©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
p21 as a Common Signaling Target of Reactive Free Radicals and Cellular Redox Stress (*)

(Received for publication, April 24, 1995; and in revised form, June 26, 1995)

Harry M. Lander (1)(§) Jason S. Ogiste (1) Kenneth K. Teng (2) Abraham Novogrodsky (3)

From the  (1)Department of Biochemistry and (2)Medicine, Cornell University Medical College, New York, New York 10021 and the (3)Felsenstein Medical Research Center, Beilinson Campus, Petah-Tikva 49100, Israel and Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Reactive free radicals have been implicated in mediating signal transduction by a variety of stimuli. We have investigated the role of p21 in mediating free radical signaling. Our studies revealed that signaling by oxidative agents which modulate cellular redox status, such as H(2)O(2), hemin, Hg, and nitric oxide was prevented in cells in which p21 activity was blocked either through expression of a dominant negative mutant or by treating with a farnesyltransferase inhibitor, as assessed by NF-kappaB binding activity. Furthermore, the NF-kappaB response to these oxidative stress stimuli was found to be enhanced when cells from the human T cell line, Jurkat, were pretreated with L-buthionine-(S,R)-sulfoximine, an inhibitor of glutathione synthesis. We directly assayed p21 and mitogen-activated protein kinase activities in Jurkat cells and found both of these signaling molecules to be activated in cells treated with the redox modulating agents. Blocking glutathione synthesis made cells 10- to 100-fold more sensitive to these agents. Finally, using recombinant p21in vitro, we found that redox modulators directly promoted guanine nucleotide exchange on p21. This study suggests that direct activation of p21 may be a central mechanism by which a variety of redox stress stimuli transmit their signal to the nucleus.


INTRODUCTION

Free radicals have been shown to play important roles in carcinogenesis by directly damaging DNA and acting as tumor promoters (1, 2, 3, 4) . Free radicals and redox stress are now thought to participate in cellular signaling(5, 6, 7, 8, 9) , and, thus, additional targets may exist. The transcription factor NF-kappaB has been demonstrated to mediate signaling by reactive oxygen (5) and reactive nitrogen(10) . The exact target of these species is unknown, although it has been postulated to be upstream of p21 and involve tyrosine phosphorylation(8, 9, 11) . We have previously identified G proteins(12) , and particularly p21(13) , as central targets by which nitric oxide transmits signals. Therefore, we explored whether p21 is a more general target for reactive free radicals and senses cellular redox status.


EXPERIMENTAL PROCEDURES

Materials

The farnesyltransferase inhibitor, alpha-hydroxyfarnesylphosphonic acid, was obtained from Biomol (Plymouth Meeting, PA), and L-buthionine-(S,R)-sulfoximine, H(2)O(2), phorbol 12-myristate 13-acetate, hemin, sodium nitroprusside, and HgCl(2) from Sigma. Purified, recombinant p21 was generously provided by Dr. Daniel Manor, Dept. of Pharmacology, Cornell University.

Cell Culture and Treatment

The rat pheochromocytoma PC12 cell lines were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 5% horse serum. The p21 mutant PC12 line (14) was generously provided by Dr. Geoffery Cooper, Dana Farber Cancer Institute, Boston, MA. The Jurkat line was maintained in RPMI 1640 containing 10% fetal bovine serum. Cells treated with inhibitors were resuspended in serum-free medium containing alpha-hydroxyfarnesylphosphonic acid (10 µM) or L-buthionine-(S,R)-sulfoximine (100 µg/ml) 24 h prior to redox stress.

NF-kappaB Electromobility Shift Assay

Nuclei were isolated, mixed, and assayed using the P-labeled NF-kappaB consensus sequence exactly as we described previously(10) . Cells treated with H(2)O(2) were also treated with phorbol 12-myristate 13-acetate (100 ng/ml), which has been demonstrated to synergize with H(2)O(2) and thus yield detectable amounts of NF-kappaB binding activity(15) . To confirm the specificity of the assay, positive samples were incubated with 100-fold excess unlabeled NF-kappaB-specific DNA as well as 100-fold excess unlabeled nonspecific DNA. In all cases, the NF-kappaB-specific DNA effectively competed with the sample, whereas the nonspecific DNA had no effect.

Quantitation of p21-associated Nucleotides

The assay used to measure the GTP/GDP ratio on immunoprecipitated p21 was essentially that of Downward et al.(16) with minor modifications(13) .

