Tumor necrosis factor-a increases MmSOD expression: protection against oxidant injury BARBARA B. WARNER, MICHAEL S. BURHANS, JEAN C. CLARK, AND JONATHAN R. WISPE Division of Neonatology, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
WARNER, BARBARA B., MICHAEL S. BURHANS, JEAN C. CLARK, AND JONATHAN R. WISPJ?. Tumor necrosis factor-cu increases Mn-SOD expression:protection against oxidant injury. Am. J. Physiol. 260 (Lung Cell. Mol. Physiol. 4): L296-L301, 1991.-Antioxidant enzymes, including superoxide dismutase, are important for protecting the lung against 0, injury. Manganese superoxide dismutase (Mn-SOD) is a superoxide anion (0; ) scavenger located in the mitochondria, a primary site of 0,. production during hyperoxia. We studied the effects of tumor necrosis factor (TNF-cu), a macrophage-derived cytokine, on Mn-SOD expression in human pulmonary adenocarcinoma cells. TNF-cu significantly increased Mn-SOD activity and mRNA in a dose- and time-dependent manner. Mn-SOD activity was increased 3-fold and mRNA Z&fold after a 48-h incubation with TNF-cu (25 rig/ml). To examine the mechanism of this increase, cells were incubated for 48 h with TNF-(U (25 ng/ ml) with or without cycloheximide (10 PM) or actinomycin D (10 pg/ml). Actinomycin D blocked the induction of Mn-SOD mRNA by TNF- CY,but cycloheximide did not. These findings suggest that the effect of TNF-cu requires gene transcription but not synthesis of new protein intermediates. To test the hypothesis that increased Mn-SOD protects against oxidative injury, pulmonary adenocarcinoma cells were incubated in TNF-cu (25 rig/ml) for 48 h and then exposed to paraquat (PQ’), an intracellular 0, generator. Cells pretreated with TNF-(1 had significantly improved survival in PQ’ compared with controls. At the LDso (6 PM) for control cells, 95% of TNF-n-treated cells survived, 85% at the LD7, (10 PM), and 77% at the LDr,,, (14 PM). Our results suggest that the induction of Mn-SOD by TNF- (Y in pulmonary adenocarcinoma cells is pretranslationally mediated and that increasing Mn-SOD activity with TNF-cu confers protection against 0, radicals. l
Superoxide dismutases (SOD) are metalloproteins that dismutate 0,. to hydrogen peroxide (H202). In eukaryotic cells, three forms of SOD exist, a cytosolic copperzinc-SOD (CuZn-SOD), an extracellular CuZn-SOD, and a mitochondrial manganese-SOD (Mn-SOD). Tumor necrosis factor CY(TNF-cu), is a 17-kDa soluble peptide secreted by macrophages and2 x lo7 U/mg protein was purchased from Boehringer-Mannheim, Indianapolis, IN. PGEM-32 and RNA transcription kits were obtained from Promega Biotech, Madison, WI. Cycloheximide, actinomycin D, paraquat, and other chemicals were purchased from Sigma Chemical, St. Louis, MO. Cell culture methods. NIH H820 human pulmonary adenocarcinoma cells were isolated at the National Cancer Institute, Bethesda, MD, and kindly provided by Dr. A. Gazdar. These cells have biochemical and histological properties similar to type II pulmonary epithelial cells with junctional complexes, apical microvilli, and multilamellar inclusion bodies. Cells were incubated at 37°C in 95% air-5% COa, in RPMI-1640 (GIBCO) supplemented with hormones and growth factors as previously described (16). Medium was changed every 4 days, and cells routinely passed once a week. Before all experiments, cells were suspended in 5 ml of fresh medium. Gene transcription was inhibited by treating cells with actinomycin D (10 pg/ml). Transcriptional analysis demonstrated that 10 pg/ml actinomycin
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Physiological
Society
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TLJMOR
NECROSIS
