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Am J Physiol Lung Cell Mol Physiol 291: L983-L992, 2006. First published June 30, 2006; doi:10.1152/ajplung.00454.2005
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Identification of a hydrogen peroxide-induced PP1-JNK1-Sp1 signaling pathway for gene regulation

Shijian Chu1,2 and Thomas J. Ferro1,2,3

1McGuire Veterans Affairs Medical Center, Richmond; and Departments of 2Physiology and 3Medicine, Virginia Commonwealth University, Richmond, Virginia

Submitted 27 October 2005 ; accepted in final form 26 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oxidative stress often results in changes in gene expression through the regulation of transcription factors. In this study, we examine how Sp1 phosphorylation is regulated by H2O2 in a human alveolar epithelial cell line (HAE). Treatment of HAE cells with H2O2 increases phosphorylation of Sp1 and activates JNK. To establish a relationship between JNK and Sp1, we show that JNK activator anisomycin increases Sp1 phosphorylation, and JNK inhibitors as well as dominant-negative JNK1 attenuate H2O2-induced Sp1 phosphorylation. Additionally, JNK1 directly phosphorylates Sp1 in vitro, reducing Sp1 binding to DNA. These results demonstrate the role of JNK in H2O2-induced Sp1 phosphorylation. Because H2O2 inhibits Ser/Thr protein phosphatase-1 (PP1), we examined the role of PP1 in the regulation of JNK. Similar to H2O2, inhibition of PP1 induces phosphorylation of Sp1 and activation of JNK in HAE cells. Inhibition of JNK activity using either inhibitors or dominant-negative mutant JNK1 suppresses PP1 inhibition-induced Sp1 phosphorylation. Furthermore, PP1 directly inactivates JNK1 in vitro. These data suggest that 1) H2O2 increases the phosphorylation level of Sp1, 2) Sp1 is a target of the JNK pathway, 3) PP1 regulates JNK activation, and 4) the "PP1-JNK" pathway plays a role in H2O2-induced Sp1 phosphorylation in lung epithelial cells.

oxidative stress; reactive oxygen species; transcription factor; kinase; phosphatase; protein phosphatase-1

OXIDATIVE STRESS HAS IMPORTANT implications for lung physiology as well as the pathogenesis of lung diseases such as chronic obstructive pulmonary disease (COPD) (34). The sources of oxidative stress in COPD patients are from both inhaled oxidants and increased amounts of reactive oxygen species (ROS) generated by various migrating and resident cells. Increased H2O2 is established as a marker of oxidative stress and has been successfully measured in breath condensate in COPD patients (29). H2O2 is known to regulate gene expression (36, 45), which could be one of the major pathways mediating cellular responses to oxidative stress. However, underlying mechanisms of these regulations are not completely understood.

Sp1 is the founding member of a family of zinc-finger transcription factors, is widely expressed in cells and tissues, and regulates a large number of genes (5, 33). The function of Sp1 can be regulated by posttranslational modifications such as glycosylation or phosphorylation (7, 12). Sp1 phosphorylation is regulated by various extracellular signals such as viral infection, growth factors, and organ development and differentiation. Changes in the level of Sp1 phosphorylation may positively or negatively modulate its trans-activation function (12).

JNK is well known for its regulation of transcription factors through phosphorylation. A classic example is the phosphorylation and activation of activator protein-1 (AP-1) when JNK is activated during inflammation (22, 44). It is generally recognized that JNK is activated by stress signals including ROS (28). Whether JNK also plays a role in growth-related and housekeeping genes through transcription factors such as Sp1 is not well understood.

The role of protein phosphatase-1 (PP1) in the regulation of Sp1 phosphorylation has been reported, and an increasing number of promoters have been shown to be regulated by PP1-related Sp1 phosphorylation (11, 15, 27, 43). PP1 dephosphorylates Sp1 in vitro. Accordingly, inhibition of PP1 leads to increased Sp1 phosphorylation and altered Sp1 transcription factor activity in various types of cells (12). However, in cells, PP1 may regulate Sp1 phosphorylation indirectly through kinases that phosphorylate Sp1. A few upstream signals such as H2O2 that regulate PP1 have been recognized (31).

In this study, we demonstrate a H2O2-initiated "PP1-JNK" pathway that regulates Sp1 phosphorylation in lung epithelial cells. In this signaling pathway, PP1 suppresses Sp1 phosphorylation through inactivation of JNK. When PP1 is suppressed, JNK becomes activated and subsequently phosphorylates Sp1. Our data for the first time establish the role of PP1 in the regulation of JNK activity, and demonstrate Sp1 as a target of the JNK pathway in lung epithelial cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells and reagents. Human alveolar epithelial cell line (HAE) is a cell line derived from alveolar cell carcinoma of a 44-yr-old female [American Type Culture Collection (ATCC) no. CRL-2107] that retained many characteristics of lung epithelial cells. H441 is a human lung epithelial cell line derived from lung adenocarcinoma (ATCC no. HTB-174). HepG2 is derived from human hepatocellular carcinoma (ATCC no. HB-8065). All cell lines were cultured as recommended by the supplier.