MAP^1 Kinase Assay

Serum-starved cells (24 h, 5 10^6) were treated for 30 min at 37 °C, pelleted, and then resuspended in 300 µl of RIPA buffer containing 20 mM Tris, pH 7.4, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 25 mM beta-glycerophosphate, 1 mM NaVO(3), 2 mM NaPP(i), 20% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin. Samples were vortexed, left on ice for 15 min, and then microfuged for 2 min. Protein A-Sepharose prebound to anti-ERK1 or ERK2 (Sant Cruz Biotechnology) was added to supernatants (5 µg/sample). After 1 h at 4 °C, samples were washed twice with RIPA buffer and twice with kinase buffer (25 mM Hepes, pH 7.4, 25 mM beta-glycerophosphate, 25 mM MgCl(2), 2 mM dithiothreitol, and 0.1 mM NaVO(3)). After the final wash, samples were resuspended in 20 µl of kinase buffer, and 1 µg of myelin basic protein was added along with 22 µl of 10 µCi/nmol [-P]ATP. After 20 min at 30 °C, 4 µl of 6 Laemmli sample buffer containing 100 mM dithiothreitol was added, and samples were boiled for 2 min. Samples were run on 15% SDS-polyacrylamide gel electrophoresis gels and were subjected to PhosphorImager analysis (Molecular Dynamics).

GTPase Assay

The GTPase assay was performed on GDP-preloaded p21 exactly as we previously described(13) .


RESULTS AND DISCUSSION

Requirement of p21for Free Radical and Redox Signaling

We investigated the role of p21 in mediating NF-kappaB transcription factor activation by reactive free radicals and redox modulators. We treated either wild type rat pheochromocytoma PC12 cells or PC12 cells expressing a dominant negative mutation in p21(14) with Hg, hemin, or H(2)O(2). We found that wild type cells responded by activating NF-kappaB after 4 h of treatment, whereas p21 defective cells showed a greatly diminished or no response (Fig. 1A). Furthermore, blocking p21 localization, and therefore function(17) , by inhibiting farnesyltransferase activity with alpha-hydroxyfarnesylphosphonic acid (10 µM)(18) , led to a 50% reduction of the NF-kappaB response to Hg and sodium nitroprusside in the human Jurkat T cell line (Fig. 1B). These data suggest that p21 activity is essential for mediating NF-kappaB activation by reactive free radicals and redox modulators.


Figure 1: Effect of p21 inhibition on free radical signaling. A, wild type (wt) PC12 cells or cells harboring a dominant negative mutation in p21 (mt) were treated with the indicated concentrations of drugs for 4 h prior to isolation of nuclei. Cells treated with H(2)O(2) were also co-treated with phorbol 12-myristate 13-acetate (PMA, 100 ng/ml). B, Jurkat T cells were untreated (-FT inh) or treated with alpha-hydroxyfarnesylphosphonic acid (+FT inh, 10 µM) for 24 h prior to addition of the indicated drugs. After a 4-h drug treatment, nuclei were isolated and assayed for NF-kappaB binding activity. Bands were quantified via PhosphorImager analysis, and their relative counts/min are indicated (Rel. CPM). Arrow denotes migration of the NF-kappaB-DNA protein complex.



Our previous work implicated G proteins, and particularly p21, as targets of the reactive nitrogen species, nitric oxide(12, 13) . Therefore, we directly assessed the activation state of p21 upon exposure of Jurkat cells to free radicals and redox modulators. We found that treatment of cells with H(2)O(2), Hg, or hemin led to recovery of an activated form of p21 as evidenced by an increase in GTP-bound p21 (Fig. 2). We then treated Jurkat cells for 24 h with L-buthionine-(S,R)-sulfoximine, a specific inhibitor of -glutamylcysteine synthetase(19) , the rate-limiting enzyme in glutathione synthesis, and thus depleted cells of this critical antioxidant. We found that depletion of intracellular glutathione with L-buthionine-(S,R)-sulfoximine made cells 10- to 100-fold more sensitive to these agents (Fig. 2). Treatment of Jurkat cells with L-buthionine-(S,R)-sulfoximine (100 µg/ml) yields cells with less than 20% of their original intracellular glutathione levels(8) . We next examined whether a known downstream effector of p21 was also activated by redox stress. We found that MAP kinase immunoprecipitated from cells treated with hemin or H(2)O(2) for 30 min had an enhanced ability to phosphorylate myelin basic protein (Fig. 3). Furthermore, pretreatment of cells with L-buthionine-(S,R)-sulfoximine greatly enhanced their MAP kinase activity (Fig. 3). The close correlation between p21 and MAP kinase activation in response to oxidative stimuli suggest that this pathway is indeed important in transmitting redox stress signals. Furthermore, downstream signaling in response to Hg and sodium nitroprusside, a nitric oxide-generating compound, was profoundly enhanced by L-buthionine-(S,R)-sulfoximine treatment, as assessed by the NF-kappaB binding assay (Fig. 4). Therefore, depletion of intracellular glutathione resulted in enhanced free radical signaling through p21, MAP kinase, and NF-kappaB. The disparity in the dose-response relationship between the ability of Hg and hemin to activate p21 and NF-kappaB may lie in the chemical nature of these reagents. Hemin is a known free radical generator which can catalytically generate oxygen-free radicals, whereas Hg, a thiol-specific reagent, will stoichiometrically bind thiols. Therefore, differences seen in dose-response relationships between cell types may likely reflect their differential intracellular thiol content. These data suggest that the p21 pathway acts to transmit cellular redox stress signals to the nucleus.