FACTOR
AND
D blocked transcription of the surfactant-associated proteins. Protein synthesis was inhibited by treating cells with 10 PM cycloheximide, which inhibited [“Slmethionine incorporation by >90% in this cell line. Cells were harvested, washed vigorously, suspended in 1 ml of 5 mM K,HPO, (pH 7.8), and sonicated on ice for 45 s. SOD activity assay. SOD activity in cell lysates was measured by inhibition of nitro blue tetrazolium reduction by 0,. (22). Mn-SOD activity is discerned from CuZn-SOD by its resistance to 5 mM potassium cyanide. The protein and DNA content of cell lysate was measured as previously described (3, 13). Mn-SOD activity was expressed per milligram protein and per nanogram DNA. RNA extraction for hybridization experiments. To isolate RNA, H820 cells were sonicated in 4 M guanidine thiocyanate and centrifuged through 5.7 M cesium chloride. The RNA was dissolved in water, phenol extracted, and precipitated with ethanol (26). The amount of RNA in aqueous solution was determined by absorbance at 260 nM. For Northern blot analysis, equal amounts of total cellular RNA were electrophoresed through a 1.2% agarose-7% formaldehyde gel and transferred to Nytran (Schleicher and Schuell, Keene, NH) filters. The filters were probed with ““P-labeled antisense RNA transcribed from PGEM-3Z (Promega) into which the Mn-SOD cDNA was subcloned (26). The filters were hybridized at 60°C for 18 h, washed stringently at 60°C and exposed to XAR film at -80°C. Rehybridization with another probe was performed after washing the membrane for 2 h at 65°C in 5 mM tris(hydroxymethyl)aminomethane chloride (pH 8.0), 0.2 mM EDTA, 0.5% sodium pyrophosphate, and 0.1~ Denhart’s solution. h/In-SOD was detected as a l.O-kb message, with additional less intense bands observed at 2.0, 2.5, and 4.0 kb. Surfactant protein A (SP-A) and ,& ac t in mRNAs were detected as 2.2- and 1.8-kb species, respectively. Relative message intensity was quantitated by scanning densitometry of the autoradiograms (Helena Laboratories). Resistance to paraquat. Equal numbers of H820 cells were incubated for 48 h in the presence or absence of 25 rig/ml of TNF -cy. Paraquat was then added in concentrations varying from 2 to 20 PM. After 48 h in paraquat, 400 nM neutral red was added directly to the cultures. As described by Finter (6), only viable cells retain the dye. Two hours after addition of dye, cells were washed three times in phosphate-buffered saline (pH 7.4), centrifuged, and lysed with 1:195% ethanol-100 mM sodium citrate (pH 4.2) and absorbance at 540 nm was measured. Statistics. Comparisons between groups were made with the Student’s t test and one-way analysis of variance. RESULTS
Effect of TNF-a on SOD actiuity. TNF-a significantly increased Mn-SOD activity in human pulmonary adenocarcinoma cells (Table 1). Mn-SOD activity was increased 24 h after addition of TNF-cu, with a further increase after 48 h. TNF-cu increased Mn-SOD activity at the lowest dose of 0.1 rig/ml, an effect sustained through 100 rig/ml. In contrast to the stimulatory effect
MN-S1_JPER,OXIDE
TABLE TNF-tu, ng/rnl
Control 0.1
1. Effect of TNF-cu on SOD activity 24 h
48 h
n
Mn-SOD
CuZn-SOD
n
Mn-SOD
7
18k6
40t21
15 3
16t5 :‘32&17” 62t37* 41t25* 44t13” 118t61*
1.0 10 25 100
L297
DISMUTASE
3 4 3
32t 10* 76t7*
30t9 20t9
6 12 7
CuZn-SOI)
50535 175t7 32kll
49t23 51t42 37k24
Values are means t SD expressed as U activit.y/mg protein; n, no. of separate experiments for each concentration of’ TNF-tu. Cells were incubated for 24 or 48 h with 0.1-100 r-g/ml TNF-tu. SOD activity was assayed by the nitro blue tetrazolium method. * Significantly diff’erent from control, 1’ < 0.05.
