ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 348, No. 2, December 15, pp. 289–294, 1997 Article No. BB970355 NFkB-Independent Transcriptional Induction of the Human Manganous Superoxide Dismutase Gene Silvia Borrello1 and Bruce Demple2 Department of Molecular and Cellular Toxicology, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02165 Received May 14, 1997, and in revised form August 8, 1997 Numerous conditions induce expression of manganese-containing superoxide dismutase (MnSOD) in mammalian cells. The reported inducers of MnSOD are all agents that activate two transcription factors, AP1 and NFkB, but several reports have suggested that MnSOD induction relies solely on NFkB. We investigated the contribution of the individual transcription factors by using antioxidants and metal chelators to modulate MnSOD transcriptional activation in response to phorbol esters or hydrogen peroxide. The results indicate substantial transcriptional induction of the MnSOD gene independent of NFkB. The metal chelator and antioxidant pyrrolidine dithiocarbamate (PDTC) at 60 or 100 mM induced the MnSOD transcript in HeLa cells while diminishing expression of the NFkB-responsive transcript IkB-a. Induction of the MnSOD mRNA by phorbol-12-myristate-13-acetate (PMA) was only slightly diminished in the presence of PDTC, which in contrast virtually eliminated induction of the NFkB-dependent transcript IkB-a by PMA. MnSOD RNA induction by H2O2 was only Ç1.5-fold, compared to a ca. 3-fold activation of IkB-a expression. Two other antioxidants, N-acetyl-L-cysteine and butylated hydroxyanisole, failed to block induction of the MnSOD transcript by PMA, which is consistent with a role for AP-1. In vitro DNA binding studies confirmed strong AP-1 activation under conditions where NFkB is blocked but the MnSOD transcript is strongly induced (e.g., PMA treatment in the presence of PDTC). q 1997 Academic Press Manganese-containing superoxide dismutase (MnSOD)3 is a mitochondrial enzyme encoded by a nu1 Permanent address: Institute of General Pathology, Catholic University, Largo F Vito, 1 00168 Rome, Italy. 2 To whom correspondence should be addressed. Fax: (617) 4320377. E-mail: demple@mbcrr.harvard.edu. 3 Abbreviations used: MnSOD, manganese-containing superoxide dismutase; LPS, lipopolysaccharide; TNF-a, tumor necrosis factor clear gene (1, 2). MnSOD helps limit superoxide levels by catalyzing the dismutation of superoxide radical to hydrogen peroxide and molecular oxygen. MnSOD is one of a battery of antioxidant enzymes that protects cells from the deleterious effects of oxygen radicals produced as by-products of oxidative metabolism and by various environmental agents (3–5). Free radical damages exert cytotoxicity by damaging all the major cellular components (proteins, lipids, nucleic acids (3, 6, 7)). MnSOD expression in mammalian cells changes in response to a variety of conditions. MnSOD levels vary greatly between hypoxic and hyperoxic conditions (8), and following cell treatment with the superoxide-generating compound paraquat (1), cytokines (4, 9), phorbol esters (10), bacterial lipopolysaccharide (LPS) (11), okadaic acid and protein synthesis inhibitors (12), and X rays (13, 14). MnSOD expression is also regulated during development and differentiation (2, 15, 16) and during neoplastic transformation (5). Indeed, MnSOD could play a role in cancer development, as suggested by observations that levels of the enzyme are generally reduced in tumor cells compared to their normal counterparts ((5) and references therein). Moreover, oxygen radicals are proposed to be involved in tumorigenesis (17, 18), while antioxidants are proposed for prevention and treatment of cancer (19, 20). Despite the numerous reports of regulated MnSOD expression, little is known about the molecular mechanisms that govern transcription of the MnSOD gene. At present two main transcription factors have been shown to be activated following exposure to pro-oxidant factors (21–25): the transcription factor NFkB, a heterodimer bound in its inactive cytoplasmic form to the a; DMEM, Dulbecco’s modified Eagle’s medium; PDTC, pyrrolidinedithiocarbamate; PMA, phorbol 12-myristate 13-acetate; NAc, N-acetyl-cysteine; BHA, butylated hydroxyanisole; CHX, cycloheximide; DESF, desferrioxamine; SDS, sodium dodecyl sulfate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay. 289 0003-9861/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved. AID