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Pathways of Induction of Peroxiredoxin I Expression in Osteoblasts

Pathways of Induction of Peroxiredoxin I Expression in Osteoblasts THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 14, Issue of April 5, pp. 12418 –12422, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. ROLES OF p38 MITOGEN-ACTIVATED PROTEIN KINASE AND PROTEIN KINASE C* Received for publication, November 30, 2001, and in revised form, December 17, 2001 Published, JBC Papers in Press, January 16, 2002, DOI 10.1074/jbc.M111443200 Baojie Li‡§ , Tetsuro Ishii , Choon Ping Tan§, Jae-Won Soh‡, and Stephen P. Goff‡**‡‡ From the ‡Department of Biochemistry and Molecular Biophysics, **Howard Hughes Medical Institute, Columbia University, College of Physicians and Surgeons, New York, New York 10032, the §Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore 117609, Singapore, and the Department of Biochemistry, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8575 Japan poly(ADP-ribosylation), depletion of cellular energy stores, and Peroxiredoxin I (Prx I) is an oxidative stress-inducible antioxidant protein with thioredoxin peroxidase activ- cell death. Lower concentration of oxidants causes apoptosis in ity. Here we report that the levels of Prx I mRNA and some cell lines, whereas high concentration usually causes protein are dramatically increased in a murine osteo- necrosis (1, 2). In addition, accumulating evidence suggests blast cell line, MC3T3-E1, by treatment with sodium ar- that reactive oxygen species such as H O play important roles 2 2 senate. We further studied the signaling pathways that in cell signaling in non-phagocytic cells. For example, platelet- control the induction of Prx I expression. The treatment derived growth factor stimulation results in the generation of of osteoblasts with arsenate activated ERK1/2, JNK, and H O , which is required for platelet-derived growth factor- 2 2 p38 MAPK. Pre-treating cells with inhibitors of p38 1 induced mitogen-activated protein kinase (MAPK) activation, MAPK abolished the induction of Prx I protein but had STAT activation, and DNA synthesis (6, 7). minimal effect on the induction of Prx I mRNA, suggest- In response to oxidants, cells up-regulate the expression of ing that p38 MAPK activity was required for post-tran- several protective products. One such protein is peroxiredoxin scriptional regulation. The inhibition of ERK1 and I (Prx I), originally identified as a 23-kDa stress-inducible ERK2 had no effect on the induction of Prx I expression. protein termed MSP23 in mouse macrophages (8) and also Furthermore, rottlerin, an inhibitor of protein kinase C known as a protein OSF-3, up-regulated during the differenti- (PKC) and calmodulin kinase III, abrogated the up- ation of an osteoblast cell line, MC3T3-E1(9). The human coun- regulation at both protein and mRNA levels. Staurospo- terpart was cloned from human breast epithelial cell line 100 rine and Go6983, inhibitors for PKC, also inhibited the (HBL100) cells and was named PAG (proliferation associated induction of Prx I, suggesting that protein kinase C is gene), because it is expressed at a higher level in transformed required for the induction by arsenate. PKC was acti- cells (10). The rat counterpart was cloned as a heme-binding vated by arsenate treatment by in vitro kinase assays. protein (HBP23) in rat liver cytosol (11). Prx family proteins The inhibition of PKC by rottlerin did not affect the activation of p38 MAPK by arsenate. These results sug- have conserved cysteine residues that are important for thiore- gest that there are two separate signaling pathways in- doxin peroxidase activity (12) and thiol radical scavenging (13). volved in the up-regulation of Prx I protein in response Recently, Prx I has also been implicated in signaling pathways. to arsenate, PKC required for transcriptional activa- It was found that human Prx I (Pag) interacts with c-Abl, a tion and p38 MAPK required for post-transcriptional non-receptor tyrosine kinase, and that overexpressed Prx I can regulation. negatively regulate the kinase activity and cytostatic activity of c-Abl (14). Prx I is up-regulated by stress agents including H O , di- 2 2 Oxidative stress is believed to contribute to the etiology of ethyl maleate, and sodium arsenite in macrophages (15). Arse- various degenerative diseases such as cerebellar ischemia, can- nate is also a potent inducer of Prx I in a human mammary cell cer, and the process of aging (reviewed in Refs. 1 and 2). line (16). The induction of the prx I gene by electrophilic agents Oxidants including superoxides and H O are generated under 2 2 and reactive oxygen species is mainly controlled by the tran- physiological conditions during mitochondrial electron trans- scription factor Nrf2 in murine peritoneal macrophages (15). In port, peroxisomal fatty acid metabolism, phagocytosis by peritoneal macrophages isolated from nrf2 knockout mice, the macrophages, and bone resorption by osteoclasts (2– 4). The induction of Prx I was diminished, and the cells were more tissue concentration of H O during inflammation can reach 2 2 susceptible to the killing effect of an electrophilic agent. The millimolar levels (5). High levels of oxidants result in DNA expression of genes encoding hemeoxygenase I and A170 was strand breaks, oxidation of lipids and proteins, activation of also diminished in the mutant macrophages. Similarly, Nrf2 is also essential for the induction of the activation of genes en- coding detoxifying enzymes such as glutathione S-transferase * The costs of publication of this article were defrayed in part by the and NAD(P)H:Quinone oxidoreductase by electrophilic agents payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to (17). Nrf2 interacts with the antioxidant responsive elements indicate this fact. and regulates the expression of those genes important for de- Recipient of Cancer Research Institute Fellowship at Columbia fense against oxidative stress. University. ‡‡ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Dept. of Biochemistry and Molec- The abbreviations used are: MAPK, mitogen-activated protein ki- ular Biophysics, HHSC1128, Columbia University, College of Physi- nase; STAT, signal transducers and activators of