of TNF-a on Mn-SOD, TNF-a had no effect on CuZnSOD activity. Normalization of Mn-SOD activity to DNA content rather than protein did not alter activity results. Cellular protein content was not affected by TNF-cu (data not shown). The effect of TNF -cy on Mn-SOD activity was not unique to H820 cells. TNF-ti also increased Mn-SOD activity in the human pulmonary adenocarcinoma cell line A549. Control cells contained 161 t 9 U Mn-SOD activity/mg protein, while cells treated with TNF-tu (25 rig/ml) for 48 h contained 401 t 132 U/mg protein (n =
3, P < 0.05). Anti-TNF-a antibodies. To confirm that the increase in Mn-SOD activity was due specifically to TNF-cu, H820 cells were incubated with TNF-cu (25 rig/ml) for 48 h in the presence or absence of monoclonal antibodies to human TNF-a (Boehringer-Mannheim, Indianapolis, IN). Anti-TNF-cw antibodies completely blocked the increase in Mn-SOD activity: control cells had 20 U MnSOD activity/mg protein, 70 U/mg protein after TNF-tu, and 19 U/mg protein after TNF-cu antibodies with TNFcy(n = 2). Effect of TNF-cu on A4n-SOD mRNA. Northern blots of total cellular mRNA from H820 cells (Figs. 1A and 3A) demonstrated a major RNA species hybridizing with the Mn-SOD cRNA. This l.O-kb message encodes for the NHz-terminal leader sequence, the active protein, and the 3’-untranslated sequence including the polyadenylation sequence (26). Other mRNA species which are specific for Mn-SOD were also observed, including a 4.0kb message which is distinct from ribosomal mRNA. These larger Mn-SOD mRNA species are present in mammalian tissue and are felt to arise from differential splicing of the primary transcript (9, 26). TNF-cu increases both the l.O- and 4.0-kb message in H820 cells. To examine the time course of Mn-SOD mRNA induction by TNF- cy,H820 cells were incubated with TNFti for 0, 6, 12, or 48 h. Total cellular RNA was analyzed for Mn-SOD and ,&actin mRNAs. Figure 1A is a representative autoradiogram, and Fig. 1B the densitometric analysis of this autoradiogram. Both the l.O- and 4.0-kb Mn-SOD mRNAs are increased at 6, 12, and 48 h. ,@ Actin mRNA is unchanged. To ensure the reproducibility of these findings H820 cells were treated with TNF-cu (25 rig/ml) for 24 or 48 h in four separate experiments, and the l.O-kb Mn-SOD mRNA quantified by densitometric analysis (Fig. 2). There was a significant increase
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L298
TUMOR
A
NECROSIS
FACTOR
AND
MN-SUPEROXIDE
DISMUTASE
n
Mn-SOD
Mn-SOD El-Actm
-4.0
0 0
24 Time
FIG. 2. In 4 separate experiments H820 cells were incubated in 25 rig/ml TNF-cu for 24 or 48 h. Total cellular RNA was recovered, electrophoresed, and transferred to Nytran. After hybridization, l.O-kb Mn-SOD and 1.8-kb p-actin mRNAs were quantitated by densitometric analysis. Results are expressed as absorption relative to control, and histograms illustrate means + SD. Mn-SOD mRNA was significantly increased over control at both 24 and 48 h (P < 0.05), and /3-actin mRNA was unchanged.
DACTIN
0
6
12
48
(hrs)
46
Hours
-.m.w. *.-‘-’ --- *--
is the densitometric analysis of this autoradiogram. Both the LOand 4.0-kb messages are increased at 0.1 ng Mn-SOD 1Kb TNF-a/ml, with progressive increases through 100 ng/ Mn-SOD 4Kb ml. Examination of the densitometric analysis demonB-Actin strates a slight increase in /?-actin mRNA over the dose range used. However, the increase in P-actin mRNA is minor compared with the increase in Mn-SOD mRNA. In contrast to the induction of Mn-SOD in H820 cells, TNF-(Y inhibited expression of SP-A, a major surfactant protein in H820 cells (Fig. 4). Effect of cycloheximide or actinomycin D on induction of Mn-SOD by TNF-CY. Cycloheximide and actinomycin
..c .. ---
0
_-- *-------------------------J( Y,‘,YYY.,.,
6
12
18
24
30
36
42
48
Time (hrs) FIG. 1. A: time course for TNF-cu induction of Mn-SOD mRNA. Cells were incubated in 25 rig/ml TNF-cu for 6, 12, or 48 h. Total cellular RNA was isolated and 40 fig RNA/sample was electrophoresed, transferred to Nytran, and hybridized with 32P-labeled Mn-SOD cRNA. After washing, membranes were reprobed for p-actin using a ‘“P-labeled cDNA. B: densitometric analysis of above autoradiogram demonstrates relative increase of 1.0 and 4.0-kb Mn-SOD mRNAs over time. P-Actin mRNA is unchanged.
