, wortmannin, LY294002, SB202190 and SP600125 and dominant negative mutants of ERK2 (DNM-ERK2), p38 (DNM-p38) and JNK1 (DNM-JNK1). The response was absent in Jnk1/ or Jnk2/ knockout cells, but was unaffected by a dominant negative mutant of the phosphatidylinositol-3 kinase (PI-3K) p85 subunit (DNM-
p85). STAT1 (Ser727) phosphorylation was also blocked in a Rsk2 cell line. In Pdk1/ cells STAT1 was not activated by UVB stimulation compared with strong activation in Pdk1+/+ cells. Our data indicate that phosphorylation of STAT1 (Ser727) occurs through PI-3K, ERKs, p38 kinase, JNKs, PDK1 and p90RSK2 in the cellular response to UVB. We also show an inhibitory effect of theaflavins and EGCG on UVB-induced STAT1 (Ser727), ERKs, JNKs, PDK1 and p90RSK2 phosphorylation.
Abbreviations: AP-1, activator protein-1; DMEM, Dulbecco's modified Eagle's medium; DNM, dominant negative mutants; EGCG, ()-epigallocatechin 3-gallate; EGF, epidermal growth factor; EMEM, Eagle's minimum essential medium; ERKs, extracellular signal-regulated kinases; FBS, fetal bovine serum; GST, glutathione S-transferase; IFN, type I interferon; JNKs, c-Jun N-terminal kinases; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MSK1, mitogen- and stress-activated protein kinase 1; NP, non-phosphorylated; p90RSK, p90 kDa ribosomal S6 kinases; PBS, phosphate-buffered saline; PDK1, phosphoinositide-dependent protein kinase 1; PI-3K, phosphatidylinositol-3 kinase; PMSF, phenylmethylsulfonyl fluoride; STATs, signal transducers and activators of transcription; TPA, 12-O-tetradecanoyl-phorbol-13-acetate
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Introduction
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Over the past few years researchers have deciphered a signaling pathway which initiates within the cytoplasm but quickly translocates to the nucleus to activate transcription of target genes. This novel signaling pathway features a group of transcription factors called signal transducers and activators of transcription (STATs). These transcription factors were described by Darnell et al. (1,2) as ligand-induced transcription factors in interferon-treated cells. Subsequent studies by a number of groups showed that STATs play a critical role in signal transduction pathways associated with several cytokines and growth factors. It is now known that many cytokines, hormones and growth factors utilize STAT signaling pathways to control a remarkable variety of biological responses, including development, differentiation, cell proliferation and survival (25).
Phosphorylation of STAT1 at Ser727 occurs in response to stress, inflammation or infection. Stress signals to STAT1 through p38 kinase, which specifically phosphorylates STAT1 at Ser727 in vitro (6,7). Serine phosphorylation of STAT1 can also occur through other signal transduction pathways (810). In macrophages, phosphorylation of STAT1 (Ser727) was induced by interferon-
, bacterial lipopolysaccharide, the proinflammatory cytokine tumor necrosis factor
and UVC irradiation, with the stronger signal resulting from UVC irradiation (11). An effect of UVA irradiation on STAT1 activity and phosphorylation in cultured human keratinocytes has been reported (12). Activation of STAT3, which is highly homologous to STAT1, by UVB or UVC irradiation occurred through phosphorylation at Ser727 (11,13,14). Phosphorylation of the Ser727 residue in the C-terminal transcriptional activation domain in both STAT1 and STAT3 enhances the transcriptional activity of these STATs (15).
The phosphorylation of STAT1 or STAT3 in vitro by extracellular signal-regulated kinases (ERKs) (16,17), p38 kinase or c-Jun N-terminal kinases (JNKs) (17,18) provides evidence that members of the mitogen-activated protein kinase (MAPK) family can induce STAT serine phosphorylation. Mitogen- and stress-activated protein kinase 1 (MSK1) and JNKs mediate phosphorylation of STAT3 in UVA-irradiated mouse epidermal JB6 cells (19). Phosphorylation of STAT3 at Ser727 by UVA or UVC irradiation was stimulated through a signaling pathway involving ATM, MAPKs and p90 kDa ribosomal S6 kinases (p90RSK) (20). Evidence showing that UVA-induced MAPK signaling pathways lead to STAT1 (Ser727) phosphorylation was also provided and indicated that phosphorylation of STAT1 at Ser727 occurs through diverse MAPK cascades including MAPK/ERK kinase (MEK), ERKs, p38 kinase, JNKs and MSK1 in the cellular response to UVA (17). Solar UV irradiation is believed to be one of the most important skin carcinogens. UVA (320400 nm) constitutes >90% of solar UV, of which all of the UVC and most of the UVB (290320 nm) are absorbed by the ozone layer of the Earth's atmosphere. Different spectra of UV (UVA, UVB and UBC) induce different signal transduction pathways (21). UV activates various signaling pathways that are either oncogenic or protective or both. Many of these pathways are mediated primarily through signaling cascades involving MAPKs, resulting in the modification of transcription factors such as activator protein-1 (AP-1), which can lead to skin cancer. In the light of rising public concern over the increased incidence of skin cancer, many experiments focus on the mechanistic data supporting a role for MAPKs in stimulated skin carcinogenesis. Progress in understanding the mechanisms of UV-induced signal transduction could lead to the use of these protein kinases as specific targets for the prevention and control of skin cancer. Whether UVB stimulates STAT1 signaling through Ser727 phosphorylation is as yet unknown (21). Here, we provide evidence showing that UVB-induced cellular signaling networks are involved in modulating phosphorylation of STAT1 Ser727. Our data indicate that in the cellular response to UVB phosphorylation of STAT1 at Ser727 is mediated through diverse MAPK cascades including phosphatidylinositol-3 kinase (PI-3K), ERKs, p38 kinase, JNKs, phosphoinositide-dependent protein kinase 1 (PDK1), the serine/threonine kinase Akt (protein kinase B) and p90RSK2.