transcription; Prx I, cians and Surgeons, 701 W. 168th St., New York, NY 100322. Tel.: peroxiredoxin I; ERK, extracellular signal-regulated kinase; JNK, c- 212-305-3794; Fax: 212-305-8692; E-mail: goff@cuccfa.ccc.columbia. Jun N-terminal kinase; CaM, calmodulin; MARCKS, myristoylated ala- edu. nine-rich protein kinase C substrate; mRNA, messenger RNA. 12418 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Signaling Pathways Controlling Prx I Induction by Arsenate 12419 The signaling pathways regulating expression of Prx I are not clear. Agents causing oxidative stress activate many sig- naling pathways including MAPKs, protein kinase C-, and nuclear factor-kB (18, 19). Three MAPKs (ERK1/2, JNK, and p38 MAPK) are activated by high concentrations of H O . The 2 2 activation of MAPK and/or PKC plays an important role in the oxidative stress-induced expression of genes such as nitric- oxide synthase (20) and ornithine decarboxylase (21). In this report we studied the signaling pathways that are involved in the induction of Prx I in osteoblasts by sodium arsenate. We demonstrate that arsenate induced the expres- sion of Prx I in an osteoblast cell line, and that this induction required the activation of both p38 MAPK and protein kinase C. FIG.1. Induction of Prx I by sodium arsenate in MC3T3-E1 MATERIALS AND METHODS cells. A, the cells growing in logarismic phase were treated with differ- Culture of Osteoblasts—The osteoblast cell line MC3T3-E1 was cul- ent amounts of sodium arsenate for 16 h, harvested, and lysed in TNE buffer. Protein concentration was determined by Bio-Rad method, and tured in -minimum essential medium (Invitrogen) containing 10% 20 g of total protein was loaded onto 10% SDS-PAGE gel. Expression fetal calf serum (Research Sera). of Prx I was analyzed by Western blot using a polyclonal antibody Treatment of Osteoblasts by Sodium Arsenate—Osteoblasts were against Prx I. B, Northern blot analysis of induction of Prx I mRNA by subcultured the day before and then treated with sodium arsenate for arsenate. Cells were treated with 0.06 mM arsenate for different times various times. For the analysis of Prx I protein, cells were treated for and collected. Total RNA was isolated and analyzed with Northern blot. 16 h and then washed with phosphate-buffered saline, collected, and the level of Prx I was analyzed by Western blot. For the analysis of RESULTS mRNA, cells were treated 12 h. Total RNA was isolated and analyzed by Northern blot. For analysis of protein kinases, cells were treated from Sodium Arsenate Induces Expression of Prx I in Osteo- 5 min to 16 h. At each time point, cells from one plate were washed and blasts—Sodium arsenate is a toxic compound that activates the lysed. To test the effect of the kinase inhibitors SB203580, PD98059, oxidative stress pathway in several cell types. Arsenate is able and rottlerin (Calbiochem) on Prx I induction, various concentrations of to induce the expression of the Prx I at the level of protein and the compounds were added to the cell cultures 1 h prior to the addition mRNA in the human breast epithelial cell line HBL100 and of sodium arsenate. Cells were further treated in medium containing murine macrophage WAR cells (16). To test the effects of arse- both sodium arsenate and SB203580, PD98059, or rottlerin for an nate on osteoblasts, a murine osteoblast cell line, MC3T3-E1, additional 16 h. The cells were then harvested for Western and North- ern analysis. was incubated with medium containing arsenate at different Western Blot Analysis of Protein Levels—Cells were washed with concentrations, and Prx I levels were analyzed by Western blot. phosphate-buffered saline and lysed in a buffer containing 50 mM Tris, Two protein species of 23 and 24 kDa in size were detected with pH 7.5, 100 mM KCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM phenyl- the antiserum (Fig. 1A). The main 23-kDa band corresponds to methylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM Prx I, and the minor 24-kDa band probably corresponds to Prx -glycerophosphate, and 10 g/ml each of aprotinin and leupeptin. III, a family protein localizing in the mitochondrial matrix. Prx Protein concentrations were determined by Bio-Rad method. The same I was strongly induced in MC3T3-E1 cells in a dose-dependent amount of protein (20 g) was fractionated by electrophoresis on a 10% manner with maximum protein levels detected at approxi- SDS-PAGE gel, transferred to a nitrocellulose membrane (Immobilon- p), probed with polyclonal anti-Prx I antibodies, and visualized using an mately 0.10 mM arsenate (Fig. 1A). The induction occurs at the ECL kit (Amersham Biosciences, Inc.). Anti-phosphorylated ERKs, p38 level of transcription, because Northern blot analysis showed MAPK, JNK, and myristoylated alanine-rich protein kinase C substrate that the levels of Prx I mRNA are dramatically increased (Fig. (MARCKS) antibodies were purchased from Cell Signaling. Anti-p38 1B). The induction was detected at 4 h after the addition of antibody was purchased from Santa Cruz Biotechnology. arsenate and reached the maximal level after 8 h. The increase Northern Blot Analysis—1  10 osteoblasts were plated onto of both mRNA and protein was completely blocked by treating 100-mm plates and treated the second day with sodium arsenate. Cells the cells with RNA polymerase II inhibitor, actinomycin D were collected, and total RNA was isolated using RNAzol (TelTest), (data not shown). The decrease in the expression of Prx I at the fractionated on a 1.5% formaldehyde-agarose gel, transferred to Nytran higher concentration of sodium arsenate may be caused by cell membrane (S & S), and probed with random primer-labeled Prx I cDNA. death. Kinase Assay for PKC—Cells were challenged with arsenate for Arsenate Activates MAPKs—Oxidative stress triggered by different times and then lysed with phosphorylation lysis buffer (50 mM H O treatment has been found to activate MAP kinase path- 2 2 HEPES, 150 mM NaCl, 200 M sodium orthovanadate, 10 mM sodium ways including ERKs, JNK, and p38 MAPK, and arsenate itself pyrophosphate, 100 mM sodium fluoride, 1 mM EDTA, 1.5 mM magne- has been shown to activate JNK in human embryonic kidney sium chloride, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 293 cells (22). To