in the l.O-kb Mn-SOD message at both 24 and 48 h, with no change in P-actin mRNA. The dose-response relationship for TNF-a! and Mn-SOD mRNA is shown in Fig. 3. Figure 3A is a representative autoradiogram, and Fig. 3B
D were used to evaluate the role of new protein synthesis and gene transcription in the induction of Mn-SOD. Figure 5A is a representative autoradiogram, and Fig. 5B is a histogram summarizing the results of three separate experiments. Cycloheximide did not prevent the increase in Mn-SOD mRNA, indicating that new protein synthesis was not required for induction of Mn-SOD mRNA by TNF-cu. Cycloheximide alone appears to increase MnSOD mRNA. Actinomycin D completely blocked the increase in Mn-SOD mRNA, indicating that gene transcription was required for induction of Mn-SOD mRNA by TNF-a. Cellular protection from paraquat. The herbicide paraquat (l,l’-dimethyl-4,4’-bipyridinium) exerts its cytotoxic effects by increasing production of 020 (5). If, as proposed, Mn-SOD has a protective role against oxygen injury by scavenging 020, then cells with increased MnSOD activity should be protected against paraquat. To test this hypothesis, H820 cells were pretreated with TNF-(U and resistance to paraquat toxicity was assessed. TNF-a (25 rig/ml) protected H-820 cells from paraquat killing, with significantly improved survival at each paraquat concentration (Fig. 6). At the LD50 (6 PM) for
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TUMOR
NECROSIS
FACTOR
AND MN-SUPEROXIDE
L299
DISMUTASE
Mn-SOD
A
SP -A
DACTIN
0.1 10 TNF Ing/mll
0
25
FIG. 4. Effect of TNF-cu on mRNA for surfactant protein A and pactin. H820 cells were incubated with TNF-ol (0.1-25 rig/ml) for 48 h. Total cellular RNA was isolated and 40 rg RNA/sample was electrophoresed, transferred to Nytran, and hybridized sequentially with 3zPlabeled surfactant protein A cDNA and p-actin cDNA.
8 ACTIN
-1.8 0
0.1
1.0
10
25
100
TNF(ng/ml) B
1 Kb Mn-SOD 4 Kb Mn-SOD ~~~~~~~~~~~~~~~~~ D-actin ----
)( ---.
20 -
CON
30
$ a, t s
I3 ACTIN
.
15-
TNF
CHX
TNF+ CHX
ACT-D
TNF+
ACT-D
B
1
T
n
Mn-SOD B-Actin
lo.-: 2 u oe
?
0 .? 3 8%
FIG. 3. A: dose response for TNF-(U induction of Mn-SOD mRNA. Cells were incubated for 48 h with 0.1-100 rig/ml of TNF-a. Total cellular RNA was isolated and 40 fig RNA/sample was separated by electrophoresis, transferred to Nytran, and hybridized with “P-labeled Mn-SOD cRNA. After washing, membranes were reprobed for /3-actin using a “P-labeled cDNA. B: densitometric analysis of above autoradiogram demonstrates relative increase of l.O-kb Mn-SOD and 4.0-kb Mn-SOD mRNAs and &actin mRNA.