Figure 2: Effect of cellular redox stress on endogenous p21 activity. Jurkat T cells, labeled with PO(4) in phosphate-free media, were treated with the indicated concentrations of drugs for 10 min prior to lysis, immunoprecipitation of p21, and analysis of bound guanine nucleotides. Some cells were treated with L-buthionine-(S,R)-sulfoximine (BSO) 24 h prior to addition of drugs. Data represent the average and standard deviation of 3 experiments, each assayed in duplicate.




Figure 3: Effect of free radicals on MAP kinase activity. Jurkat T cells were either untreated or treated with L-buthionine-(S,R)-sulfoximine (BSO) 24 h prior to addition of drugs. Cells were treated with drugs for 30 min, and then their cytosol was analyzed for MAP kinase activity in an in vitro kinase assay. Data represent the average and standard deviation of 3 experiments.




Figure 4: Effect of glutathione depletion on free radical signaling. Jurkat T cells were either untreated or treated with L-buthionine-(S,R)-sulfoximine (BSO) 24 h prior to addition of drugs. Cells were treated with drugs for 4 h, and then their nuclei were analyzed for NF-kappaB binding activity. SNP, sodium nitroprusside (a nitric oxide-generating compound). Arrow denotes migration of the NF-kappaB-DNA protein complex.



In Vitro Studies

We have previously demonstrated that NO could directly activate p21in vitro by interacting with a critical Cys residue(13) . Therefore, we examined whether H(2)O(2), hemin, and Hg could directly induce guanine nucleotide exchange on pure, recombinant p21. We found that p21, which was preloaded with GDP, had an enhanced ability to hydrolyze [-P]GTP in the presence of these agents (Fig. 5). These data suggest that p21 is a direct target of reactive free radicals and thus may be responsible for sensing cellular redox status.


Figure 5: Effect of free radicals on p21in vitro. p21 (1 µM) was preloaded with GDP and then mixed with [-P]GTP and the indicated concentration of drug for 10 min. Hydrolyzed PO(4) was quantified as described under ``Experimental Procedures.'' Control GTPase rates were 27.3 ± 2.1 fmol/min/mg. Data represent the average and standard deviation of 3 experiments, each assayed in duplicate.



We have previously reported the mitogenic properties of iron compounds such as hemin on lymphocytes and implicated free radical generation as a mechanism of activation(20, 21) . HgCl(2), a thiol-binding agent, was also shown to be mitogenic to lymphocytes(22, 23) . The exact target of free radicals responsible for initiating the mitogenic event was unknown. Furthermore, the oncogenicity of free radicals is known to be mediated through both physical damage to DNA and their properties as tumor promoters(1, 2, 3, 4) . The recent identification of free radicals as second messengers (5, 6, 7, 8, 9) suggests that some of the pathophysiological consequences of free radical generation may be due to effects on the enzymes controlling signaling pathways. Our data suggest that free radicals can activate p21 and lead to a nuclear signal. Mutated and activated forms of p21 have been found in many human cancers (24) and, thus, it is possible that the apparent sensitivity of p21 to free radicals and redox status, which we have identified here, provide an additional mechanism by which free radicals promote oncogenesis.