The polyphenols from green and black tea, ()-epigallocatechin 3-gallate (EGCG) and theaflavins, respectively, are generally considered to be the most effective components of tea in inhibiting carcinogenesis. The molecular mechanism of their antitumor effects might be related to their ability to block signal transduction pathways leading to carcinogenesis (22,23). We previously showed that tea polyphenols inhibit UVB-induced AP-1 activation in mouse epidermal JB6 cells (24). UVB-induced phosphorylation of ERKs and JNKs was also inhibited by theaflavins and EGCG (24). Green tea enhances UVB-induced increases in p53 and p21 proteins and apoptosis, suggesting that green tea affects other pathways in addition to AP-1 (25). The polyphenols from tea inhibited 12-O-tetradecanoyl-phorbol-13-acetate (TPA)- and epidermal growth factor (EGF)-induced c-Jun phosphorylation and JNK activation (26). EGCG and theaflavins block phosphorylation of nuclear factor
B and/or degradation of I
B
(27). We showed that EGCG or theaflavins inhibit UVB-induced PI-3K activation in mouse epidermal JB6 Cl41 cells (28). UVB-induced phosphorylation and activation of the PI-3K downstream effectors Akt and ribosomal p70 S6 kinase (p70S6K) was also suppressed by the polyphenols (28). In the present study we determined the effect of tea polyphenols, theaflavins and EGCG on UVB-induced STAT1 (Ser727) phosphorylation. Our findings indicate that these tea polyphenols inhibit UVB-induced STAT1 (Ser727) phosphorylation and that the inhibition was mediated by ERKs, JNKs, PDK1 and p90RSK.
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Materials and methods
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Materials
Eagle's minimum essential medium (EMEM), fetal bovine serum (FBS) and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine and G418 sulfate were from Life Technologies Inc. (Grand Island, NY); the PI-3K inhibitors LY29402 and wortmannin were from BioMol Research Laboratories Inc. (Plymouth Meeting, PA); the MEK1/2 specific inhibitor UO126 and the JNK specific inhibitor SP600125 were from Calbiochem (La Jolla, CA); phosphorylated and non-phosphorylated p44/42 MAPK, p38 kinase, JNKs, PDK1, p90RSK and STAT1 antibody kits were from Cell Signaling Technology Inc. (Beverly, MA). Dominant negative mutants (DNM) of p38, ERK2 and JNK1 were prepared and identified as described previously (2931); wild-type and DNM-Akt were from Upstate Biotechnology Inc. (Lake Placid, NY); the DNM of subunit
p85 was prepared as described previously (32,33); Jnk1/ and Jnk2/ knockout mice were a gift from Dr Richard Flavell, Yale University (34); mouse stem cell lines defective for (Pdk1/) and wild-type for (Pdk1+/+) PDK1 were from Dr D.R.Alessi (35); the wild-type and Rsk2 cell lines were from the NIGMS Human Genetic Cell Repository (Coriell Institute for Medical Research, Camden, NJ; http://locus,umdnj.edu) and cultured as described previously (20); theaflavins (a mixture of theaflavin, theaflavin 3-gallate, theaflavin 3'-gallate, theaflavin 3,3'-digallate and unknowns, accounting for 21, 30, 15, 28 and 6%, respectively) were gifts from the Thomas J. Lipton Co. (Englewood Cliffs, NJ); EGCG (purity 95%), the p38 kinase inhibitor SB202190 and the MEK1 inhibitor PD98059 were from Sigma.
UVB irradiation
UVB irradiation was carried out in a UVB chamber with a transluminator emitting UVB light protons and fitted with an Eastman Kodak Co. Kodacel K6808 filter that eliminates all wavelengths below 290 nm. This lamp is one of the most frequently used UVB sources for the study of carcinogenesis. Irradiation energy was measured using a UVX radiometer from UVP (UVX-31).
Cell culture
Mouse epidermal JB6 promotion-sensitive Cl41 cells and stable transfectants with CMV-neo (Cl41) or DNM cell lines for JNK1 (DNM-JNK1) (31), p38 kinase (DNM-p38) (29), the p85 subunit of PI-3K (DNM-
p85) (32,33) or ERK2 (DNM-ERK2) (30) were established. They were cultured in monolayers using EMEM supplemented with 5% heat-inactivated FBS, 2 mM L-glutamine and 25 µg/ml gentamicin at 37°C in humidified air with 5% CO2. Before each experiment, transfectants were selected with G418 and tested with phospho-specific antibodies.
Phosphorylation of STAT1, p38, ERKs and JNKs, PDK1 and p90RSK
Western blotting analysis of phosphorylated STAT1, p38 kinase, ERKs, JNKs, PDK1 and p90RSK2 proteins was carried out using the respective phospho-specific antibodies. Equal numbers of JB6 Cl41 cells (7 x 105) were cultured for 1215 h in 10 cm diameter dishes. After 7080% confluence was reached the cells were starved for 24 h (48 h for PDK1 and RSK) in EMEM containing 0.1% FBS. Cells were treated with UVB irradiation (4 kJ/m2), harvested and then disrupted in 200 µl of RIPA buffer [1x phosphate-buffered saline (PBS), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, 1 mM aprotinin and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. The samples were sonicated and centrifuged at 14 000 r.p.m. for 15 min. The quantity of protein was measured by the Bradford method (36) and normalized to controls. The samples were heated to 95°C for 5 min, cooled on ice and centrifuged at 14 000 r.p.m. for 5 min. Then the samples (2030 µg protein) were resolved by 1012% SDSPAGE. Immunoblot analysis was carried out using specific antibodies against phosphorylated sites of STAT1 (Ser727), ERKs (p44/p42), JNKs, PDK1 and p90RSK (Thr359/Ser363). Some transfer membranes were washed with stripping buffer (7 M guanidine hydrochloride, 50 mM glycine, pH 10.8, 0.05 mM EDTA, 0.1 M KCl and 20 mM ß-mercaptoethanol) and reproved with other primary phospho-specific and non-phospho-specific antibodies. Antibody-bound proteins were detected by chemiluminescence (ECF Western Blotting Kit; Amersham Pharmacia Biotech) and analyzed using a Storm 840 Scanner (Molecular Dynamics, Sunnyvale, CA). Non-irradiated cell samples were used as negative controls.