control cells, 95% of TNF-a-treated cells survived, 85% at the LDT5 (10 PM), and 77% at the LDgo (14 PM). DISCUSSION
TNF-(U increased Mn-SOD activity and mRNA in a time- and dose-dependent manner in a human pulmo-
10
0 control
TNF
CHX
CHX+
TNF
ActD
ActD
+ TNF
5. A: effect of cycloheximide and actinomycin D on induction of Mn-SOD mRNA by TNF-ol. Cells were incubated for 24 h with medium (Con), 25 rig/ml TNF-(Y (TNF), 10 PM cycloheximide (Chx), cycloheximide plus TNF-cu (TNF + Chx), 10 rg/ml actinomycin D (Act D), or actinomycin D plus TNF-cu (TNF + Act D). Total cellular RNA was isolated, separated by electrophoresis, and transferred to Nytran. Blots were hybridized with [“*P]Mn-SOD cRNA and then /3-[“‘Plactin cDNA, with resulting representative autoradiogram. B: densitometric analysis of l.O-kb Mn-SOD and 1.8-kb P-actin mRNAs was done on autoradiograms from 3 separate experiments. Histogram values represent means f SD of these 3 experiments. Results are expressed as absorbance relative to control. Mn-SOD mRNA was increased in cells treated with TNF-cu, cycloheximide, and TNF-a plus cycloheximide (P < 0.05). fl-Actin was unchanged. FIG.
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TIJMOR
NECROSIS
FACTOR
AND
control tnf 100
20
Kh 20
10 Paraquat
30
(uM)
FI(:. 6. Effect of TNF-tu on parayuat killing. Cells were preincuhat.ed for 48 h in presence or absence of’ 25 rig/ml TNF-tu. Paraquat (2-20 FM) was then added for 48 h. Cell viability was measured by neutral red uptake and retention. Values represent means t SD. TNF-tu improved survival (P < 0.05) at each paraquat concentrat,ion.
nary adenocarcinoma cell line, NIH H820. These results confirm work of other investigators using different cell lines (1, 24, 28). The enhancement of Mn-SOD in H820 cells was not due to a generalized stimulatory effect of TNF-cr on cellular proteins; SP-A mRNA was decreased, whereas P-actin and CuZn-SOD were unchanged. The increase in Mn-SOD was specific to TNF-a, anti-TNFcy antibodies blocked the increase in Mn-SOD activity. Induction of h/In-SOD by TNF-cu appears to be pretranslational. Actinomycin D blocked the increase in Mn-SOD mRNA by TNF- cy, indicating that gene transcription is required. TNF-cu regulates transcription of other proteins including collagenase, jun (2), the HLA complex (19), and macrophage-specific colony-stimulating factor (21). Cycloheximide did not block the induction of Mn-SOD mRNA by TNF-cu, suggesting that synthesis of new protein intermediates is not required. Cycloheximide alone increased Mn-SOD mRNA, an effect reported for other messages. Cycloheximide increases m,yc (12), c-fos (20), and the EGF receptor (10) mRNAs by increasing message half-life. In combination with TNF-a, cycloheximide increased Mn-SOD mRNA more than either alone. This apparent synergy may be due to separate mechanisms. For example, TNF-cu may increase Mn-SOD mRNA transcription, whereas cycloheximide stabilizes the cytoplasmic message. Cellular events involved in the regulation of Mn-SOD gene expression are unclear. In prokaryotic cells, MnSOD is induced by oxygen, redox-active compounds such as viologen or quinones, and heat shock, all oxidative stresses (8, 19). In eukaryotic cells, oxygen also may regulate Mn-SOD expression. Rats exposed to hyperoxia have increased Mn-SOD activity in whole lung (4) and alveolar type II cells (7). Mn-SOD activity is also increased in mammalian cell lines after exposure to paraquat (11) and X-irradiation (16). One common pathway of oxygen, paraquat, and X-irradiation is the production
MN-SUPEROXIDE
DISMUTASE