Recombinant PP1{alpha} and ERK2 were purchased from New England Biolabs (Beverly, MA). Recombinant PP2A, JNK1, and p38 MAP kinase were purchased from Upstate (Charlottesville, VA). Recombinant Elk-1 protein was purchased from Cell Signaling Technology (Beverly, MA). Okadaic acid, okadaic acid methyl ester, fostriecin, and calyculin A were purchased from Sigma (St. Louis, MO). Anisomycin, dicumarol, and SP600125 were from Calbiochem (San Diego, CA). Antibodies against Elk-1, phospho-c-Jun, and total and phosphorylated MAP kinases (ERK1/2, JNK, and p38) were purchased from Cell Signaling Technology. Anti-Sp1 antibody was from Santa Cruz (Santa Cruz, CA).

Immunoblotting and quantitation. Immunoblotting experiments were essentially performed as described before (6), using 8 and 10% discontinuous polyacrylamide gels and polyvinylidene difluoride (PVDF) membranes. Membranes were processed according to protocols provided by the manufacturers of antibodies. Band intensities were quantified by digital scanning using a LAS-1000 Luminescent Image Analyzer (Fuji Photo Film, Valhalla, NY) and Advanced Image Data Analyzer software (Raytest, Straubenhardt, Germany).

Immunoprecipitation. HAE cells were washed with cold PBS and collected by centrifugation. The entire procedure was performed at 4°C. The harvested cells were incubated for 1 h in cell lysis buffer containing 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 1% NP-40, 1 mM PMSF, and 1% proteinase inhibitor cocktail (Sigma). After being pushed through a 21-gauge syringe needle four times, cell debris were pelleted and discarded. The supernatant was cleared by incubation with protein A-agarose beads (Roche) for 1 h. The beads were then discarded by centrifugation. The supernatant was incubated with antibody for 1 h. Protein A-agarose beads were added and incubated overnight. The beads were washed four times with PBS.

In vitro phosphorylation and dephosphorylation. For in vitro phosphorylation of Sp1 and Elk-1 by ERK2, the experiments were performed using the kinase buffer supplied by New England Biolabs. The kinase buffer for in vitro phosphorylation using JNK1 contained 50 mM Tris-Cl at pH 7.5, 1 mM EGTA, 1 mM DTT, 10 mM MgCl2, and 0.01% Brij. ATP was added to a final concentration of 1 mM where indicated. Incubations of the phosphorylation reactions were carried out at 30°C for 15 min.

Dephosphorylation reactions by PP1 were performed at 30°C for 15 min according to the protocol recommended by the New England Biolabs. Dephosphorylation reactions by PP2A were incubated in a buffer containing 50 mM Tris-Cl, pH 7.0, 0.1 mM CaCl2, 60 mM beta-mercaptoethanol, and 1 mM MgCl2. Phosphorylated kinases were detected using immunoblotting with anti-phospho-kinase antibodies from Cell Signaling Technology.

JNK kinase assay. A JNK assay kit from Cell Signaling Technology was used to determine JNK kinase activity. For JNK kinase activity in whole cell lysate, the manufacturer's protocol was followed. When recombinant JNK1 was used to study the effect of PP1 on JNK activity, the assay was performed in two steps. In the first step, JNK1 was incubated with various concentrations of PP1 at 30°C for 15 min in a buffer containing 50 mM Tris-Cl, pH 7.0, 0.1 mM Na2-EDTA, 5 mM DTT, 0.01% Brij-35, and 1 mM MnCl2. In the second step, JNK1 in the reactions was isolated, and kinase activity was determined just as described above for using whole cell lysate.

Stable transfection of cells. The FuGene6 kit from Roche was used for transfection. Transfected HAE cells were selected by resistance to G418 (200 µg/ml). Single colonies were picked using cloning discs. Picked colonies were further incubated in the presence of G418 for >30 days and four passages.

Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was performed as described earlier, with some modifications (11). Before adding to the protein-DNA reaction mix, nuclear extract was incubated with JNK1 with or without ATP for in vitro phosphorylation. The phosphorylation reactions were set up in 30-µl-vol reactions containing 50 mM Tris-Cl at pH 7.5, 1 mM EGTA, 1 mM DTT, 10 mM MgCl2, 0.01% Brij, 17 µl of HAE nuclear extract, and 0.4 units of JNK1. ATP was added to a final concentration of 1 mM where indicated. The phosphorylation reactions were incubated at 30°C for 30 min. For each protein-DNA binding reaction, 4 µl of the phosphorylation reaction were added as nuclear extract. The oligo probe derived from the cyclin D3 gene promoter had the sequence 5'-TGCGGCCCCGCCCCTTAGAACG-3' from –134 through –113 (42). The underlined sequence is the predicted GC box (Sp1 binding site). DNA-protein binding was performed in a reaction buffer containing 25 mM HEPES, pH 7.5, 50 mM KCl, 20 µM ZnSO4, 11 mM beta-mercaptoethanol, 0.05% NP-40, 10% glycerol, and 10 µg/ml poly(dI-dC).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
H2O2 induces phosphorylation of Sp1 and activation of JNK. We treated HAE lung epithelial cells with H2O2 and found that the mobility of Sp1 was markedly retarded in a time- and dose-dependent fashion, an indication of increased Sp1 phosphorylation (10, 23). Figure 1, A and B, shows examples of repeated independent experiments of dose- and time-dependent responses, respectively. Treatment of HAE with 5 mM H2O2 for 24 h resulted in cell death. However, treatments with 3 mM H2O2 for 8, 24, and 48 h did not change cell viability (data not shown). In contrast, a marked Sp1 band shift became apparent after 2 h at a concentration of 0.5 mM (Fig. 1A), and a similar change was detected 15 min after incubation with 2 mM H2O2 (Fig. 1B), suggesting a response not caused by cell death. A similar band shift was found in another lung epithelial cell line, H441, and a hepatocellular carcinoma cell line, HepG2 (Fig. 1C). In support of a change in phosphorylation, incubation of Sp1 isolated from H2O2-treated HAE cells with PP1 was able to reverse the mobility shift (Fig. 1D). Sp1 can be phosphorylated through a variety of kinases. Among others, MAP kinases have been shown to phosphorylate Sp1 in lung epithelial cells (32). We therefore postulated that H2O2 induces Sp1 phosphorylation in lung epithelial cells through activation of MAP kinases. As the first step in our investigation, we examined whether JNK plays a role in the H2O2-induced response in HAE cells. Using purified c-Jun as the substrate, we found that H2O2 activated JNK by ~10-fold in HAE cells in 2.5 h (Fig. 2). The simultaneous occurrence of JNK activation and Sp1 phosphorylation during H2O2 treatment suggests a potential link between the two events.


Figure 1
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  		Fig. 1. H2O2 induces Sp1 phosphorylation in lung epithelial cells. A: human alveolar epithelial (HAE) cells were treated with 0–2 mM H2O2 for 2 h as labeled (top). Whole cell lysates were prepared and examined in immunoblots using anti-Sp1 antibody. The intensity ratios of the top and bottom bands of Sp1 are plotted in the line graph. The experiment was repeated 4 times. The cells used were 24–30 h after passing and were 80–95% confluent. B: HAE cells of the same growth condition as in A were treated with 2 mM H2O2 (labeled at top) for 0–2 h. Whole cell lysates were examined in immunoblot using anti-Sp1 antibody. The intensity ratios of the Sp1 doublet are plotted similarly to A. The experiment was repeated 3 times. C: H441 and HepG2 cells were treated with H2O2 as labeled (top). Sp1 was examined using the same anti-Sp1 antibody in immunoblots. Each experiment was repeated twice. D: HAE cells were treated with 2.5 mM H2O2 for 2.5 h. Sp1 was isolated from the treated cells by immunoprecipitation. Immunoprecipitated Sp1 was then incubated with various doses of protein phosphatase-1 (PP1) (as labeled on top) and examined by immunoblotting using anti-Sp1 antibody. The ratios of top-to-bottom band intensity of the Sp1 doublet are plotted below. The experiment was repeated twice.

 

Figure 2
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  		Fig. 2. H2O2 activates JNK in HAE cells. HAE cells were treated with H2O2 as marked at top. JNK was isolated from the HAE cells and tested for its kinase activity using purified c-Jun as the substrate. Changes in phosphorylation of c-Jun were revealed by immunoblotting using an antibody recognizing phosphorylated c-Jun (p-c-Jun). The relative band intensities of p-c-Jun are plotted in the line graph.