ABB 0355 / 6b44$$$501 11-12-97 08:23:25 arcal 290 BORRELLO AND DEMPLE inhibitory subunit IkB (26), and the transcription factor AP-1, consisting of homo- or heterodimers of proteins of the c-Fos and c-Jun families (27). NFkB controls the regulation of numerous acute-phase genes of the immune and inflammatory response, and AP-1 regulation has been implicated in cell growth and differentiation events (26). The two factors share various physiological activators and even some steps in the mechanisms of their activation (26). Cytokines such as TNF-a and some interleukins, physical agents (ultraviolet light and ionizing radiation), tumor-promoting agents, H2O2 , and bacterial LPS have been reported to activate the DNA-binding or the transactivation capacity of both NFkB and AP-1. These are the same conditions that induce mammalian MnSOD expression. Thus, it is possible that one or both of these redoxresponsive transcription factors is involved in MnSOD regulation. The aim of the present work was to determine experimentally which of these regulatory proteins might activate transcription of the MnSOD gene in response to different stimuli. Cellular exposure to various antioxidants strongly activates AP-1, while simultaneously blocking NFkB activation by oxidants (28). Metal chelators (e.g., dithiocarbamates or desferrioxamine) and radical scavengers (e.g., thiols) are both effective in this regard. We have used these compounds to distinguish the contribution of NFkB from that of other factors such as AP-1 to transcriptional regulation of MnSOD. cDNA probes. The cDNA encoding human MnSOD (2) was a generous gift of Dr. Daret St. Clair (University of Kentucky Medical Center, Lexington). A plasmid containing the full-length cDNA for the NFkB inhibitory subunit IkB-a was a gift of Dr. James Chen (Myogenic Laboratories, Boston, MA). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe has been used previously in this laboratory (31). Human MnSOD, IkB-a, and GAPDH cDNA probes were radiolabeled using a DNA random-primer labeling system (GIBCO-BRL). Electrophoretic mobility shift assay (EMSA). HeLa cells were incubated in 125-cm2 flasks with PDTC, PMA, H2O2 , and their combinations as indicated in the figure legends. Nuclear extracts were prepared following the method of Dignam et al. (32), modified according to Lee et al. (33). Binding reactions were performed for 20 min at 07C with 5 mg total extract protein in 10 ml of 10 mM TrisHCl, pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol, 1 mM MgCl2, 50 ng/ml poly(dI-dC) (Boehringer Mannheim) containing 104 cpm of 32P-labeled synthetic oligonucleotides with consensus sequences for AP-1 or NFkB (Promega), labeled using T4 polynucleotide kinase and [g-32P]ATP (Ç3000 ci/mmol; NEN products). The double-stranded oligonucleotide probes for AP-1 or NFkB binding were purchased from Promega Corp. (Middleton, WI) and had the sequences: AP-1 5*-CGCTTGATGAGTCAGCCGGAA-3* (TRE) 3*-GCGAACTACTCAGTCGGCCTT-5* NFkB 5*-AGTTGAGGGGACTTTCCCAGGC-3* (kB) 3*-TCAACTCCCCTGAAAGGGTCCG-5*. EMSA of DNA–protein complexes was performed in native 4.5% polyacrylamide gels in 0.51 TBE (30) at 100 V. The dried gels were exposed to film overnight at 0807C. MATERIALS AND METHODS Cell cultures and treatment. Human cervical carcinoma (HeLaS3) and human colon carcinoma (HT29) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 8 and 10% fetal calf serum, respectively. The cells were grown in 75-cm2 flasks to semiconfluence and treated with 0.02–0.1 mM pyrrolidine-dithiocarbamate (PDTC), 10 ng/ml phorbol 12-myristate 13-acetate (PMA), 0.25 mM hydrogen peroxide (H2O2), 30 mM N-acetyl-cysteine (NAC), 0.20 mM butylated hydroxyanisole (BHA), 0.04 mM cycloheximide (CHX; added from a 40 mM stock dissolved in phosphate-buffered saline (29)), 0.1 mM desferrioxamine (DESF), or 0.1 mM hemin. In the experiments in which H2O2 was used, DMEM was substituted by iron-free MEM. Where treatment by multiple agents is indicated, they were present concurrently. Growth media were purchased from GIBCO-BRL (Gaithersburg, MD) and other chemicals from the Sigma Chemical Company (St. Louis, MO). RNA extraction and Northern blotting. Total cellular RNA was isolated from guanidinium isothiocyanate-lysed cells and purified using a phenol:chloroform extraction method (29), and 10- to 20-mg samples (estimated by absorbance measurements at 260 nm) were electrophoresed in 1% agarose gels in 0.5 mM formaldehyde, 