of reactive oxygen metabolites, supporting the hypothesis that the cellular oxidative state regulates Mn-SOD expression. TNF-a increases production of oxygen metabolites in several models. In cell culture, TNF-a increases 0,. (15, 23) and malondialdehyde (14), a free radical lipid peroxidation product. Anaerobic conditions or exogenous antioxidants (14, 29) significantly decrease TNF-c-w cytotoxicity, again suggesting that cellular effects of TNF-cu are mediated by oxygen metabolism. Whether the induction of Mn-SOD by TNF-cw involves reactive oxygen metabolites remains to be clarified. Under hyperoxic conditions, there is increased production of reactive oxygen metabolites including 0,. , HzOz and hydroxyl radical. Although oxygen radicals arise from multiple subcellular sources, mitochondria contribute at least 25% of whole lung oxygen radical production (27). This observation is consistent with the histological response of mitochondrial enlargement and inner mitochondrial membrane swelling seen early in pulmonary oxygen injury. Mn-SOD, strategically located in the mitochondrial matrix, may provide an important initial defense against 0, . To examine the protective effect of Mn-SOD against oxidant injury, H820 cells were pretreated with TNF-cw to increase Mn-SOD and then exposed to paraquat. Paraquat, a dipyridylium herbicide, increases mitochondrial production of 0~0. In our study, TNF-cu pretreatment significantly protected human pulmonary adenocarcinoma cells against paraquat-induced cell death. These results confirm the findings of Krall et al. (ll), who demonstrated that Mn-SOD protected HeLa cells against paraquat cytotoxicity. Generalizations from these findings must be made with caution. only in the mitochondria, Paraquat produces 0, whereas hyperoxia increases production in a number of subcellular organelles. Thus cellular injury associated with hyperoxia is likely to be more complex than with paraquat. Future experiments will directly determine whether increased Mn-SOD protects against oxygen injury in vitro and in vivo. TNF-a may also be protecting against paraquat through mechanisms unrelated to MnSOD. Wong et al. (28) reported that catalase and glutathione system enzymes were unchanged by TNF-a. However, TNF-a affects many cell processes, some of which could alter the cytotoxicity of paraquat. NIH H820 are similar to type II alveolar epithelial cells. The type II cell is important in oxygen injury because it contains a major portion of pulmonary SOD (7) and serves as a progenitor cell for repopulating the damaged alveolar epithelial lining. The current findings raise the possibility that inflammatory mediators may directly interact with respiratory epithelial cells to affect important protective and functional components of the lung. l
l
The authors wish to thank discussions and critical review This work was supported Blood Institute Grant HL-39605 Research Foundation. Address reprint requests to Received
5 February,
Jeffrey A. Whitsett for his many helpful of this paper. in part by National Heart, Lung, and to J. R. Wispi! and Children’s Hospital J. R. Wisp&
1990; accepted
in final
form
5 September
1990.
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TUMOR
NECROSIS
FACTOR
AND
MN-SUPEROXIDE