test whether sodium arsenate activates these 10 g of aprotinin/ml). Cell lysate was separated to cytosolic and par- pathways in osteoblasts, MC3T3-E1 cells were exposed to so- ticulate fractions by centrifugation for 30 min at 100,000  g, and the dium arsenate in the culture medium. The cells were harvested particulate was dissolved in the same buffer plus Triton X-100. The and lysed after various times of treatment, and the levels of particulate fractions were immunoprecipitated with an antibody against PKC using protein G-Sepharose (Amersham Biosciences, Inc.). active kinases were analyzed by Western blot. Antibodies that The immunocomplexes were washed three times with phosphorylation specifically recognize the activated forms of ERKs, JNK, and lysis buffer and two times with kinase buffer (25 mM Tris-HCl, pH 7.4, p38 MAPK were used to determine the kinase activation (Fig. 5mM MgCl , 0.5 mM EGTA, 1 mM dithiothreitol, 20 g of phosphati- 2A). The level of activated ERKs, rapidly increased to the dylserine, 20 M ATP) and were resuspended in 30 l of kinase buffer maximum level within 10 min, started to decline within 30 min containing 5 g of histone H1, and 20 Ci of [ P]ATP was added. The and then stayed at a level slightly higher than the basal level reaction was incubated for 30 min at room temperature and was ter- for a few hours. JNK was also activated within 10 min and minated by the addition of SDS sample buffer. Proteins were analyzed declined after approximately 30 min, although the activation by SDS-PAGE, and the phosphorylated form of histone H1 was detected by autoradiography. was much weaker. Osmotic stress was found to activate JNK to 12420 Signaling Pathways Controlling Prx I Induction by Arsenate FIG.3. A, ERKs are not required for Prx I induction by sodium arsenate. MC3T3-E1 cells were pretreated with different amounts of PD98059 for 1 h, and sodium arsenate was added to a final concentra- tion of 0.06 mM. Cells were further incubated for 16 h, and expression of FIG.2. A, activation of ERKS, JNK, and p38 MAPK in MC3T3-E1 Prx I was analyzed as described in Fig. 1. B, inhibition of ERKs by cells by arsenate. Equal numbers of osteoblasts from the same passages PD98059. Cells were pretreated with different concentrations of arse- were plated and treated with 0.06 mM sodium arsenate for different nate for 1 h, and arsenate was added. After 10 min, cells were collected, times. Cells were then harvested and lysed. 60 g of total proteins were and activation of ERKs was analyzed as described in Fig. 2. analyzed by Western blot using antibodies that specifically recognize activated ERKs, JNK, and p38 MAPK, respectively. The same blot was stripped and reprobed with different antibodies. B, activation of JNK by osmotic stress. Cells were treated with 500 mM sorbitol for different times just before collection. Activation of JNK was analyzed with West- ern blot using anti-phosphorylated JNK antibodies. a much higher extent (Fig. 2B), indicating that arsenate is not a strong activator of the JNK pathway under these settings. In contrast to the rapid initial increase in ERK and JNK activa- tion, p38 MAPK showed no rapid increase but was activated only in a slow time course. The levels of active p38 MAPK gradually increased to a maximum (Fig. 2A) even after 16 h of treatment (data not shown). The level of p38 MAPK protein itself as monitored by Western blot did not change upon sodium FIG.4. A, inhibition of p38 MAPK suppresses the induction of Prx I arsenate treatment. Thus, the three kinases examined in these protein by arsenate. Experiments were carried out as described in Fig. experiments showed distinct patterns of induction. 3A. B, Northern blot analysis showing that Prx I mRNA was not affected by the inhibition of p38 MAPK. MC3T3-E1 cells were treated as Inhibition of p38 MAPK Abolishes Induction of Prx I Pro- in A, and total RNA was isolated. 20 g of total RNA was fractionated tein—To determine whether or not the MAPK pathways were on a 1.2% formaldehyde-agarose gel, and the mRNA level of Prx I was important for the induction of Prx I by arsenate, cells were analyzed by Northern blot using a random primer-labeled probe. 18 S pretreated with kinase inhibitors for 1 h before adding sodium ribosomal RNA was used as loading control. arsenate. PD98059, which specifically inhibits the ERKs, had no effect on the induction of Prx I even at a concentration of 40 M (Fig. 3A), suggesting that the induction of Prx I did not controlling the induction of Prx I by arsenate, we used an require the ERK pathways. Fig. 3B shows that PD98059 inhib- inhibitor specific for PKC, rottlerin (23). Rottlerin also in- ited the activation of ERKs by arsenate in a dosage-dependent hibits CaM kinase III, but the only known substrate for CaM manner. Even at 5 M, the level of activated ERKs was reduced kinase III is elongation factor 2, and neither elongation factor to the basal level. In contrast, SB203580, a specific inhibitor for 2 nor CaM kinase III has been implicated in oxidative stress p38 MAPK, potently inhibited the induction of Prx I in a responses. Osteoblasts were pretreated with different con- dose-dependent manner (Fig. 4A). At 5 M concentration, centrations of rottlerin for 1 h, and then 0.06 mM arsenate SB203580 dramatically reduced the levels of Prx I, and at 30 was added. After 16 h, cells were collected, and the Prx I M, the amount of Prx I protein was reduced to basal levels protein levels were determined by Western blot analysis. Fig. indicating a complete inhibition. This is the range of concen- 5A shows that 5 M rottlerin completely blocked the induc- tration that is effective for the inhibition of p38 in a large tion of Prx I protein. To exclude a role for CaM kinase III in number of tested cell lines. A second specific inhibitor for p38 the induction, two other PKC inhibitors, staurosporine and MAPK, SB202190, could also block the induction of Prx I at a Go6983, were similarly tested. Both were able to inhibit Prx concentration of 2 M (data not shown). To determine whether I induction. Staurosporine, a general PKC inhibitor, inhib- the inhibition occurred at the level of transcription, Northern ited the induction of Prx I in a dosage-dependent manner, blot analysis was carried out. The levels of Prx I mRNA