Preparation and analysis
Embryo fibroblasts from normal Jnk1/ or Jnk2/ knockout mice were isolated and prepared according to established procedures (34). Mouse stem cell lines defective for PDK1 (Pdk1/) and corresponding wild-type cell lines were carefully cultured according to a previous report (35). The wild-type and RSK2 cell lines were cultured as described previously (20). Cells were established in culture in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C. For analysis of protein phosphorylation, the cells were starved by replacing growth medium with serum-free DMEM for 24 h, at which time they were exposed to UVB. The cells were lysed with SDS sample buffer and protein concentration in the cell lysates was determined by the Bradford method (36). Equal amounts of protein were loaded onto a 10% SDSpolyacrylamide gel and levels of phosphorylated and non-phosphorylated proteins were determined by western blot analysis.
Analysis of STAT1 (Ser727) phosphorylation with phospho-specific antibodies
Cells (70 x 104) were seeded into 10 cm dishes and cultured for 1215 h until the cells reached 80% confluence. The CMV-neo Cl41, DNM-ERK2, DNM-JNK1, DNM-p38 and DNM-
p85 cells were starved for 24 h in MEM containing 0.1% FBS, 2 mM L-glutamine and 25 µg/ml gentamicin. After treatment with UVB (4 kJ/m2) and incubation for 30 min after UVB, the cells were washed once with ice-cold PBS and lysed in 200 µl of RIPA buffer. The lysed samples were scraped into 1.5 ml tubes and sonicated for 510 s. Samples containing equal amounts of protein were loaded into each lane of a 10% SDSpolyacrylamide gel for electrophoresis and subsequently transferred onto an Immobilon P transfer membrane. Phosphorylated STAT1 (Ser727) was selectively detected by western immunoblotting using a chemiluminescent detection system and phospho-specific antibodies against STAT1 phosphorylation at Ser727. Non-phosphorylated STAT1 was used as a control to verify equal protein loading.
Immunoprecipitation assay
After culturing for 1224 h the experimental cells were starved for 24 h in DMEM containing 1% FBS. The cells were harvested at the indicated times following irradiation and lysed in 250 µl of immunoprecipitation buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% v/v Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 1 mM PMSF). The clarified supernatant fractions containing equal amounts of protein were subjected to immunoprecipitation followed by western blot analysis. An antibody against total STAT1 was used for immunoprecipitation of STAT1 protein and the immune complex beads were washed with PBS.
Assay for in vitro phosphorylation by protein kinases
Samples containing glutathione S-transferase (GST) pull-down STAT1 proteins (17) were incubated at 30°C for 60 min with active RSK1, RSK2 or Akt (Upstate Biotechnology) in kinase buffer (50 mM TrisHCl, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol and 0.01 % Brij 35) (Cell Signaling) containing 5 mM ATP or 1 µCi [
-32P]ATP. The reactions were stopped by adding 5x SDS sample buffer, then phosphorylation of STAT1 was analyzed by autoradiography. Total STAT1 was utilized as an internal control to verify equal protein loading.
Inhibition of STAT1 (Ser727) phosphorylation by theaflavins and EGCG
JB6 Cl41 cells were cultured to 80% confluence. The cells were starved in EMEM containing 0.1% FBS for 24 h at 37°C. Then the medium was changed to fresh EMEM containing 0.1% FBS and the cells were incubated for another 6 h at 37°C. Before the cells were irradiated with UVB they were incubated with or without EGCG or theaflavins for 1 h. Then the cells were exposed to UVB (4 kJ/m2) and subsequently incubated for an additional 30 min at 37°C in the presence of tea polyphenols. The cells were then lysed with RIPA buffer. Immunoblot analysis was performed with antibodies against STAT1 phosphorylated at Ser727, ERKs, JNKs, p90RSK and PDK1 or non-phospho-specific antibodies against the total non-phosphorylated proteins.
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Results
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Phosphorylation of STAT1 at Ser727 is induced by UVB
A recent report indicated that irradiation of JB6 Cl41 lines with UVA stimulated STAT1 activation through Ser727 phosphorylation (12,17). On the other hand, experiments with UVC irradiation showed that Ser727 phosphorylation was also required for activation of STAT1 signaling (11,14,39,40). However, UVB may be the most important human skin carcinogen. But whether UVB stimulates STAT1 signaling through Ser727 phosphorylation is as yet unknown. This question has been investigated herein using western blot analysis with specific antibodies to detect STAT1 phosphorylation at Ser727. Our data show a strong phosphorylation of STAT1 at Ser727 induced in a dose- (Figure 1A) and time-dependent (Figure 1B) manner following UVB exposure of mouse epidermal tumor promotion-sensitive JB6 Cl41 cells. STAT1 phosphorylation at Ser727 began 5 min after irradiation with UVB (4 kJ/m2), increased to maximal induction at 30 min and then decreased back to basal level (Figure 1B). In further experiments we used irradiation with UVB (4 kJ/m2) and harvested cells 30 min after irradiation. Cells do not undergo apoptosis at this UVB dose and time of harvest. Investigators typically use UVB exposures ranging from a low dose of 50 J/m2 to a maximal dose of 8000 J/m2, with most falling between 250 and 4000 J/m2 (21). We have consistently used 4000 J/m2 (4 kJ/m2) as the optimal dose for studying UVB-induced signal transduction (24,28,37).