20 mM 3-[N-morpholino]-propanesulfonic acid, and 1 mM EDTA (30) followed by blotting onto positively charged nylon membranes (Boehringer Mannheim). Hybridization was performed at 427C in 10% (w/ v) dextran sulfate, 51 SSPE (30), 50% formamide, 1% SDS containing 106 cpm/ml of [a-32P]dCTP-labeled probe (see below). Blots were washed twice at 427C in 0.3 M NaCl, 0.03 M sodium citrate, pH 7.0, and 0.1% SDS, and once at 507C, 0.03 M NaCl, 0.003 sodium citrate, pH 7.0, 0.1% SDS. For reprobing, bound radioactive probe was removed from the blots by washing in boiling 0.5% SDS. Quantitation of the intensities of the bands was carried out using a scanning densitometer (Visage system; Millipore Corp., Milford, MA). AID ABB 0355 / 6b44$$$501 11-12-97 08:23:25 RESULTS We examined the expression of the MnSOD gene (SOD2) transcript in HeLa cells treated with various AP-1 and NFkB activators in the presence and absence of antioxidants. In HeLa cells treated for 5 h with the metal chelator/antioxidant PDTC alone, the MnSOD message was up-regulated up to fourfold over the range 20–100 mM PDTC (Fig. 1). We monitored the mRNA encoding the inhibitory subunit IkB-a, which is positively regulated by NFkB (34), as a measure of the NFkB status in the same cells. These measurements confirmed the inhibiting effect of dithiocarbamates such as PDTC (28) on the in vivo activity of NFkB (Fig. 1). In contrast, AP-1 has been reported to be activated by PDTC and other metal chelators (35). The addition of 0.1 mM PDTC during treatment of HeLa cells with the tumor promoter PMA only partly decreased the MnSOD mRNA induction exerted by PMA alone (Fig. 2). H2O2 , which by itself gave a weak induction of the transcript in HeLa cells, limited the MnSOD gene activation by PMA and completely prevented the induction by PDTC. As seen by others (e.g., 36), in these latter experiments, two MnSOD-specific transcripts of Ç1 and Ç4 kb were observed that usually behaved in a parallel fashion. However, H2O2 treatment had greater effects on the 4-kb than on the 1-kb arcal NFkB-INDEPENDENT INDUCTION OF HUMAN MnSOD GENE FIG. 1. The effect of PDTC on MnSOD and IkB-a gene expression in HeLa cells. Treatments consisted of 5-h incubations with the indicated concentrations of PDTC. (A) Northern blots of total RNA were hybridized with 32P-labeled DNA probes for the MnSOD, IkB-a, and GAPDH genes. Only the 1-kb MnSOD-specific RNA (indicated) was visible in this exposure. (B) Densitometric quantitation of the Northern blots, with the MnSOD transcript (upper) and IkB-a transcript (lower) normalized to the GAPDH message; the ratios in untreated cells are defined as 100. transcript in these experiments (Fig. 2); perhaps the H2O2 treatment inhibited RNA processing to some degree. Northern blot analysis of RNA from the colon carcinoma line HT29 revealed both transcripts and a similar regulatory pattern to that seen in HeLa cells, except that the HT29 cells responded more dramatically to H2O2 (Fig. 2). With a treatment time of 5 h, the amounts of the 291 MnSOD and IkB-a transcripts were quantified and normalized to the levels of the control GAPDH transcript. The results confirmed that PMA, H2O2, and, more dramatically, PMA combined with H2O2 increased the mRNA level of IkB-a, while 0.06 mM PDTC blocked induction of IkB-a expression in response to H2O2 (Fig. 3). In the same Northern blot, MnSOD mRNA was upregulated Ç2.5-fold by PDTC (0.06 mM), in contrast to the effect on IkB-a mRNA (Ç2-fold decreased). Activation of NFkB by a 5-h exposure to 0.25 mM H2O2 , indicated by the induction of the IkB-a transcript, was insufficient to induce MnSOD expression, and the H2O2 treatment actually limited MnSOD induction by PMA, while activating that of IkB-a (Fig. 3). We determined the activation of the transcription factors AP-1 and NFkB in nuclear extracts from HeLa cells by means of in vitro DNA binding assays (EMSA). Figure 4 shows that, while PMA alone increased the DNA binding activity of both NFkB and AP-1, 0.1 mM PDTC exerted a clear induction of AP-1 binding to DNA but nearly eliminated NFkB activation. When administered simultaneously with PMA, PDTC interfered with AP-1 activation and completely blocked activation of NFkB (Fig. 4). H2O2 at 0.25 mM strongly activated NFkB and augmented the effects of PMA. AP-1 was only partially activated by H2O2 (compared to PMA treatment alone), and H2O2 interfered to some degree with the ability of either PMA or PDTC to activate