 
JNK mediates H2O2-induced Sp1 phosphorylation in HAE cells. To establish a causative relationship between JNK activation and Sp1 phosphorylation, we examined Sp1 doublet in HAE cells treated with JNK activators and inhibitors. Anisomycin, a JNK activator (21), increased the intensity of the top band and decreased the intensity of the bottom band of the Sp1 doublet in 3 h in a dose-dependent manner (Fig. 3A), suggesting an increase in Sp1 phosphorylation. We then treated HAE cells with H2O2 in the presence and absence of the JNK inhibitors dicumarol and SP600125 (3, 14). The experiments were repeated three times, and the results showed that H2O2-induced Sp1 phosphorylation was suppressed in cells pretreated with JNK inhibitors (Fig. 3B). This suggests that inhibition of JNK interrupts H2O2-induced signal transduction pathways leading to Sp1 phosphorylation. To further confirm the role of JNK in H2O2-induced Sp1 phosphorylation, we suppressed JNK activity in HAE cells by stably transfecting the cells with a dominant-negative mutant JNK1 construct (Fig. 3C) (24). Both untransfected and transfected cells were then treated with H2O2. As shown in Fig. 3D, compared with 0-h treatment, 0.25-h treatment shifted the intensity of the Sp1 doublet in untransfected cells but not in transfected cells. At the time points beyond 0.5 h, the top band of the Sp1 doublet became the predominant band in untransfected cells, but both bands showed nearly equal intensity in transfected cells. These results demonstrate that JNK activity is necessary in H2O2-induced Sp1 phosphorylation.


Figure 3
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  		Fig. 3. JNK mediates H2O2-induced Sp1 phosphorylation in HAE cells. A: HAE cells were incubated with JNK activator anisomycin (Aniso) at concentrations indicated for 3 h. Sp1 phosphorylation was examined by immunoblotting. B: HAE cells were treated with 2.5 mM H2O2 for 2.5 h in the presence and absence of either 200 µM SP600125 or 100 µM dicumarol. Sp1, phosphorylated JNK, and total JNK were examined by immunoblotting. C: expression of JNK in untransfected HAE and HAE stably transfected with a dominant-negative JNK1 (HAE/DNJNK) was examined by immunoblotting using anti-JNK and anti-beta-actin antibodies. D: untransfected HAE cells and HAE cells stably transfected with a dominant-negative JNK1 expression vector were treated with 2 mM H2O2 for 0–2 h. Whole cell lysates were examined for Sp1 phosphorylation in immunoblots.

 
JNK1 directly phosphorylates Sp1. We next examined whether JNK1 plays a direct role in Sp1 phosphorylation using in vitro phosphorylation assays. Sp1 was isolated from HAE cells by immunoprecipitation. The isolated Sp1 was then incubated with various concentrations of recombinant JNK1 in kinase buffers with or without ATP. Our results show that, in the presence of ATP, the gel migration of the Sp1 protein was increasingly retarded along with increasing JNK1 concentration, whereas no change was detected in the absence of ATP, even at the highest concentration of JNK1 (Fig. 4A), suggesting that JNK1 directly phosphorylates Sp1. As a control, addition of recombinant ERK2 did not result in any changes in the protein migration of Sp1 (Fig. 4B, top). The kinase activity of ERK2 is demonstrated by replacing Sp1 with the recombinant transcription factor Elk-1 (25), which showed an impressive up-shift of the apparent molecular weight at the high end of the same concentration range of ERK2 (Fig. 4B, bottom). These results suggest that Sp1 is a direct substrate of JNK1 but not ERK2.


Figure 4
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  		Fig. 4. JNK1 directly phosphorylates Sp1. A: Sp1 isolated from HAE cells by immunoprecipitation was incubated with JNK1 at various concentrations (labeled at top) in phosphorylation reactions. As a control, the reaction in the last lane was incubated in the absence of ATP. Sp1 in the reactions was examined by immunoblotting. B: Sp1 immunoprecipitated from HAE cells was incubated with ERK2 at concentrations marked at top. Sp1 was examined by immunoblotting (top). A positive control experiment was performed using recombinant Elk-1 as the substrate, because Elk-1 is phosphorylated by ERK2. Elk-1 was examined in immunoblot using an anti-Elk-1 antibody (bottom).

 
Inhibition of PP1 induces phosphorylation of Sp1 and activation of JNK. PP1 is one of the major mammalian Ser/Thr protein phosphatases. Several lines of evidence show that oxidative stress may play a role in the regulation of PP1 activity. Predicted redox-sensitive cysteine residues are present in PP1 near the phosphatase active site that could be potentially oxidized (20). Furthermore, PP1 activity is inhibited in cultured cells on H2O2 treatment (31). PP1 regulates Sp1 phosphorylation in many types of cells including lung epithelial cells (11, 15, 27, 43). Although PP1 directly dephosphorylates Sp1 in vitro, in cells, it may suppress Sp1 phosphorylation through indirect mechanisms such as inhibition of kinases. Because JNK phosphorylates Sp1, we tested the hypothesis that PP1 plays a role in H2O2-induced Sp1 phosphorylation through inactivation of JNK. In this scenario, when PP1 activity is inhibited by H2O2, JNK is activated, which subsequently phosphorylates Sp1.