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Fig. 1. A strong dose-dependent and time-dependent phosphorylation of STAT1 (Ser727) is induced in UVB-irradiated cells and the MEK/ERK cascades have a role in UVB-stimulated phosphorylation of STAT1 (Ser727). After starvation for 24 h, JB6 Cl41 cells were or were not irradiated with UVB at the indicated doses (A) or with UVB at 4 kJ/m2 (B). The cells were then harvested at 30 min (A) or at the indicated times (B) after UVB stimulation. STAT1 proteins in the cell lysates were separated by 10% SDSPAGE followed by western blot analysis with specific antibodies against STAT1 phosphorylated at Ser727 or against total STAT1. Non-irradiated samples served as negative controls. (C) Inhibition of UVB-stimulated phosphorylation of STAT1 (Ser727) by MEK1 inhibition. After pretreatment with PD98059 or UO126 at the indicated doses, JB6 cells were or were not irradiated with UVB (4 kJ/m2) and then harvested 30 min following irradiation. (D) A decrease was observed in UVB-induced STAT1 (Ser727) phosphorylation in DNM-ERK2 cells. The cell lines stably expressing an empty vector (CMV-neo) or a dominant negative mutant of ERK2 (DNM-ERK2) were harvested at the indicated times after irradiation with UVB at 4 kJ/m2. The cell lysates were used to determine the phosphorylation levels of STAT1 (Ser727) and p90RSK (Thr359/Ser363), as well as total levels of STAT1 and non-phosphorylated (Np)-p90RSK. These data are representative of at least three independent experiments.
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UVB-stimulated phosphorylation of STAT1 (Ser727) is blocked by ERKs and MEK inhibitors
Recently, Ser727 phosphorylation of STAT1 (17) and STAT3, which is highly homologous to STAT1, by UVA was determined to require stress-activated protein kinase/ERK kinase 1, MAPK kinase 4 (41,42) and MEK kinase 1 (43). In our present experiments PD98059 or UO126, two specific inhibitors of MEK1 and/or MEK2, markedly inhibited UVB-induced phosphorylation of STAT1 (Ser727) (Figure 1C). Secondly, a DNM of ERK2 (Figure 1D) showed a marked decrease in UVB-induced STAT1 (Ser727) phosphorylation. These experiments suggested that ERK2 is involved in the UVB-induced phosphorylation of STAT1 (Ser727). p90RSK is known to be a downstream substrate for ERKs (38) and UVB-stimulated phosphorylation at Thr359/Ser363 in p90RSK was also substantially inhibited (Figure 1D) in DNM-ERK2 cells compared with the control CMV-neo cells. The levels of non-phosphorylated (NP)-p90RSK were unaffected by DNM-ERK2 expression.
p38 kinase is required for mediating UVB-stimulated STAT1 (Ser727) phosphorylation
We treated JB6 cells with SB202190, a specific inhibitor of p38 kinase (44,45). Results from this experiment showed that UVB-induced Ser727 phosphorylation of STAT1 was suppressed by pretreatment with SB202190 (Figure 2A). In addition, UVB-induced phosphorylation of p38 kinase was substantially inhibited by DNM-p38 kinase (Figure 2B). Data showed that stimulation of Ser727 phosphorylation of STAT1 with UVB was prevented in a dose- and time-dependent (Figure 2B and C) manner by expression of DNM-p38 compared with control JB6 cells expressing the empty CMV-neo vector. These results suggest that p38 kinase may be involved in the regulation of Ser727 phosphorylation of STAT1 in UVB-irradiated JB6 cells.

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Fig. 2. Inhibition of p38 kinase blocks UVB-induced STAT1 (Ser727) phosphorylation and inhibition of JNKs impairs UVB-induced STAT1 phosphorylation at Ser727. (A) JB6 cells were or were not preincubated for 1 h with SB202190 at the indicated doses and then were or were not irradiated with UVB (4 kJ/m2) and harvested 30 min following irradiation. (B and C) JB6 cells were stably transfected with vector (CMV-neo) or a dominant negative mutant of p38 kinase (DNM-p38). The two cell lines were harvested at 30 min (B) or at the indicated times (C) following irradiation with UVB at the indicated doses (B) or at 4 kJ/m2 (C). (D and E) JB6 cell lines stably expressing an empty vector (CMV-neo) or a dominant negative mutant of JNK1 (DNM-JNK1) were harvested at 30 min (D) or at the indicated times (E) after irradiation with UVB at the indicated doses (D) or at 4 kJ/m2 (E). (F) After pretreatment with SP600125 at the indicated doses, JB6 cells were or were not irradiated with UVB (4 kJ/m2) and then harvested 30 min following irradiation. (G) After starvation in serum-free DMEM, the cells from wild-type (Jnk+/+) or JNK knockout mice (Jnk1/ or Jnk2/) were or were not irradiated with UVB (4 kJ/m2) and were then harvested 15 or 30 min following irradiation. Subsequently, the cell lysates were assayed for phosphorylated Ser727 of STAT1 or total STAT1. These data are representative of at least three independent experiments.
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JNKs are required for STAT1 (Ser727) phosphorylation
Here, we used cell lines with a DNM of JNK1 (DNM-JNK1) (31) or murine embryonic fibroblast Jnk1/ and Jnk2/ knockout cells to assess whether JNKs play a role in phosphorylation of STAT1 induced by UVB. Our data showed that stimulation of Ser727 phosphorylation of STAT1 with UVB was almost prevented in a dose- and time-dependent (Figure 2D and E) manner by expression of DNM-JNK1 compared with control JB6 cells expressing the empty CMV-neo vector. Results from this experiment also showed that UVB-induced Ser727 phosphorylation of STAT1 was suppressed by pretreatment with SP600125 (Figure 2F). Furthermore, Ser727phosphorylation was also attenuated in Jnk1/ and Jnk2/ knockout cells compared with wild-type Jnk+/+ cells (Figure 2G). These results suggest that STAT1 phosphorylation at Ser727 may require JNKs pathway activation when cells are exposed to UVB.