AP1 (Fig. 4). The effect of CHX was tested to assess whether stimulation of MnSOD transcription required new protein synthesis. Figure 5 shows that, in HeLa cells, this inhibitor increased the level of MnSOD mRNA markedly but increased IkB-a expression only slightly (relative FIG. 2. The effect of 3-h treatments with PMA, PDTC, H2O2 , PMA / H2O2 , and PDTC / H2O2 on expression of human MnSOD gene. The treatment concentrations were 10 ng/ml PMA, 0.1 mM PDTC, and 0.25 mM H2O2 . Both the 4- and the 1-kb transcript of the human MnSOD gene are evident in HeLa (left) and in HT29 (right) cells. Northern blots were rehybridized with GAPDH-radiolabeled probe to check for loading (see lower panels). AID ABB 0355 / 6b44$$$501 11-12-97 08:23:25 arcal 292 BORRELLO AND DEMPLE NFkB. Several points support this conclusion. First, MnSOD mRNA was induced in response to PDTC in two different cell types, HeLa cells (derived from a cervical carcinoma) and HT29 cells (from a colon carcinoma), and this activation was characterized by a concentration dependence up to 0.1 mM PDTC. In contrast, the activity of NFkB, monitored as the level of IkB-a transcript (34, 38), was suppressed by the PDTC concentrations that induce MnSOD mRNA. Second, the effects of H2O2, alone or in combination with PMA, were consistent with the involvement of AP-1 in MnSOD gene induction (28). Although H2O2 alone can activate both transcription factors, the relative AP-1 activation by PMA was decreased by H2O2, while PMA and H2O2 acted synergistically on NFkB. The behavior of MnSOD gene expression under these conditions was parallel to AP-1 rather than to NFkB. A similar conclusion applies to the joint effects of CHX and PDTC (Fig. 5). Third, the EMSA analysis performed in parallel to the Northern blot experiments directly confirmed the contrasting regulation of NFkB and AP-1 activities suggested by the foregoing analysis. However, although cotreatment with PDTC and PMA was reported to activate AP-1 more effectively than PMA alone (28), we found this combination to exert a weaker effect. These discrepancies might be ascribed to differences in dose and time of PMA addition. Most importantly, treatment of HeLa cells with 0.1 mM PDTC plus 10 ng/ml PMA induced FIG. 3. Relative expression of MnSOD transcript (top; sum of the 4- and 1-kb bands) and IkB-a transcript (bottom) in HeLa cells after 5-h treatments with PMA, H2O2, PDTC, PMA / H2O2, or PDTC / H2O2 . The concentrations of the agents were as described for Fig. 2, except that PDTC was used at 0.06 mM. to the GAPDH control mRNA). CHX also acted synergistically with PMA. Another antioxidant, butylated hydroxyanisole, activated MnSOD expression slightly, again without stimulating IkB-a expression, and was not blocked in its action by PDTC (Fig. 5). Taken together, these data indicate significant up-regulation of the MnSOD transcript independent of NFkB, although activated NFkB contributes to induced MnSOD expression in some circumstances. Since PDTC is a metal chelator and there are wellestablished connections between iron metabolism and oxidative stress (37), we tested whether MnSOD induction is linked to intracellular iron content. Desferrioxamine, a more specific iron chelator, increased MnSOD mRNA levels, while the iron-delivery protein hemin was without apparent effect (Fig. 6). Neither agent prevented the induction of MnSOD mRNA by PMA (Fig. 6). DISCUSSION The data reported here show that induction of MnSOD gene transcription can occur independently of AID ABB 0355 / 6b44$$$501 11-12-97 08:23:25 FIG. 4. Activation of DNA binding by NFkB and AP-1 in HeLa cells. Treatments were for 1 h with PMA, PDTC, H2O2 , and their combinations as described for Fig. 2. Nuclear extracts were prepared and EMSA was performed as described under Materials and Methods. The TRE probe contains three consensus AP-1 binding sites; the kB probe contains three consensus NFkB binding sites. arcal NFkB-INDEPENDENT INDUCTION OF HUMAN MnSOD GENE FIG. 5. Effect of CHX on induction of the MnSOD and IkB-a mRNAs. CHX (0.04 mM) and BHA (0.20 mM) were added during treatment of HeLa cells with PMA or PDTC as described for Fig. 2, followed by RNA isolation and analysis by Northern blotting sequentially with the MnSOD, IkB-a, and GAPDH probes. MnSOD mRNA (Fig. 2), while almost completely inhibiting the NFkB DNA binding activity (Fig. 4) (28). The foregoing observations do not exclude a role for NFkB in MnSOD gene regulation