were and at 100 nM, the inhibition was close to complete. Because only slightly reduced by SB203580 compared with the levels of rottlerin is a drug that only affects PKC of the protein protein (Fig. 4B). These results suggest that the inhibition of kinase C family, these results taken together suggest that Prx I induction by the drug may involve both transcriptional PKC is required for the induction of Prx I by arsenate and post-transcriptional mechanisms, but that the major effect treatment. Northern blot analysis (Fig. 5C) shows that the is post-transcriptional. inhibition occurred also at the level of mRNA. These data Inhibition of PKC Prevents Induction of Prx I—To deter- suggest that the activation of a PKC kinase activity is nec- mine whether PKC plays a role in the signaling pathways essary for the up-regulation of the Prx I mRNA in response to Signaling Pathways Controlling Prx I Induction by Arsenate 12421 FIG.6. A, activation of protein kinase C by arsenate. B, inhibition of PKC by rottlerin. Treatment osteoblasts with arsenate resulted in the phosphorylation of MARCKS, and the phosphorylation was inhibited by pretreatment of cells with rottlerin for 1 h. Phosphorylated MARCKS was detected by Western blot analysis using polyclonal antibodies against phosphorylated MARCKS. FIG.7. Activation of p38 MAPK by arsenate was not affected by FIG.5. A, rottlerin suppresses the induction of Prx I at the level of the inhibition of PKC. Osteoblasts were pretreated with 5 M rott- protein. A different concentration of rottlerin was used to pretreat lerin for 1 h, and then arsenate was added to the cultures. At different osteoblasts for 1 h, and then arsenate was added to a final concentration time points, a plate was washed, cells were collected, and activated p38 of 0.06 mM for 16 h. The levels of Prx I protein were analyzed by MAPK was analyzed by Western blot as described in Fig. 2. Western blot as described in Fig. 1. B, staurosporine and Go6983 inhibit the induction of Prx I. C, rottlerin inhibits the expression of Prx I mRNA. arsenate treatment. On the other hand, wortmannin, a spe- cific inhibitor of phosphatidylinositol 3-kinase, has no effect on Prx I induction (data not shown). To test whether arsenate activates PKC in osteoblasts, cells were treated with arsenate and were collected at different time points, PKC was immunoprecipitated with a monoclonal an- tibody, and an in vitro kinase assay was performed using H1 histone protein as substrate. Fig. 6A shows that arsenate rap- idly activates PKC. Within 30 min, PKC activation reached the maximal level, and the kinase activity remained high even 4 h after stimulation. Furthermore, arsenate treatment also led to the phosphorylation of a PKC substrate, MARCKS, and FIG.8. Signaling pathways involved in the induction of Prx I the phosphorylation was inhibited by rottlerin in a dosage-de- by arsenate in osteoblasts. pendent manner (Fig. 6B) as was the inhibition of Prx I induc- tion. These data support the conclusion that the activation of DISCUSSION PKC is required for Prx I induction. Activation of p38 MAPK Is Not Affected by Rottlerin—The The experiments presented above show that arsenate causes experiments presented above suggest that the induction of Prx a dramatic increase in Prx I protein and mRNA in a murine I by arsenate treatment required the activation of both p38 osteoblast cell line, MC3T3-E1. Osteoblasts may be exposed to MAPK and protein kinase . To determine whether the activa- significant levels of oxidants generated by osteoclasts during tion of PKC was upstream of p38 MAPK in response to arse- bone remodeling (24) and may have a particular need for a nate treatment, we pretreated osteoblasts for 1 h with 5 M strong response to oxidative stress. In fact, oxidative stress has rottlerin, a concentration sufficient to block the induction of also been found to induce the expression of genes such as Hic5 Prx I. We then added 0.06 mM arsenate, and at different time (hydrogen peroxide inducible clone 5), c-jun,c-fos, and Erg-1 in points, we collected cells and made cell lysates. The activation MC3T3-E1 (reviewed in Ref. 25). There is some difference of p38 MAPK was determined by Western blot analysis using between osteoblasts and other cell lines in terms of Prx I the antibody that specifically recognizes activated p38 MAPK induction by arsenate. In WAR and HBL100 cells, the induc- (Fig. 7). We did not detect any significant difference in the tion of Prx I mRNA by arsenate is dramatic, whereas the activation of p38 MAPK with or without rottlerin. Similarly, induction at the protein level is much less, indicating that there the activation of ERK1/2 was not affected by the presence of may be a level of regulation after transcription that is missing rottlerin (data not shown). Because the activation of p38 MAPK from these two cell lines. is a much slower process than the activation of PKC (30 min The induction of Prx I expression by arsenate is associated versus 2 h), it is unlikely that p38 MAPK works upstream of with the activation of multiple MAPK pathways, including the PKC. These data suggest that the activation of PKC and p38 ERK, JNK, and p38 pathways. We find that p38 activation but MAPK was by parallel and independent pathways. not ERK is required for Prx I expression. The role for p38 12422 Signaling Pathways Controlling Prx I Induction by Arsenate MAPK seems to act mainly at the post-transcriptional level, transcriptional regulation, and PKC is involved in the tran- because the inhibition of p38 MAPK only slightly suppressed scriptional activation (Fig. 8). the mRNA level of Prx I while almost completely blocking the Acknowledgments—We thank M. Tondravi for helpful discussions increase of Prx I protein levels. An example of similar regula- and Sharon Boast, Kenia de los Santos, and Hang-In Ian for technical tion is the induction of vascular cell adhesion molecule 1 in assistance. human umbilical vein endothelial cells by tumor necrosis factor REFERENCES . The inhibition of p38 MAPK by an inhibitor, which com- pletely abolished the tumor necrosis factor -induced vascular 1. Halliwell, B., and Gutteridge, J. M. (1990) Methods Enzymol. 186, 1– 85 2. Beckman, K. B., and Ames, B. N. (1998) Physiol. Rev. 78, 547–581 cell adhesion molecule 1 expression did not affect the accumu- 3. Garrett, I. R., Boyce, B. F., Oreffo, R. O., Bonewald, L., Poser, J., and Mundy, lation of vascular cell adhesion molecule 1 mRNA. In the same G. R. (1990) J. Clin. Invest. 85, 632– 639 4. Key, L. L., Jr., Wolf, W. C., Gundberg, C. M., and Ries, W. L. (1994) Bone (NY) cells, the induction of intercellular adhesion molecule 1 15, 431– 436 (ICAM-1) by tumor necrosis factor  was not affected by the 5. Lee, Y. 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Pathways of Induction of Peroxiredoxin I Expression in Osteoblasts