Treating cells with a combination of inhibitors of ERKs, p38 kinase and JNKs suggest that p38 kinase is most important in signaling to STAT1
Cells were treated with the MEK inhibitor PD98059 (12.5 µM), the p38 kinase inhibitor SB202190 (0.2 µM), the JNK inhibitor SP600126 (1.0 µM) or a combination of these inhibitors (Figure 3). After 1 h treatment with inhibitor, cells were treated with UVB (4 kJ/m2) and incubated for 30 min, then harvested for assessment of STAT1 phosphorylation. Treatment with SB202190 markedly decreased STAT1 phosphorylation and SP600125 and PD98059 had small inhibitory effects. The results of combining the inhibitors suggests that p38 kinase may be most important in mediating UVB-induced STAT1 (Ser727) phosphorylation (Figure 3).

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Fig. 3. Phosphorylation of STAT1 at Ser727 is induced by UVB irradiation and antagonized by PD98059, SB202190 and SP600125. After 24 h in 0.5% serum medium and in the absence () or presence of 25 µM PD98059, 0.2 µM SB202190 or 2.0 µM SP600125 or a combination of PD98059/SB202190, PD98059/SP600125 and SB202190/SP600125, extracts of JB6 cells were prepared and probed by western blotting for phosphorylation of STAT1 (Ser727). Subsequently, the cell lysates were assayed for phosphorylated Ser727 of STAT1 or total STAT1. These data are representative of at least three independent experiments.
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PI-3 kinase has a role in the mediation of UVB-induced signaling to STAT1 (Ser727)
We used a JB6 cell line expressing a DNM of the PI-3K p85 subunit (DNM-
p85) (32,33) to study the role of the PI-3K pathway in UVB-stimulated phosphorylation of STAT1 at Ser727. The results showed an enhancement of UVB-induced phosphorylation of STAT1 at Ser727 in cells expressing DNM-
p85 (Figure 4A and Ba), in comparison with the corresponding induction in control CMV-neo cells. On the other hand, phosphorylation of Akt in cells expressing DNM-
p85 decreases (Figure 4Bb). This suggests that DNM-
p85 inhibits UVB induced Akt phosphorylation but not other downstream PI-3K signaling. Results also showed that UVB-induced phosphorylation of STAT1 at Ser727 was inhibited by pretreatment of JB6 cells with wortmannin (46) or LY294002 (47), selective inhibitors of PI-3K (Figure 4C). These data demonstrate that Ser727 phosphorylation of STAT1 may be mediated mainly by UVB activation of a PI-3K-independent signaling pathway. Further, a negative signaling pathway possibly involving the PI-3K p85 subunit alone, not in combination with the p110 catalytic subunit, might regulate the STAT1 phosphorylation response.

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Fig. 4. PI-3K or the p85 subunit mediate UVB-induced signaling to STAT1 (Ser727) and Akt1, PDK1 and RSK2 are involved in modulating phosphorylation of STAT1 (Ser727). (A and B) Cell lines stably expressing CMV-neo or a DNM- p85 were harvested at 30 min (A) or at the indicated times (B) following irradiation with UVB at the indicated dose (A) or at 4 kJ/m2 (B). After UVB stimulation STAT1 proteins in the cell lysates were separated by 10% SDSPAGE followed by western blot analysis with specific antibodies against STAT1 phosphorylated at Ser727 (A and Ba), Akt (Bb) or total STAT1. Non-irradiated samples served as negative controls. (C) After pretreatment with wortmannin or LY294002 at the indicated doses, JB6 cells were irradiated with UVB (4 kJ/m2) and then harvested 30 min following irradiation. The experimental cells were starved for 48 h in 0.1% FBS (D) (Wt-Akt and DNM-Akt), serum-free medium (E) (Pdk1+/+ and Pdk1/) or a medium containing 0.5% FBS (F) (wild-type and Rsk2). The cells were harvested at the indicated times following irradiation with UVB (4 kJ/m2) and total and phosphorylated (Ser727) levels of STAT1 were determined. These data are representative of at least three independent experiments.
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PDK1, Akt and p90RSK, downstream kinases of PI-3K, may have a role in the mediation of UVB-induced signaling to STAT1 (Ser727)
A recent report indicated that RSK2 is involved in the phosphorylation of STAT3 (20). Here, we further assess whether downstream targets of PI-3K, including PDK1, Akt and RSK2, are involved in mediating the UVB-induced STAT1 (Ser727) phosphorylation response. Our results show that UVB-induced Ser727 phosphorylation of STAT1 was enhanced in wild-type Akt cells (Wt-Akt) and decreased in DNM-Akt cells (Figure 4D). We further showed that UVB-stimulated STAT1 phosphorylation of Ser727 was significantly attenuated in mouse PDK1 knockout (Pdk1/) stem cells compared with wild-type Pdk1+/+ cells (Figure 4E). In addition, RSK2-deficient (Rsk2) cells from a CoffinLowry Syndrome patient also had an inhibitory effect on UVB-induced STAT1 (Ser727) phosphorylation (Figure 4F). No change in total levels of STAT1 protein expression was observed in these cell lines. Thus, these data indicate that Akt, PDK1 and RSK2 are required for mediating UVB-stimulated phosphorylation of STAT1 at Ser727.