REFERENCES 1. ASOH, K., Y. WATANABE, H. MIZOGUCHI, M. MAWATAI, M. ONO, K. KOHNO, AND M. KUWANO. Induction of manganese superoxide dismutase by tumor necrosis factor in human breast cancer MCF7 cell line and its TNF-resistant variant. Biochim. Biophys. Acta 162: 794-801, 1989. 2. BRENNER, D. A., M. OHARA, P. ANGEL, M. CHOJKIER, AND M. KARIN. Prolonged activation ofjun and collagenase genes by tumor necrosis factor N. Nature Lond. 337: 661-663, 1989. 3. BRUNK, C. F., K. C. JONES, AND T. W. JAMES. Assay for nanogram quantities of DNA in cellular homogenates. Anal Biochem. 92: 497-500, 1979. 4. CRAPO, J. D., B. C. BARRYS, J. A. FOSCUE, AND J. SHELBURNE. Structural and biochemical changes in rat lungs occurring during exposure to lethal and adaptive doses of oxygen. Am. Reu. Respir. Dis. 122: 123-143, 1980. 5. FARRINGTON, J. A., J. EBERT, E. J. LAND, AND K. FLETCHER. Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implication for the mode of action of bipyridyl herbicides. Biochim. Biophys. Acta 314: 372-381, 1973. 6. FINTER, N. B. Dye uptake methods for assessing viral cytopathogenicity and their application to interferon assays. J. Gen. ViroL. 5: 419-427, 1969. 7. FORMAN, H. J., AND A. B. FISHER. Antioxidant enzymes of rat granular pneumocytes. Constitutive levels and effect of hyperoxia. Lab. Invest. 45: l-6, 1981. a. HASSAN, H. Biosynthesis and regulation of superoxide dismutases. Free Radical Biol. Med. 5: 377-385, 1988. 9. HO, Y., AND J. D. CRAPO. Isolation and characterization of cornplementary DNAs encoding human manganese-containing superoxide dismutase. FEBS Lett. 229: 256-260, 1988. 10. KESAVAN, P., P. DAS, J. KERN, AND M. DAS. Regulation of stability and synthesis of EGF-receptor mRNAs encoding for intact and truncated receptor forms. Oncogene 5: 483-488, 1990. 11. KRALL, J., A. C. BAGLEY, G. T. MULLENBACH, R. A. HALLEWELL, AND R. E. LYNCH. Superoxide mediates the toxicity of paraquat for cultured mammalian cells. J. Biol. Chem. 263: 1910-1914, 1988. 12. KRONKE, M., C. SCHLUTER, AND K. PFIZENMAIER. Tumor necrosis factor inhibits myc expression in HL-60 cells at the level of mRNA transcription. Proc. Natl. Acad. Sci. USA 84: 469-473, 1987. 13. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. Protein measurement with the Folin phenol reagent. J. BioZ. Chem. 193: 265-275, 1951. 14. MATTHEWS, N., M. L. NEALE, S. K. JACKSON, AND J. M. STARK. Tumor cell killing by tumor necrosis factor: inhibition by anaerobic conditions, free radical scavengers and inhibitors of arachidonic metabolism. Immunology 62: 153-155, 1987. 15. MEIER, B., H. H. RADEKE, S. SELLS, M. YOUNES, H. SIES, K. RESCH, AND G. G. HABERNEHL. Human fibroblasts release reactive
16 ’ 17
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oxygen species in response to interleukin-1 or tumor necrosis factor (Y. Biochem. J. 263: 539-545, 1989. OBERLY, L. W., D. K. ST. CLAIR, A. P. AUTOR, AND T. D. OBERLEY. Increase in manganese superoxide dismutase activity in the mouse heart after X-irradiation. Arch. Biochem. Biophys. 254: 69-80, 1987. O’REILLY, M. A., A. F. GAZDAR, J. C. CLARK, T. J. PILOT-MATIAS, S. E. WERT, W. M. HULL, AND J. A. WHITSETT. Glucocorticoids regulate surfactant protein biosynthesis in a pulmonary adenocarcinema cell line. Am. J. Physiol. 257 (Lung CelZ Mol. Physiol. 1): L385-L392, 1989. PFIZENMAIER, K., P. SCHEURICH, C. SCHLUTER, AND M. KRONKE. Tumor necrosis factor enhances HLA-A,B,C. and HLA-DR gene expression in human tumor cells. J. Immunol. 138: 975-980, 1987. PRIVALLE, C. T., AND I. FRIDOVICH. Induction of superoxide dismutase in Escherichia coli by heat shock. Proc. Natl. Acad. Sci. USA 84: 2723-2726, 1987. RHAMSDORF, H. J., A. SCHONTHAL, P. ANGEL, M. LITFIN, U. RUTCHR, AND P. HERRLICK. Post-transcriptional regulation of c/OS mRNA expression. Nucleic Acids Res. 15: 1643-1659, 1987. SHERMAN, M., B. L. WEBER, R. DATTA, AND D. W. KUFE. Transcriptional and post-transcriptional regulation of macrophage-specific colony stimulating factor gene expression by tumor necrosis factor. J. CLin. Inuest. 85: 442-47, 1990. SPITZ, D. R., AND L. W. OBERLY. An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal. Biochem. 