To confirm that inhibition of PP1 leads to Sp1 phosphorylation, we tested different protein phosphatase inhibitors [okadaic acid (OA), calyculin A, and fostriecin] in cultured HAE cells. These experiments were repeated two to five times with similar results. As shown in Fig. 5, OA at 100 nM, but not lower concentrations, induced a remarkable increase in Sp1 phosphorylation in 1.5 h. The effective concentration of OA supports a role for PP1 (IC50 = 20 nM) but not PP2A (IC50 = 0.1–1 nM) (13, 19, 39). As a negative control, okadaic acid methyl ester (OAME), an OA derivative with no protein phosphatase inhibitor activity (30), did not result in any changes in protein mobility of Sp1. A second protein phosphatase inhibitor, calyculin A, with similar inhibitory effect for PP1 (IC50 = 2 nM) and PP2A (IC50 = 0.5–1.0 nM) (13), also shifted the gel mobility of Sp1 at the concentration of 10 nM. In contrast, fostriecin, an inhibitor highly selective for PP2A (IC50 = 3.2 nM) but not PP1 (IC50 = 131 nM) (40), had no effect on Sp1 mobility at 100 nM. These results support the role of PP1 in H2O2-induced Sp1 phosphorylation.


Figure 5
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  		Fig. 5. Inhibition of PP1 induces Sp1 phosphorylation in HAE cells. HAE cells were treated with various concentrations and time periods of okadaic acid (OA), OA methyl ester (OAME), calyculin A (CalyA), or fostriecin (Fostr, 100 nM) as indicated. Whole cell lysates were prepared and analyzed in immunoblots using anti-Sp1 antibody.

 
To investigate an indirect role of PP1 in Sp1 phosphorylation through JNK, we tested the phosphorylation state of JNK in cells where PP1 was inhibited. We found that both OA and calyculin A induced phosphorylation of JNK under the same conditions that they induced Sp1 phosphorylation, whereas the inactive OA derivative OAME did not have any effect (Fig. 6A). Each experiment was repeated two or three times. Consistently, OA and calyculin A treatments activated JNK activity, as shown by an increase in the phosphorylation of c-Jun (Fig. 6B). The dose-dependence study showed that OA activated JNK activity under exactly the same condition as it induced phosphorylation of Sp1 and JNK (Fig. 6C), indicating a role for PP1 in the regulation of JNK activity. These results indicate that, similar to H2O2 treatment, inhibition of PP1 induces activation of JNK and phosphorylation of Sp1.


Figure 6
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  		Fig. 6. Inhibition of PP1 induces phosphorylation and activation of JNK in HAE cells. A: HAE cells were incubated for 2 h with OA, OAME, or calyculin A as indicated. Whole cell lysates were analyzed in immunoblots for phosphorylated JNK (top) and total JNK (bottom). B: HAE cells were untreated (Cont) or treated with calyculin A (10 nM, 2 h) or OA (100 nM, 2 h). JNK isolated from cells was incubated with recombinant c-Jun in phosphorylation reactions. Phosphorylated c-Jun (p-c-Jun) in the reactions was determined by immunoblotting using an anti-phospho-c-Jun antibody. C: HAE cells were treated with different concentrations of OA for 2 h. Cellular JNK was isolated, and the same assay as in B was performed to determine JNK kinase activity.

 
PP1 directly dephosphorylates JNK. To determine the role of PP1 in the signal transduction cascade, we first tried to determine whether JNK is a PP1 substrate. In vitro dephosphorylation experiments were performed using recombinant active JNK1, ERK2, and p38 MAP kinase as substrates. We found that phosphorylated JNK1 and p38 MAP kinase were efficiently dephosphorylated by PP1. In contrast, PP1 was not able to dephosphorylate ERK2 (Fig. 7). These results suggest that PP1 directly and selectively dephosphorylates JNK1.


Figure 7
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  		Fig. 7. PP1 directly dephosphorylates JNK1. In vitro dephosphorylation of recombinant JNK1, ERK2, and p38 MAP kinase by PP1. The kinases were incubated with various amounts of PP1 as labeled at top. JNK1, ERK2, and p38 were examined in immunoblots with antibodies against phosphorylated and total kinases as marked. The bands were scanned and the ratios of phosphorylated/total (p/total) kinases were calculated. The relative ratios of p/total kinases are plotted in the line graph. The ratios from no-PP1 controls were set at 1.