RSK1, RSK2 and Akt kinases phosphorylate STAT1 proteins in vitro
Phosphorylation of STAT1 (Ser727) catalyzed by ERKs, JNKs and p38 kinase, as well as by MEK1 and MSK1, may occur in the presence of an unidentified cofactor or downstream kinase (17). Here, we examined whether RSK1, RSK2 and Akt are responsible for phosphorylating STAT1 protein substrates. Kinase experiments utilizing as substrates combined STAT1 proteins from GST pull-down (17) and immunoprecipitation followed by autoradiographic analysis revealed that the immunoprecipitated STAT1 proteins from cell lysates were, to different degrees, phosphorylated by active RSK1, RSK2 and Akt (Figure 5).

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Fig. 5. Autoradiographic analysis of STAT1 phosphorylation induced by protein kinases in vitro. Wild-type STAT1 proteins from GST pull-down (GST-STAT1) (17) or immunoprecipitation (IP-STAT1) was used as a substrate of activated protein kinases, including RSK1 (200 mU), RSK2 (150 U) and Akt (100 mU). The kinase reactions were carried out in a kinase buffer containing [ -32P]ATP and then the reactive products were subjected to 8% PAGE resolution followed by autoradiography.
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UVB-induced phosphorylation of STAT1 (Ser727) is inhibited by theaflavins or EGCG
The polyphenols from green tea and black tea, EGCG and theaflavins, respectively, are generally considered to be the most effective tea components for inhibition of carcinogenesis. Tea polyphenols inhibit UVB-induced AP-1 activation in mouse epidermal JB6 cells (24) and the UVB-activated PI-3K pathway (28). We investigated the effect of tea polyphenols on UVB-induced activation of STAT1 (Ser727). Theaflavins and EGCG markedly inhibited UVB-stimulated phosphorylation of STAT1 (Ser727) (Figure 6A and B).

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Fig. 6. Theaflavins and EGCG inhibit UVB-induced phosphorylation of STAT1 (Ser727). JB6 Cl41 cells were treated with theaflavins (A) or EGCG (B) at the doses indicated and then irradiated with UVB (4 kJ/m2). Then total and phosphorylated (Ser727) levels of STAT1 were determined. These data are representative of at least three independent experiments.
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Theaflavins and EGCG inhibit UVB-induced activation of ERKs, PDK1, JNKs and p90RSK
Recently, we showed that theaflavins and EGCG inhibited UVB-induced AP-1 activation and the inhibitory effect of theaflavins on AP-1 was stronger than that of EGCG (27). Because STAT1 (Ser727) has been identified as a target of several kinases, including ERKs, JNKs, PDK1 and p90RSK, we analyzed the inhibitory effects of theaflavins and EGCG on UVB-induced phosphorylation of these kinases. As shown in Figure 7, we found that pretreatment of cells with these polyphenols blocked UVB-induced phosphorylation of ERKs (Figure 7A), JNK2 (Figure 7B), PDK1 (Figure 7C) and p90RSK (Thr359/Ser363) (Figure 7D and E). These data suggest that the inhibitory effects of theaflavins and EGCG on UVB-induced STAT1 (Ser727) phosphorylation are possibly modulated by blocking the phosphorylation of ERKs, JNKs, PDK1 and p90RSK.

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Fig. 7. Theaflavins and EGCG inhibit UVB-induced Ser727 phosphorylation of kinases that mediate phosphorylation of STAT1. JB6 Cl41 cells were treated with theaflavins or EGCG at the doses indicated and then irradiated with UVB at 4 kJ/m2. Then phosphorylation of ERKs (A), JNKs (B), PDK1 (C) and p90RSK (Thr359/Ser363) (D and E) was determined, as was NP-ERKs, NP-JNKs, NP-PDK and NP-p90RSK. These data are representative of at least three independent experiments.
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Discussion
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UV light affects various cellular functions through activation of intracellular signaling systems, including the MAPK family of proteins (48). Serine phosphorylation (Ser727) was shown to be essential for activation of STAT1 (11,14), following stimulation of cells with diverse stresses, including UVA (19) and UVC irradiation (11,13). These findings demonstrate that signaling pathways may regulate STAT1 signaling activation by Ser727 phosphorylation. Early studies showed that Ser727 phosphorylation of STAT1 might be associated with activation of ERKs (16,49), p38 kinase (11,14,50,51) or JNKs (52). Irradiation of JB6 cells with UVA or UVC induces a stronger Ser727 phosphorylation of STAT1 and this phosphorylation occurs through diverse MAPK cascades in the cellular response to UVA (17). In vitro kinase assays using purified full-length and pull-down STAT3 as the substrate show that ERK, JNK and p38 kinase can phosphorylate STAT3 (16,18). In vitro kinase assays using STAT1 proteins combined from immunoprecipitation and GST pull-down assays as substrates indicate that active ERK1, JNK1, p38 kinase, MEK1 and MSK1 stimulated phosphorylation of STAT1 (Ser727) indirectly through an unidentified factor or a downstream kinase (17). We found that exposure of JB6 cells to UVB irradiation induced a dose- and time-dependent phosphorylation of STAT1 at Ser727. However, upstream kinase pathways leading to phosphorylation of STAT1 (Ser727) are not understood. Here, we used selective kinase inhibitors and specific DNM kinases to further analyze the roles of MAPKs and PI-3K, PDK1, Akt and p90RSK in the regulation of UVB-stimulated STAT1 (Ser727) phosphorylation.