A common mechanism by which cells respond to redox signals and initiate gene expression has been postulated(9, 11, 15) , although the precise target remains unknown. Our data indicate that, under normal conditions, p21 may represent the cellular sensor for redox stress. Studies are currently underway to identify the exact molecular alteration on p21 generated by reactive free radicals.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI37637 (to H. M. L.) and the American Health Assistance Foundation (to K. K. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Cornell University Medical College, New York, NY 10021. Tel.: 212-746-6462; Fax: 212-746-8789.

(^1)
The abbreviation used is: MAP, mitogen-activated protein kinase.


REFERENCES

  1. Ames, B. N., Shigenaga, M. K., and Gold, L. S. (1993) Environ. Health Perspect. 101,35-44 [Medline] [Order article via Infotrieve]
  2. Halliwell, B., and Gutteridge, J. M. C. (1989) Free Radicals in Biology and Medicine , 2nd Ed, Oxford University Press, New York
  3. Adelson, R., Saul, R. L., and Ames, B. N. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,2706-2710 [Abstract]
  4. Muehlmatter, D., Larsson, R., and Cerutti, P. (1988) Carcinogenesis 9,239-247 [Abstract]
  5. Schreck, R., Rieber, P., and Baeuerle, P. A. (1991) EMBO J. 10,2247-2258 [Abstract]
  6. Yan, S. D., Schmidt, A. M., Anderson, G. M., Zhang, J., Brett, J., Zou, Y. S., Pinsky, D., and Stern, D. (1994) J. Biol. Chem. 269,9889-9897 [Abstract/Free Full Text]
  7. Fialkow, L., Chan, C. K., Rotin, D., Grinstein, S., and Downey, G. P. (1994) J. Biol. Chem. 269,31234-31242 [Abstract/Free Full Text]
  8. Staal, F. J. I., Anderson, M. T., Staal, G. E., Herzenberg, L. A., and Herzenberg, L. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,3619-3622 [Abstract]
  9. Anderson, M. T., Staal, F. J. T., Gitler, C., Herzenberg, L. A., and Herzenberg, L. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,11527-11531 [Abstract/Free Full Text]
  10. Lander, H. M., Sehajpal, P., Levine, D. M., and Novogrodsky, A. (1993) J. Immunol. 150,1509-1516 [Abstract/Free Full Text]
  11. Devary, Y., Rosette, C., DiDonato, J. A., and Karin, M. (1993) Science 261,1442-1445 [Medline] [Order article via Infotrieve]
  12. Lander, H. M., Sehajpal, P. K., and Novogrodsky, A. (1993) J. Immunol. 151,7182-7187 [Abstract/Free Full Text]
  13. Lander, H. M., Ogiste, J. O., Pearce, S. F. A., Levi, R., and Novogrodsky, A. (1995) J. Biol. Chem. 270,7017-7020 [Abstract/Free Full Text]
  14. Szeberenyi, J., Cai, H., and Cooper, G. M. (1990) Mol. Cell. Biol. 10,5324-5332 [Medline] [Order article via Infotrieve]
  15. Schreck, R., and Baeuerle, P. A. (1994) Methods Enzymol. 234,151-163 [Medline] [Order article via Infotrieve]
  16. Downward, J., Graves, J. D., Warne, P. H., Rayter, S., and Cantrell, D. (1990) Nature 346,719-723 [CrossRef][Medline] [Order article via Infotrieve]
  17. Gibbs, J. B., Oliff, A., and Kohl, N. E. (1994) Cell 77,175-178 [Medline] [Order article via Infotrieve]
  18. Gibbs, J. B., Pompliano, D. L., Moser, S. D., Rands, E., Lingham, R. B., Singh, S. B., Scolnick, E. M., Kohl, N. E., and Oliff, A. (1993) J. Biol. Chem. 268,7617-7620 [Abstract/Free Full Text]
  19. Griffith, O. W., and Meister, A. (1979) J. Biol. Chem. 254,7558-7560 [Abstract]
  20. Stenzel, K. H., Rubin, A. L., and Novogrodsky, A. (1981) J. Immunol. 127,2469-2475 [Abstract/Free Full Text]
  21. Lander, H. M., Levine, D. M., and Novogrodsky, A. (1993) Biochem. J. 291,281-297 [Medline] [Order article via Infotrieve]
  22. Warner, G. L., and Lawrence, D. A. (1986) Cell Immunol. 101,425-434 [Medline] [Order article via Infotrieve]
  23. Reardon, C. L., and Lucas, D. O. (1987) Immunobiology 175,455-469 [Medline] [Order article via Infotrieve]
  24. Bos, J. L. (1989) Cancer Res. 49,4682-4689 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.