 
PP2A is another major Ser/Thr protein phosphatase in eukaryotic cells. As mentioned above, the effect of PP2A can be readily distinguished from that of PP1 by using OA and fostriecin (4, 13, 39, 40). However, we still examined a possible role for PP2A in the dephosphorylation of JNK1/ERK2, because we suspected that dephosphorylation of JNK1/ERK2 by Ser/Thr phosphatases is subtype specific. As shown in Fig. 8, there is a stunning contrast between JNK1 and ERK2 as PP2A substrates. PP2A efficiently dephosphorylated ERK2 but poorly dephosphorylated JNK1. Phosphorylated ERK2 completely disappeared after incubation with 0.1 units of PP2A, but 0.5 units of PP2A only slightly weakened the signal of phosphorylated JNK1. Taken together, the data shown in Figs. 7 and 8 indicate that PP1 is by far a more efficient phosphatase for JNK1 compared with PP2A. In addition, JNK1 and ERK2 could be a useful pair of substrates in distinguishing phosphatase activities of PP1 and PP2A, which most currently available assays are unable to differentiate.


Figure 8
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  		Fig. 8. PP2A dephosphorylates ERK2 but not JNK1. In vitro experiments were used to examine the role of PP2A in the dephosphorylation of JNK1 and ERK2. Concentrations of PP2A are labeled at top. Phosphorylated and total JNK1 and ERK2 were examined in immunoblots. The bands were scanned, and the p/total ratios were calculated and plotted as described in Fig. 7.

 
PP1 inactivates JNK. Because PP1 is a Ser/Thr phosphatase, PP1 can only dephosphorylate Thr183 of active JNK, which is phosphorylated at both Thr183 and Tyr185 (17). The disappearance of phosphorylated JNK in immunoblots after PP1 treatment (Fig. 7) can be explained by the fact that the anti-p-JNK antibody recognizes only dually phosphorylated JNK, i.e., the reactivity is markedly reduced when Thr183 is dephosphorylated (unpublished data; Chris Salvadore, Cell Signaling Technology). Whether the dephosphorylation of Thr183 inhibits JNK kinase activity became a critical question, because this would determine whether it is possible for JNK to play a role in PP1-regulated phosphorylation of Sp1. Therefore, we examined the kinase activity of JNK in relation to PP1 treatment. In this study, recombinant active JNK1 was treated with PP1 and examined for its ability to phosphorylate c-Jun. As shown in Fig. 9, PP1 inhibited JNK1 kinase activity in a dose-dependent fashion, and this inhibition was blocked by OA and calyculin A. These results demonstrate that PP1-mediated dephosphorylation of JNK1 results in inhibition of JNK1 activity.


Figure 9
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  		Fig. 9. PP1 inactivates JNK1 activity. Recombinant JNK1 was first incubated with various concentrations of PP1. At the highest concentration of PP1 (1.25 U/reaction), parallel reactions were performed in the presence of OA (100 nM) and calyculin A (10 nM). JNK1 was then isolated and incubated with recombinant c-Jun. Phosphorylated c-Jun was determined by immunoblotting.

 
JNK mediates PP1 inhibition-induced Sp1 phosphorylation in HAE cells. Because inhibition of PP1 appears to induce similar cellular effects as those induced by H2O2, i.e., activation of JNK and phosphorylation of Sp1, and inhibition of JNK suppresses H2O2-induced effects, we examined whether inhibition of JNK also blocks OA-induced effects. With two independent experiments, we found that OA-induced phosphorylation of Sp1 was suppressed in cells pretreated with the JNK inhibitors dicumarol and SP600125 (Fig. 10A). To further verify the role of JNK in PP1 inhibition-induced Sp1 phosphorylation, we tested HAE cells untransfected and stably transfected with the same dominant-negative JNK expression vector (24). The cells were treated with OA at various concentrations for 2–3 h. As shown in Fig. 10B, the OA-induced shift in Sp1 mobility was remarkably reduced in the stably transfected cells compared with untransfected cells. Taken together, these results again support a role for JNK in PP1 inhibition-induced Sp1 phosphorylation in HAE cells.


Figure 10
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  		Fig. 10. OA-induced Sp1 phosphorylation in HAE cells requires the presence of active JNK. A: to determine the role of JNK in OA-induced Sp1 phosphorylation, JNK inhibitors SP600125 (200 µM) and dicumarol (100 µM) were added to HAE cells 30 min before the addition of OA (100 nM for 2 h). After incubation, the cells were collected, and whole cell lysates were analyzed in immunoblots for Sp1 and phosphorylated and total JNK with respective antibodies as labeled at right. B: HAE cells were stably transfected with an expression vector coding for dominant-negative JNK1. Control and transfected cells (HAE and HAE/DNJNK, respectively) were treated with OA at various concentrations for different lengths of time, as marked at top. Whole cell lysates were analyzed in immunoblots using anti-Sp1 antibody.