Journal of Biological ChemistryApr 1, 2002

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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 14, Issue of April 5, pp. 12418 –12422, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. ROLES OF p38 MITOGEN-ACTIVATED PROTEIN KINASE AND PROTEIN KINASE C* Received for publication, November 30, 2001, and in revised form, December 17, 2001 Published, JBC Papers in Press, January 16, 2002, DOI 10.1074/jbc.M111443200 Baojie Li‡§ , Tetsuro Ishii , Choon Ping Tan§, Jae-Won Soh‡, and Stephen P. Goff‡**‡‡ From the ‡Department of Biochemistry and Molecular Biophysics, **Howard Hughes Medical Institute, Columbia University, College of Physicians and Surgeons, New York, New York 10032, the §Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore 117609, Singapore, and the Department of Biochemistry, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8575 Japan poly(ADP-ribosylation), depletion of cellular energy stores, and Peroxiredoxin I (Prx I) is an oxidative stress-inducible antioxidant protein with thioredoxin peroxidase activ- cell death. Lower concentration of oxidants causes apoptosis in ity. Here we report that the levels of Prx I mRNA and some cell lines, whereas high concentration usually causes protein are dramatically increased in a murine osteo- necrosis (1, 2). In addition, accumulating evidence suggests blast cell line, MC3T3-E1, by treatment with sodium ar- that reactive oxygen species such as H O play important roles 2 2 senate. We further studied the signaling pathways that in cell signaling in non-phagocytic cells. For example, platelet- control the induction of Prx I expression. The treatment derived growth factor stimulation results in the generation of of osteoblasts with arsenate activated ERK1/2, JNK, and H O , which is required for platelet-derived growth factor- 2 2 p38 MAPK. Pre-treating cells with inhibitors of p38 1 induced mitogen-activated protein kinase (MAPK) activation, MAPK abolished the induction of Prx I protein but had STAT activation, and DNA synthesis (6, 7). minimal effect on the induction of Prx I mRNA, suggest- In response to oxidants, cells up-regulate the expression of ing that p38 MAPK activity was required for post-tran- several protective products. One such protein is peroxiredoxin scriptional regulation. The inhibition of ERK1 and I (Prx I), originally identified as a 23-kDa stress-inducible ERK2 had no effect on the induction of Prx I expression. protein termed MSP23 in mouse macrophages (8) and also Furthermore, rottlerin, an inhibitor of protein kinase C known as a protein OSF-3, up-regulated during the differenti- (PKC) and calmodulin kinase III, abrogated the up- ation of an osteoblast cell line, MC3T3-E1(9). The human coun- regulation at both protein and mRNA levels. Staurospo- terpart was cloned from human breast epithelial cell line 100 rine and Go6983, inhibitors for PKC, also inhibited the (HBL100) cells and was named PAG (proliferation associated induction of Prx I, suggesting that protein kinase C is gene), because it is expressed at a higher level in transformed required for the induction by arsenate. PKC was acti- cells (10). The rat counterpart was cloned as a heme-binding vated by arsenate treatment by in vitro kinase assays. protein (HBP23) in rat liver cytosol (11). Prx family proteins The inhibition of PKC by rottlerin did not affect the activation of p38 MAPK by arsenate. These results sug- have conserved cysteine residues that are important for thiore- gest that there are two separate signaling pathways in- doxin peroxidase activity (12) and thiol radical scavenging (13). volved in the up-regulation of Prx I protein in response Recently, Prx I has also been implicated in signaling pathways. to arsenate, PKC required for transcriptional activa- It was found that human Prx I (Pag) interacts with c-Abl, a tion and p38 MAPK required for post-transcriptional non-receptor tyrosine kinase, and that overexpressed Prx I can regulation. negatively regulate the kinase activity and cytostatic activity of c-Abl (14). Prx I is up-regulated by stress agents including H O , di- 2 2 Oxidative stress is believed to contribute to the etiology of ethyl maleate, and sodium arsenite in macrophages (15). Arse- various degenerative diseases such as cerebellar ischemia, can- nate is also a potent inducer of Prx I in a human mammary cell cer, and the process of aging (reviewed in Refs. 1 and 2). line (16). The induction of the prx I gene by electrophilic agents Oxidants including superoxides and H O are generated under 2 2 and reactive oxygen species is mainly controlled by the tran- physiological conditions during mitochondrial electron trans- scription factor Nrf2 in murine peritoneal macrophages (15). In port, peroxisomal fatty acid metabolism, phagocytosis by peritoneal macrophages isolated from nrf2 knockout mice, the macrophages, and bone resorption by osteoclasts (2– 4). The induction of Prx I was diminished, and the cells were more tissue concentration of H O during inflammation can reach 2 2 susceptible to the killing effect of an electrophilic agent. The millimolar levels (5). High levels of oxidants result in DNA expression of genes encoding hemeoxygenase I and A170 was strand breaks, oxidation of lipids and proteins, activation of also diminished in the mutant macrophages. Similarly, Nrf2 is also essential for the induction of the activation of genes en- coding detoxifying enzymes such as glutathione S-transferase * The costs of publication of this article were defrayed in part by the and NAD(P)H:Quinone oxidoreductase by electrophilic agents payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to (17). Nrf2 interacts with the