Inhibition of phosphorylation by PD98059 or UO126 is thought to be a specific method for assessing the involvement of the ERKs pathway. Compelling evidence that ERKs are STAT kinases has been provided for STAT3 (40,52), which is highly homologous to STAT1. Phosphorylation of STAT1 at Ser727 occurs through ERKs in the cellular response to UVA (17). Presence of the MEK1/2 inhibitors PD98059 and UO126 significantly reduced STAT1 phosphorylation induced by UVA, suggesting that MEKs are involved in the process (17). The enzyme co-immunoprecipitated with STAT1 and dominant-negative ERK cells decreased transcription in response to interferon-
(49). In the case of interferon-
, a dominant negative tyrosine kinase (Pyk2) suppressed ERKs activation and, at the same time, Ser727 phosphorylation of STAT1 (53). In our study we used PD98059 or UO126, specific inhibitors of MEK1 and/or MEK2. We showed that pretreatment of cells with these inhibitors markedly inhibited UVB-induced phosphorylation of STAT1 at Ser727. Also, we showed a decrease in UVB-induced STAT1 (Ser727) phosphorylation in DNM-ERK2 cells. These experiments strongly suggest that MEK or ERK2 is involved in the UVB-induced phosphorylation of STAT1 (Ser727). Among the substrates of ERKs are p90RSK (43,51). ERK activation and subsequent phosphorylation of the p90RSK1 C-terminal catalytic loop stimulates phosphotransferase activity in the p90RSK1 N-terminal kinase domain (54). The use of specific kinase inhibitors indicates that UV-induced phosphorylation and activation of p90RSK2 is mediated by the MAPK/ERK pathway (55). In this study we showed that UVB-stimulated phosphorylation at Thr359/Ser363 in p90RSK was significantly inhibited in DNM-ERK2 cells. Therefore DNM-ERK2 is a useful tool for identifying ERKs-dependent substrates such as p90RSK.
Recent results suggest that STAT3 is a target of JNKs, which may regulate STAT3 activity via both Ser727 phosphorylation-dependent and -independent mechanisms (13). In JB6 cells UVA-stimulated Ser727 phosphorylation of STAT3 and its DNA binding activity may be mediated by ERKs and p38 kinase, as well as JNKs (19). Prevention of JNK activation by DNM-JNK1 and in Jnk1/ or Jnk2/ cells resulting in inhibition of Ser727 phosphorylation of STAT3 and its DNA binding activity suggests that Ser727 phosphorylation was induced by active JNKs in vitro (19). Similar to results for STAT3, UVA-induced Ser727 phosphorylation of STAT1 was diminished by PD169316, an inhibitor of JNKs, and STAT1 phosphorylation was blocked by DNM-JNK1 and in Jnk1/ and Jnk2/ cells (17). In vitro kinase assays using STAT1 proteins combined from immunoprecipitation and GST pull-down assays as substrates showed that active ERK1, JNK1 and p38 kinase stimulate phosphorylation of STAT1 (Ser727) (17). In this study our data indicate that the JNKs signaling networks are required to mediate serine phosphorylation of STAT1 stimulated by UVB irradiation. Our experimental evidence shows that UVB stimulation of Ser727 phosphorylation of STAT1 in JB6 cells was inhibited by treatment with SP600125 and a DNM of JNK1 and was also abolished by deficiency of JNK1 or JNK2. These results suggest that JNKs are involved in mediating not only phosphorylation of STAT3 (Ser727) but also STAT1 (Ser727).
STAT1 (Ser727) phosphorylation in response to cellular stress (UV irradiation), inflammatory signals (bacterial lipopolysaccharide) or proinflammatory cytokines (tumor necrosis factor-
) is inhibited by SB203580, a specific p38 kinase inhibitor (11). Two groups independently reported activation of the p38 kinase pathway by type I interferon (IFN)-
/ß (39) and demonstrated an interaction between IFN-
signaling and the p38 kinase pathway that leads to increased transcriptional activation by STAT1 independently of phosphorylation at Ser727 (38). DNM-p38 inhibits EGF-stimulated phosphorylation of STAT1 at Ser727 (51). These results indicate that EGF-induced phosphorylation of STAT1 at Ser727 is at least partially dependent on p38 kinase and that STAT1 is involved in JB6 cell transformation promoted by EGF (51). In this study we have shown that UVB-stimulated phosphorylation of STAT1 was inhibited by expression of DNM-p38 compared with the corresponding control CMV-neo cells. We showed a similar inhibitory effect after preincubation of JB6 cells with SB202190, another specific p38 kinase inhibitor. These observations and previous observations (17) demonstrate that p38 kinase is required for STAT1 (Ser727) phosphorylation in JB6 cells after UVB irradiation. Results of studies with combinations of ERKs, JNKs and p38 kinase inhibitors suggest that the p38 kinase pathway may be most important in UVB-induced phosphorylation of STAT1 (Ser727).
PI-3K enzymes are present in all cell types and their activities have been shown to be necessary for many different cell regulatory pathways. PI-3K, a heterodimer that is composed of a catalytic subunit (p110) and a regulatory subunit (p85) (56), functions as a key upstream regulatory enzyme for a number of signaling pathways that are deregulated in carcinogenesis (57,58). To date, whether the PI-3K signaling network modulates STAT1 phosphorylation is unknown. The relationship between PI-3K, STAT1 and UVB irradiation is unclear. In our study, pretreatment of cells with wortmannin or LY294002 blocked UVB-induced phosphorylation of STAT1. These experiments indicated that PI-3K is involved in UVB-induced phosphorylation of STAT1 (Ser727). On the other hand, an enhancement in UVB-induced phosphorylation of STAT1 (Ser727) was observed in a JB6 cell line expressing DNM-
p85, in comparison with the corresponding induction in control CMV-neo cells. This suggests that the STAT1 phosphorylation response may be regulated by a negative signaling pathway involving the PI-3K p85 subunit alone, not in combination with the p110 catalytic subunit.