 
JNK1-mediated phosphorylation of Sp1 in HAE cell nuclear extract decreases Sp1-DNA binding. The effect of Sp1 phosphorylation on its transcription factor function has been well recognized (12). Phosphorylation of Sp1 may have either positive or negative effects on its DNA binding and trans-activity. JNK-mediated phosphorylation of transcription factors such as AP-1, however, is known to activate gene transcription (22). To demonstrate the physiological significance of JNK1-mediated Sp1 phosphorylation, we examined the ability of HAE nuclear Sp1 to bind DNA when phosphorylated in vitro by JNK1. A parallel phosphorylation reaction in the absence of ATP was used as a control, because JNK1 is unable to phosphorylate Sp1 without ATP (Fig. 4A). The cyclin D3 promoter is regulated by Sp1, and nonphosphorylated Sp1 has a greater affinity for the promoter DNA (43). Whether JNK1 plays a role in Sp1 binding to the cyclin D3 promoter has not been studied. We used an Sp1 binding site-containing oligonucleotide probe derived from the cyclin D3 promoter in this study. As shown in Fig. 11, Sp1 phosphorylated by JNK1 ("+ATP" lanes) had a much weaker interaction with the cyclin D3 promoter probe compared with the original Sp1 ("no ATP" lanes), suggesting a negative effect of JNK1-induced phosphorylation on DNA binding in HAE nuclear extract.


Figure 11
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  		Fig. 11. JNK1-mediated phosphorylation of Sp1 reduces Sp1 binding to its DNA target. Electrophoretic mobility shift assay was performed using HAE nuclear extract and an oligonucleotide probe derived from the cyclin D3 gene promoter containing an Sp1 binding site. Before adding to the DNA binding reactions, the nuclear extract was incubated with JNK1 in phosphorylation reactions in the presence (+ATP) and absence (no ATP) of ATP. For each reaction, the same amounts of nuclear extract and oligonucleotide probe were used. Antibodies used in supershift are marked at top. Original positions of protein-DNA complexes containing Sp1 and Sp3 are labeled at right.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Stimulation of cells with ROS such as H2O2 often results in changes in gene expression. Growing evidence has shown that the changes in gene expression could be due to H2O2-initiated regulation of transcription factors (35, 37). Phosphorylation/dephosphorylation is one of the most effective means for the regulation of transcription factors, which could be achieved through a variety of kinases and phosphatases. JNK was first reported to phosphorylate and activate transcription factors more than a decade ago (22). In addition to a small group of well-characterized JNK-activated transcription factors such as c-Jun, activating transcription factor-2, and Elk-1, an increasing number of other transcription factors have been reported as JNK targets. Because H2O2 activates JNK in certain cells (28), JNK may play a role in H2O2-induced regulation of gene expression. Indeed, H2O2-induced and JNK-mediated regulation of transcription has been reported in a recent study in which the transcription factor FOXO4 is activated by H2O2 through the small GTPase Ral and JNK (18).

Phosphorylation of Sp1 can be mediated by many kinases. However, the only indication of a role of JNK in Sp1 phosphorylation was reported in a study of the human urokinase (urokinase-type plasminogen activator; uPA) gene promoter, where expression of dominant-negative JNK in a human adenocarcinoma cell line reduced uPA promoter activity and Sp1 phosphorylation (2). However, it is unclear whether JNK plays a direct or indirect role in Sp1 phosphorylation and whether the reduced promoter activity is a direct result of the inhibition of JNK-mediated Sp1 phosphorylation.

Our initial results show that H2O2 induces Sp1 phosphorylation and JNK activation, suggesting that JNK mediates H2O2-induced phosphorylation of Sp1 (Figs. 1 and 2). We subsequently investigated this possible signaling pathway using JNK activators and inhibitors and a dominant-negative JNK1 mutant. Activator anisomycin, inhibitors dicumarol and SP600125, and dominant-negative mutant JNK1 all generated expected results in HAE cells (Fig. 3), supporting a role for JNK in H2O2-induced Sp1 phosphorylation. In addition, in vitro phosphorylation results demonstrate that JNK1, but not ERK2, directly phosphorylates Sp1 (Fig. 4). As a result, Sp1 from HAE cell nuclear extract, when phosphorylated by JNK1, shows a compromised ability to bind DNA (Fig. 11).

Like other MAP kinases, JNK is maintained at low activity in unstimulated cells. JNK is activated by stress signals through dual phosphorylation at Thr183 and Tyr185 by mitogen-activated protein kinase kinase-4 (MKK4), MKK7, and other upstream molecules (16, 41). In general, the low activities of MAP kinases in unstimulated cells could be due to the lack of activation from upstream kinases or the presence of phosphatases. The MAP kinases are dephosphorylated by a group of phosphatases known as MAP kinase phosphatases (MKPs), which include tyrosine-specific, serine/threonine-specific, and dual-specificity MKPs (9). Little is known regarding whether PP1 inactivates MAP kinases.