antioxidant responsive elements indicate this fact. and regulates the expression of those genes important for de- Recipient of Cancer Research Institute Fellowship at Columbia fense against oxidative stress. University. ‡‡ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Dept. of Biochemistry and Molec- The abbreviations used are: MAPK, mitogen-activated protein ki- ular Biophysics, HHSC1128, Columbia University, College of Physi- nase; STAT, signal transducers and activators of transcription; Prx I, cians and Surgeons, 701 W. 168th St., New York, NY 100322. Tel.: peroxiredoxin I; ERK, extracellular signal-regulated kinase; JNK, c- 212-305-3794; Fax: 212-305-8692; E-mail: goff@cuccfa.ccc.columbia. Jun N-terminal kinase; CaM, calmodulin; MARCKS, myristoylated ala- edu. nine-rich protein kinase C substrate; mRNA, messenger RNA. 12418 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Signaling Pathways Controlling Prx I Induction by Arsenate 12419 The signaling pathways regulating expression of Prx I are not clear. Agents causing oxidative stress activate many sig- naling pathways including MAPKs, protein kinase C-, and nuclear factor-kB (18, 19). Three MAPKs (ERK1/2, JNK, and p38 MAPK) are activated by high concentrations of H O . The 2 2 activation of MAPK and/or PKC plays an important role in the oxidative stress-induced expression of genes such as nitric- oxide synthase (20) and ornithine decarboxylase (21). In this report we studied the signaling pathways that are involved in the induction of Prx I in osteoblasts by sodium arsenate. We demonstrate that arsenate induced the expres- sion of Prx I in an osteoblast cell line, and that this induction required the activation of both p38 MAPK and protein kinase C. FIG.1. Induction of Prx I by sodium arsenate in MC3T3-E1 MATERIALS AND METHODS cells. A, the cells growing in logarismic phase were treated with differ- Culture of Osteoblasts—The osteoblast cell line MC3T3-E1 was cul- ent amounts of sodium arsenate for 16 h, harvested, and lysed in TNE buffer. Protein concentration was determined by Bio-Rad method, and tured in -minimum essential medium (Invitrogen) containing 10% 20 g of total protein was loaded onto 10% SDS-PAGE gel. Expression fetal calf serum (Research Sera). of Prx I was analyzed by Western blot using a polyclonal antibody Treatment of Osteoblasts by Sodium Arsenate—Osteoblasts were against Prx I. B, Northern blot analysis of induction of Prx I mRNA by subcultured the day before and then treated with sodium arsenate for arsenate. Cells were treated with 0.06 mM arsenate for different times various times. For the analysis of Prx I protein, cells were treated for and collected. Total RNA was isolated and analyzed with Northern blot. 16 h and then washed with phosphate-buffered saline, collected, and the level of Prx I was analyzed by Western blot. For the analysis of RESULTS mRNA, cells were treated 12 h. Total RNA was isolated and analyzed by Northern blot. For analysis of protein kinases, cells were treated from Sodium Arsenate Induces Expression of Prx I in Osteo- 5 min to 16 h. At each time point, cells from one plate were washed and blasts—Sodium arsenate is a toxic compound that activates the lysed. To test the effect of the kinase inhibitors SB203580, PD98059, oxidative stress pathway in several cell types. Arsenate is able and rottlerin (Calbiochem) on Prx I induction, various concentrations of to induce the expression of the Prx I at the level of protein and the compounds were added to the cell cultures 1 h prior to the addition mRNA in the human breast epithelial cell line HBL100 and of sodium arsenate. Cells were further treated in medium containing murine macrophage WAR cells (16). To test the effects of arse- both sodium arsenate and SB203580, PD98059, or rottlerin for an nate on osteoblasts, a murine osteoblast cell line, MC3T3-E1, additional 16 h. The cells were then harvested for Western and North- ern analysis. was incubated with medium containing arsenate at different Western Blot Analysis of Protein Levels—Cells were washed with concentrations, and Prx I levels were analyzed by Western blot. phosphate-buffered saline and lysed in a buffer containing 50 mM Tris, Two protein species of 23 and 24 kDa in size were detected with pH 7.5, 100 mM KCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM phenyl- the antiserum (Fig. 1A). The main 23-kDa band corresponds to methylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM Prx I, and the minor 24-kDa band probably corresponds to Prx -glycerophosphate, and 10 g/ml each of aprotinin and leupeptin. III, a family protein localizing in the mitochondrial matrix. Prx Protein concentrations were determined by Bio-Rad method. The same I was strongly induced in MC3T3-E1 cells in a dose-dependent amount of protein (20 g) was fractionated by electrophoresis on a 10% manner with maximum protein levels detected at approxi- SDS-PAGE gel, transferred to a nitrocellulose membrane (Immobilon- p), probed with polyclonal anti-Prx I antibodies, and visualized using an mately 0.10 mM arsenate (Fig. 1A). The induction occurs at the ECL kit (Amersham Biosciences, Inc.). Anti-phosphorylated ERKs, p38 level of transcription, because Northern blot analysis showed MAPK, JNK, and myristoylated alanine-rich protein kinase C substrate that the levels of Prx I mRNA are dramatically increased (Fig. (MARCKS) antibodies were purchased from Cell Signaling. Anti-p38 1B). The induction was detected at 4 h after the addition of antibody was purchased from Santa Cruz Biotechnology. arsenate and reached the maximal level after 8 h. The increase Northern Blot Analysis—1  10 osteoblasts were plated onto of both mRNA and protein was completely blocked by treating 100-mm plates and treated the second day with sodium arsenate. Cells the cells with RNA polymerase II inhibitor, actinomycin D were collected, and total RNA was isolated using RNAzol (TelTest), (data not shown). The decrease in the expression of Prx I at the fractionated on a 1.5% formaldehyde-agarose gel, transferred to Nytran higher concentration of sodium arsenate may be caused by cell membrane (S & S), and probed with random primer-labeled Prx I cDNA. death. Kinase Assay for PKC—Cells were challenged with arsenate for Arsenate Activates MAPKs—Oxidative