One of the targets of PI-3K is Akt. The PI-3K/Akt signaling pathway regulates many normal cellular processes, including cell proliferation, survival, growth and motility, processes that are critical for tumorigenesis (59,60). In cells Akt resides in the cytosol in a low activity conformation. Upon cellular stimulation Akt is activated through recruitment to cellular membranes by PI-3K lipid products and phosphorylation by PDK1 (61). Extensive biochemical studies have clearly demonstrated that PDK1 is the Akt upstream kinase and that PDK1 also phosphorylates a number of other kinases, including p70S6 kinase and protein kinase C (28,61,62). In this study we have shown that the PDK1/Akt pathway is involved in modulating the UVB-induced STAT1 (Ser727) phosphorylation response. Our results indicate that UVB-stimulated STAT1 phosphorylation at Ser727 was significantly attenuated in mouse PDK1 knockout (Pdk1/) stem cells compared with wild-type PDK1 cells. Our data also show that UVB-induced Ser727 phosphorylation of STAT1 is enhanced in wild-type Akt cells and decreased in DNM-Akt cells. Thus, these data indicate that PDK1 and Akt are required to mediate UVB-stimulated Ser727 phosphorylation of STAT1. The expressed STAT1 proteins obtained from a GST pull-down assay were primarily characterized by western blotting with a GST antibody. This experimental evidence supports the idea that the wild-type GSTSTAT proteins are also phosphorylated by Akt, p90RSK1 and p90RSK2.
p90RSK2 is a growth factor-stimulated protein kinase with two kinase domains. The C-terminal kinase of p90RSK2 is activated by ERKs and the N-terminal kinase is activated by PDK1 (63,64). A novel regulatory mechanism was suggested based on phosphoserine-mediated recruitment of PDK1 to p90RSK2, leading to coordinated phosphorylation and activation of PDK1 and p90RSK2 (65). p90RSK2 is involved in mediation of UVA-induced STAT1 serine phosphorylation (17). p90RSKs are also potential upstream kinases for modulating phosphorylation of STAT3 (20). In the present study a Rsk2 cell line from a CoffinLowry syndrome patient decreased UVB-induced STAT1 (Ser727) phosphorylation. Thus, our data indicate that RSK2 is required to mediate UVB-stimulated phosphorylation of STAT1 at Ser727 and that the PDK1/p90RSK2 pathway might be activated through a PI-3K-independent signaling pathway.
A model of the signaling networks that modulate Ser727 phosphorylation of STAT1 in the cellular response to UVB is summarized in Figure 8.
The polyphenols from green tea and black tea, EGCG and theaflavins, are generally considered to be the most effective tea components in the inhibition of carcinogenesis (22,23,66,67). Tea polyphenols have also been shown to inhibit UV-induced carcinogenesis in animal models (68,69). Inhibition of MAPK and AP-1 activities by EGCG and other tea polyphenols has been demonstrated in JB6 and H-ras-transformed cells (24,26,70). Tea components target specific cell signaling pathways responsible for regulating cell growth, survival and cell transformation (24,57,71,72). In this study we have shown that phosphorylation of STAT1 (Ser727) occurs through MAPK pathways, including ERKs, JNKs and downstream kinases of PI-3K, such as PDK1, Akt and p90RSK2. In addition, our study has demonstrated that these kinases are required for UVB-induced STAT1 (Ser727) phosphorylation. Therefore, to further analyze the mechanism of the inhibitory effect of tea polyphenols on UVB-induced phosphorylation of STAT1 (Ser727), we investigated their influence on UVB-induced phosphorylation of MAPKs and downstream kinases of PI-3K. Earlier results suggest that theaflavins inhibit UVB-induced AP-1 activity through ERK- and JNK-dependent pathways and thus may be more potent inhibitors than EGCG, which inhibits only the ERK-dependent pathway (24). Chen et al. (73) have provided additional evidence supporting the finding that in human keratinocytes EGCG blocks UVB-induced expression of c-Fos, which, through inhibition of p38 kinase activation, is associated with UVB-induced AP-1 DNA binding and transactivation (74). In the present study we have shown that treatment of cells with EGCG and theaflavins after UVB irradiation markedly blocked UVB-induced STAT1 (Ser727) phosphorylation and UVB-induced phosphorylation of ERKs. Pretreatment of cells with theaflavins markedly blocked UVB-induced phosphorylation of JNKs, but pretreatment of cells with EGCG markedly blocked UVB-induced phosphorylation of JNK2 only. EGCG and theaflavins inhibited activation of PI-3K and also attenuated activation of Akt and p70S6 kinase, downstream effectors of PI-3K (28). Because PI-3K and its downstream effectors are considered to play a critical role in carcinogenesis, we also studied the inhibitory effect of EGCG and theaflavins on UVB-induced phosphorylation of PDK1 and p90RSK, other downstream kinases of PI-3K. Our results indicate that pretreatment of cells with EGCG or theaflavins significantly inhibits UVB-induced PDK1 and p90RSK phosphorylation, as well as phosphorylation of ERKs and JNKs. Inhibition by these polyphenols was only observed when the EGCG and theaflavin treatment of cells preceded UVB irradiation, not when it was after UVB irradiation. These results provide insights into the biological actions of tea polyphenols on UVB-induced carcinogenesis and the molecular basis for the development of new chemopreventive agents.
In summary, based on these observations (Figure 8) we conclude that the main signaling pathways of UVB-induced STAT1 phosphorylation at Ser727 are mediated through PI-3K, p38 kinase, ERKs, JNKs, PDK1, p90RSK and Akt, p38 kinase, MEKs/ERKs and JNKs. EGCG and theaflavins block UVB-induced STAT1 (Ser727) phosphorylation through the inhibition of ERKs, JNKs, PDK1 and p90RSK. This information provides more definitive answers regarding the causes and effects of STAT serine phosphorylation.
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Acknowledgments
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This work was supported by The Hormel Foundation and National Institutes of Health grants CA81064 and CA77646.
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Received May 4, 2004;
revised October 20, 2004;
accepted November 5, 2004.