On the basis of our results and previous findings, we propose that PP1 inactivates JNK in HAE cells. This is because H2O2 has been reported to inhibit PP1 (31), and H2O2 activates JNK in HAE cells (Fig. 2). More direct evidence supporting this interaction includes the fact that inhibition of PP1 leads to phosphorylation and activation of JNK (Fig. 6) and that PP1 directly dephosphorylates and inactivates JNK in vitro (Figs. 7 and 9). This mechanism is further supported by the previous finding that a Cys-x-x-Cys site is present in close proximity to the phosphatase active site, which could be potentially oxidized (20). Similar to our results and in support of a role for phosphatases in H2O2-induced activation of JNK, Kamata et al. (26) recently reported that H2O2 contributes to TNF{alpha}-induced JNK activation through inhibition of MKPs. However, because H2O2-induced cellular responses could be cell type specific (1, 28), different signaling events may participate in different cell systems.

We have reported in our earlier studies that PP1 directly dephosphorylates Sp1 in vitro (11). This direct dephosphorylation appears to be not as important an event as inhibition of JNK, because the kinase inhibitor experiments and transfection with the dominant-negative JNK1 both demonstrate that JNK is indispensable for the H2O2- and OA-induced Sp1 phosphorylation (Figs. 3 and 10). Therefore, the role of PP1 in Sp1 phosphorylation is most likely indirect in live cells through inhibition of JNK.

PP2A is the other Ser/Thr protein phosphatase with the possibility of being inhibited by protein phosphatase inhibitors. However, an identical high concentration of OA, 100 nM, is required for JNK phosphorylation (Fig. 6A), JNK activation (Fig. 6C), and Sp1 phosphorylation (Fig. 5). None of the above effects was seen when 10 nM OA was used. In addition, fostriecin, a more selective PP2A inhibitor, did not have any effect on Sp1 phosphorylation (Fig. 5). All of these results support a role for PP1 but not PP2A (19). Furthermore, JNK1 appears to be a very poor substrate of PP2A compared with PP1 (Figs. 7 and 8). There is one report showing PP2A-mediated dephosphorylation of JNK (38). However, the phosphatase activity appeared to be from PP1, because it was inhibited only by high concentrations of OA. Although PP2A was shown to co-immunoprecipitate with JNK, whether PP1 interacted with JNK was not examined, which has been reported in other cells (8). It is also possible that PP1 and PP2A regulate JNK activity in a cell type-specific fashion. PP2A is therefore less likely to play a major role in JNK1-mediated Sp1 phosphorylation in the HAE cells.

Functional implications of Sp1 phosphorylation vary widely in the literature. Phosphorylation of Sp1 may result in both increased and decreased DNA binding capabilities depending on the sequence of the DNA targets and other unknown factors (12). JNK is traditionally known to phosphorylate and activate transcription factors. Our results, however, indicate that JNK-mediated phosphorylation of Sp1 may result in a much reduced ability of Sp1 to bind DNA (Fig. 11). Although the EMSA result is shown using a cyclin D3 promoter sequence, we have tested five other oligo probes in similar experiments, and all of them showed reduced DNA binding on JNK1-mediated phosphorylation. This indicates that, at least in HAE cells, JNK-mediated phosphorylation may result in a decrease in the function of transcription factors.

In summary, our results show that 1) JNK is activated by treatment with H2O2 and inhibition of PP1 in HAE cells (Figs. 2 and 6), 2) PP1 dephosphorylates JNK (Fig. 7), 3) dephosphorylation of JNK by PP1 results in inhibition of JNK activity (Fig. 9), 4) JNK1 directly phosphorylates Sp1 (Fig. 4), and 5) inhibition of JNK in HAE cells by inhibitors or dominant-negative JNK1 results in blockage of H2O2- and OA-induced Sp1 phosphorylation (Figs. 3 and 10). These results suggest the presence of a PP1-JNK signaling mechanism that regulates the phosphorylation state of Sp1 in HAE cells.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the McGuire Research Institute, a March of Dimes Foundation Research Grant, and a Department of Veterans Affairs Merit Review Grant.


   ACKNOWLEDGMENTS
 
We thank Dr. Stephen T. Sawyer of Virginia Commonwealth University for the kind gift of the dominant-negative JNK expression vector.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Chu, McGuire VA Medical Center, 1201 Broad Rock Blvd., Richmond, VA 23249 (e-mail: schu{at}hsc.vcu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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