stress triggered by different times and then lysed with phosphorylation lysis buffer (50 mM H O treatment has been found to activate MAP kinase path- 2 2 HEPES, 150 mM NaCl, 200 M sodium orthovanadate, 10 mM sodium ways including ERKs, JNK, and p38 MAPK, and arsenate itself pyrophosphate, 100 mM sodium fluoride, 1 mM EDTA, 1.5 mM magne- has been shown to activate JNK in human embryonic kidney sium chloride, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 293 cells (22). To test whether sodium arsenate activates these 10 g of aprotinin/ml). Cell lysate was separated to cytosolic and par- pathways in osteoblasts, MC3T3-E1 cells were exposed to so- ticulate fractions by centrifugation for 30 min at 100,000  g, and the dium arsenate in the culture medium. The cells were harvested particulate was dissolved in the same buffer plus Triton X-100. The and lysed after various times of treatment, and the levels of particulate fractions were immunoprecipitated with an antibody against PKC using protein G-Sepharose (Amersham Biosciences, Inc.). active kinases were analyzed by Western blot. Antibodies that The immunocomplexes were washed three times with phosphorylation specifically recognize the activated forms of ERKs, JNK, and lysis buffer and two times with kinase buffer (25 mM Tris-HCl, pH 7.4, p38 MAPK were used to determine the kinase activation (Fig. 5mM MgCl , 0.5 mM EGTA, 1 mM dithiothreitol, 20 g of phosphati- 2A). The level of activated ERKs, rapidly increased to the dylserine, 20 M ATP) and were resuspended in 30 l of kinase buffer maximum level within 10 min, started to decline within 30 min containing 5 g of histone H1, and 20 Ci of [ P]ATP was added. The and then stayed at a level slightly higher than the basal level reaction was incubated for 30 min at room temperature and was ter- for a few hours. JNK was also activated within 10 min and minated by the addition of SDS sample buffer. Proteins were analyzed declined after approximately 30 min, although the activation by SDS-PAGE, and the phosphorylated form of histone H1 was detected by autoradiography. was much weaker. Osmotic stress was found to activate JNK to 12420 Signaling Pathways Controlling Prx I Induction by Arsenate FIG.3. A, ERKs are not required for Prx I induction by sodium arsenate. MC3T3-E1 cells were pretreated with different amounts of PD98059 for 1 h, and sodium arsenate was added to a final concentra- tion of 0.06 mM. Cells were further incubated for 16 h, and expression of FIG.2. A, activation of ERKS, JNK, and p38 MAPK in MC3T3-E1 Prx I was analyzed as described in Fig. 1. B, inhibition of ERKs by cells by arsenate. Equal numbers of osteoblasts from the same passages PD98059. Cells were pretreated with different concentrations of arse- were plated and treated with 0.06 mM sodium arsenate for different nate for 1 h, and arsenate was added. After 10 min, cells were collected, times. Cells were then harvested and lysed. 60 g of total proteins were and activation of ERKs was analyzed as described in Fig. 2. analyzed by Western blot using antibodies that specifically recognize activated ERKs, JNK, and p38 MAPK, respectively. The same blot was stripped and reprobed with different antibodies. B, activation of JNK by osmotic stress. Cells were treated with 500 mM sorbitol for different times just before collection. Activation of JNK was analyzed with West- ern blot using anti-phosphorylated JNK antibodies. a much higher extent (Fig. 2B), indicating that arsenate is not a strong activator of the JNK pathway under these settings. In contrast to the rapid initial increase in ERK and JNK activa- tion, p38 MAPK showed no rapid increase but was activated only in a slow time course. The levels of active p38 MAPK gradually increased to a maximum (Fig. 2A) even after 16 h of treatment (data not shown). The level of p38 MAPK protein itself as monitored by Western blot did not change upon sodium FIG.4. A, inhibition of p38 MAPK suppresses the induction of Prx I arsenate treatment. Thus, the three kinases examined in these protein by arsenate. Experiments were carried out as described in Fig. experiments showed distinct patterns of induction. 3A. B, Northern blot analysis showing that Prx I mRNA was not affected by the inhibition of p38 MAPK. MC3T3-E1 cells were treated as Inhibition of p38 MAPK Abolishes Induction of Prx I Pro- in A, and total RNA was isolated. 20 g of total RNA was fractionated tein—To determine whether or not the MAPK pathways were on a 1.2% formaldehyde-agarose gel, and the mRNA level of Prx I was important for the induction of Prx I by arsenate, cells were analyzed by Northern blot using a random primer-labeled probe. 18 S pretreated with kinase inhibitors for 1 h before adding sodium ribosomal RNA was used as loading control. arsenate. PD98059, which specifically inhibits the ERKs, had no effect on the induction of Prx I even at a concentration of 40 M (Fig. 3A), suggesting that the induction of Prx I did not controlling the induction of Prx I by arsenate, we used an require the ERK pathways. Fig. 3B shows that PD98059 inhib- inhibitor specific for PKC, rottlerin (23). Rottlerin also in- ited the activation of ERKs by arsenate in a dosage-dependent hibits CaM kinase III, but the only known substrate for CaM manner. Even at 5 M, the level of activated ERKs was reduced kinase III is elongation factor 2, and neither elongation factor to the basal level. In contrast, SB203580, a specific inhibitor for 2 nor CaM kinase III has been implicated in oxidative stress p38 MAPK, potently inhibited the induction of Prx I in a responses. Osteoblasts were pretreated with different con- dose-dependent manner (Fig. 4A). At 5 M concentration, centrations of rottlerin for 1 h, and then 0.06 mM arsenate SB203580 dramatically reduced the levels of Prx I, and at 30 was added. After 16 h, cells were collected, and the Prx I M, the amount of Prx I protein was reduced to basal levels protein levels were determined by Western blot analysis. Fig. indicating a complete inhibition. This is the range of concen- 5A shows that 5 M rottlerin completely blocked the induc- tration that is effective for the inhibition of p38 in a large tion of Prx I protein. To exclude a role for CaM kinase III in number of tested cell lines. A second specific inhibitor for