in some circumstances. For example, Warner et al. (36) showed induction of MnSOD mRNA in TNF-a-treated pulmonary adenocarcinoma cells that closely paralleled NFkB activation. It has also been reported (39) that NFkB and AP-1 can synergize to activate transcription in vivo through either kB or AP-1 response elements. In our experiments, both PMA and CHX activated MnSOD gene expression more strongly than any other treatment, and both agents are activators of both transcription factors. The effect of CHX also suggests that MnSOD can be up-regulated by agents without overt pro-oxidant effects. Protein synthesis inhibitors can cause superinduction of AP-1 activity by stabilization of c-Fos mRNA and elimination of autorepression of c-Fos transcription (27). CHX also activates the two main families of c-Jun kinases, the MAP and SAP kinases (40). It might appear paradoxical that an antioxidant enzyme, whose activation has classically been associated with pro-oxidant stimuli, can be induced by substances typically classed as antioxidants (e.g., thiols and metal chelators). This phenomenon could have some clinical relevance, as in ischemia–reperfusion. The ischemic condition might parallel some antioxidant effects, with AID ABB 0355 / 6b44$$$501 11-12-97 08:23:25 293 cells needing to potentiate their antioxidant defenses in order to counteract the oxidative stress that can follow during reperfusion. At the cellular level, hypoxia followed by reoxygenation may constitute a similar situation. Whether agents such as PDTC act exclusively as antioxidants is open to debate, however. Dithiocarbamates have been reported to inhibit the in vivo activity of some antioxidant enzymes (41) and to transport redox-active copper into some cell types (42). In this way, such compounds could actually generate a pro-oxidant state consistent with the expected logic of MnSOD induction. In other circumstances, metal chelation and the radical-scavenging potential of PDTC would support its activity as an antioxidant. It should be noted in this context that the Elk-1 transcription factor is proposed to respond directly to both pro- and antioxidant conditions (43). The possible linkage of cellular iron content to MnSOD gene expression is indicated by the inducing effect of desferrioxamine. Perhaps the metal-chelating capacity of PDTC acts in this way. Such a link could reflect the logic of averting oxidative damage potentiated by the iron-dependent Fenton reaction. An alternative possibility is suggested by recent observations in bacteria indicating that superoxide can damage protein iron – sulfur centers to liberate free iron (44, 45). A reciprocal relationship between cellular iron load and the redox status may also be true for mammalian cells (37). Our results showing MnSOD induction independent of NFkB contrast with several recent reports suggesting exclusive NFkB dependence (36, 46, 47). The data presented here are consistent with a substantial transcriptional contribution by AP-1 in HeLa cells treated with PMA or other activating agents. In fact, recent sequence analysis of the 5*-flanking region of the human MnSOD gene revealed only Sp1 and AP-1 consensus binding sites (48). Perhaps the potential for FIG. 6. Effect of iron status on MnSOD RNA expression in HeLa cells. HeLa cells were cultured for 24 h with 0.1 mM hemin or 0.1 mM desferrioxamine, followed by treatment with PMA (10 ng/ml) for 3 h. Total RNA was harvested and analyzed by Northern blotting with the MnSOD probe. arcal 294 BORRELLO AND DEMPLE synergistic activity of NFkB and AP-1 (39) mediates induction through these sites by activation of either transcription factor. ACKNOWLEDGMENTS We are particularly grateful to Dr. Daret K. St. Clair for her generous gift of human MnSOD cDNA and for practical advice, and to Dr. James Chen for the human IkB-a cDNA. We are indebted to our colleagues for discussions, and especially to Dr. Lynn Harrison for advice and help in using the cell lines. We thank Ms. Clotilde Castellani (Rome) and Mrs. Karen O’Connor (Boston) for preparation of the manuscript. This work was supported by NIH Grant CA37831 to B.D. S.B. was the recipient of a short-term visiting fellowship from the Fogarty Center of NIH. REFERENCES 1. Krall, J., Bagley, A. C., Mullenbach, G. I., Hallewell, A., and Lynch, R. E. (1988) J. Biol. Chem. 263, 1910–1914. 2. St. Clair, D. K., Oberley, T. D., Muse, K. 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