p38 the induction, two other PKC inhibitors, staurosporine and MAPK, SB202190, could also block the induction of Prx I at a Go6983, were similarly tested. Both were able to inhibit Prx concentration of 2 M (data not shown). To determine whether I induction. Staurosporine, a general PKC inhibitor, inhib- the inhibition occurred at the level of transcription, Northern ited the induction of Prx I in a dosage-dependent manner, blot analysis was carried out. The levels of Prx I mRNA were and at 100 nM, the inhibition was close to complete. Because only slightly reduced by SB203580 compared with the levels of rottlerin is a drug that only affects PKC of the protein protein (Fig. 4B). These results suggest that the inhibition of kinase C family, these results taken together suggest that Prx I induction by the drug may involve both transcriptional PKC is required for the induction of Prx I by arsenate and post-transcriptional mechanisms, but that the major effect treatment. Northern blot analysis (Fig. 5C) shows that the is post-transcriptional. inhibition occurred also at the level of mRNA. These data Inhibition of PKC Prevents Induction of Prx I—To deter- suggest that the activation of a PKC kinase activity is nec- mine whether PKC plays a role in the signaling pathways essary for the up-regulation of the Prx I mRNA in response to Signaling Pathways Controlling Prx I Induction by Arsenate 12421 FIG.6. A, activation of protein kinase C by arsenate. B, inhibition of PKC by rottlerin. Treatment osteoblasts with arsenate resulted in the phosphorylation of MARCKS, and the phosphorylation was inhibited by pretreatment of cells with rottlerin for 1 h. Phosphorylated MARCKS was detected by Western blot analysis using polyclonal antibodies against phosphorylated MARCKS. FIG.7. Activation of p38 MAPK by arsenate was not affected by FIG.5. A, rottlerin suppresses the induction of Prx I at the level of the inhibition of PKC. Osteoblasts were pretreated with 5 M rott- protein. A different concentration of rottlerin was used to pretreat lerin for 1 h, and then arsenate was added to the cultures. At different osteoblasts for 1 h, and then arsenate was added to a final concentration time points, a plate was washed, cells were collected, and activated p38 of 0.06 mM for 16 h. The levels of Prx I protein were analyzed by MAPK was analyzed by Western blot as described in Fig. 2. Western blot as described in Fig. 1. B, staurosporine and Go6983 inhibit the induction of Prx I. C, rottlerin inhibits the expression of Prx I mRNA. arsenate treatment. On the other hand, wortmannin, a spe- cific inhibitor of phosphatidylinositol 3-kinase, has no effect on Prx I induction (data not shown). To test whether arsenate activates PKC in osteoblasts, cells were treated with arsenate and were collected at different time points, PKC was immunoprecipitated with a monoclonal an- tibody, and an in vitro kinase assay was performed using H1 histone protein as substrate. Fig. 6A shows that arsenate rap- idly activates PKC. Within 30 min, PKC activation reached the maximal level, and the kinase activity remained high even 4 h after stimulation. Furthermore, arsenate treatment also led to the phosphorylation of a PKC substrate, MARCKS, and FIG.8. Signaling pathways involved in the induction of Prx I the phosphorylation was inhibited by rottlerin in a dosage-de- by arsenate in osteoblasts. pendent manner (Fig. 6B) as was the inhibition of Prx I induc- tion. These data support the conclusion that the activation of DISCUSSION PKC is required for Prx I induction. Activation of p38 MAPK Is Not Affected by Rottlerin—The The experiments presented above show that arsenate causes experiments presented above suggest that the induction of Prx a dramatic increase in Prx I protein and mRNA in a murine I by arsenate treatment required the activation of both p38 osteoblast cell line, MC3T3-E1. Osteoblasts may be exposed to MAPK and protein kinase . To determine whether the activa- significant levels of oxidants generated by osteoclasts during tion of PKC was upstream of p38 MAPK in response to arse- bone remodeling (24) and may have a particular need for a nate treatment, we pretreated osteoblasts for 1 h with 5 M strong response to oxidative stress. In fact, oxidative stress has rottlerin, a concentration sufficient to block the induction of also been found to induce the expression of genes such as Hic5 Prx I. We then added 0.06 mM arsenate, and at different time (hydrogen peroxide inducible clone 5), c-jun,c-fos, and Erg-1 in points, we collected cells and made cell lysates. The activation MC3T3-E1 (reviewed in Ref. 25). There is some difference of p38 MAPK was determined by Western blot analysis using between osteoblasts and other cell lines in terms of Prx I the antibody that specifically recognizes activated p38 MAPK induction by arsenate. In WAR and HBL100 cells, the induc- (Fig. 7). We did not detect any significant difference in the tion of Prx I mRNA by arsenate is dramatic, whereas the activation of p38 MAPK with or without rottlerin. Similarly, induction at the protein level is much less, indicating that there the activation of ERK1/2 was not affected by the presence of may be a level of regulation after transcription that is missing rottlerin (data not shown). Because the activation of p38 MAPK from these two cell lines. is a much slower process than the activation of PKC (30 min The induction of Prx I expression by arsenate is associated versus 2 h), it is unlikely that p38 MAPK works upstream of with the activation of multiple MAPK pathways, including the PKC. These data suggest that the activation of PKC and p38 ERK, JNK, and p38 pathways. We find that p38 activation but MAPK was by parallel and independent pathways. not ERK is required for Prx I expression. The role for p38 12422 Signaling Pathways Controlling Prx I Induction by Arsenate MAPK seems to act mainly at the post-transcriptional level, transcriptional regulation, and PKC is involved in the tran- because the inhibition of p38 MAPK only slightly suppressed scriptional activation (Fig. 8). the mRNA level of Prx I while almost completely blocking the Acknowledgments—We thank M. Tondravi for helpful discussions increase of Prx I protein levels. 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