179: 8-18, 1989. TSUJIMOTO, M., S. YOKOTA, J. VILCEK, AND G. WEISSMAN. Tumor necrosis factor provokes superoxide anion generation from neutrophils. Biochem. Biophys. Res. Commun. 137: 1094-1100, 1986. VISNER, G. A., W. C. DOUGALL, J. M. WILSON, I. A. BARR, AND H. S. NICK. Regulation of manganese superoxide dismutase by lipopolysaccharide, interleukin-1, and tumor necrosis factor. J. BioZ. Chem. 265: 2856-2863, 1990. WHITE, C. W., P. GHEZZ, S. MCMAHON, C. A. DINARELLO, AND J. E. REPINE. Cytokines increase rat lung antioxidant enzymes during exposure to hyperoxia. J. Appl. Physiol. 66: 1003-1007, 1989. WISP& J. R., J. C. CLARK, M. S. BURHANS, K. E. KROPP, T. R. KORFHAGEN, AND J. A. WHITSETT. Synthesis and processing of the precursor for human mangano-superoxide dismutase. Biochim. Biophys. Acta 994: 30-36, 1989. WISP& J. R., AND R. J. ROBERTS. Molecular basis of pulmonary oxygen toxicity. CLin. Perinat. 14: 651-666, 1987. WONG, G. H., AND D. V. GOEDDEL. Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science Wash. DC 242: 941-943, 1988. ZIMMERMAN, R. J., B. J. MARATINO, JR., A. CHAN, P. LANDRE, AND J. L. WINKELHAKE. The role of oxidant injury in tumor cell sensitivity to recombinant human tumor necrosis factor in vivo. J. Immunol. 142: 1405-1409, 1989.
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WARNER, BARBARA B., MICHAEL S. BURHANS, JEAN C. CLARK, AND JONATHAN R. WISPJ?. Tumor necrosis factor-cu increases Mn-SOD expression:protection against oxidant injury. Am. J. Physiol. 260 (Lung Cell. Mol. Physiol. 4): L296-L301, 1991.-Antioxidant enzymes, including superoxide dismutase, are important for protecting the lung against 0, injury. Manganese superoxide dismutase (Mn-SOD) is a superoxide anion (0; ) scavenger located in the mitochondria, a primary site of 0,. production during hyperoxia. We studied the effects of tumor necrosis factor (TNF-cu), a macrophage-derived cytokine, on Mn-SOD expression in human pulmonary adenocarcinoma cells. TNF-cu significantly increased Mn-SOD activity and mRNA in a dose- and time-dependent manner. Mn-SOD activity was increased 3-fold and mRNA Z&fold after a 48-h incubation with TNF-cu (25 rig/ml). To examine the mechanism of this increase, cells were incubated for 48 h with TNF-(U (25 ng/ ml) with or without cycloheximide (10 PM) or actinomycin D (10 pg/ml). Actinomycin D blocked the induction of Mn-SOD mRNA by TNF- CY,but cycloheximide did not. These findings suggest that the effect of TNF-cu requires gene transcription but not synthesis of new protein intermediates. To test the hypothesis that increased Mn-SOD protects against oxidative injury, pulmonary adenocarcinoma cells were incubated in TNF-cu (25 rig/ml) for 48 h and then exposed to paraquat (PQ’), an intracellular 0, generator. Cells pretreated with TNF-(1 had significantly improved survival in PQ’ compared with controls. At the LDso (6 PM) for control cells, 95% of TNF-n-treated cells survived, 85% at the LD7, (10 PM), and 77% at the LDr,,, (14 PM). Our results suggest that the induction of Mn-SOD by TNF- (Y in pulmonary adenocarcinoma cells is pretranslationally mediated and that increasing Mn-SOD activity with TNF-cu confers protection against 0, radicals. l
Superoxide dismutases (SOD) are metalloproteins that dismutate 0,. to hydrogen peroxide (H202). In eukaryotic cells, three forms of SOD exist, a cytosolic copperzinc-SOD (CuZn-SOD), an extracellular CuZn-SOD, and a mitochondrial manganese-SOD (Mn-SOD). Tumor necrosis factor CY(TNF-cu), is a 17-kDa soluble peptide secreted by macrophages and2 x lo7 U/mg protein was purchased from Boehringer-Mannheim, Indianapolis, IN. PGEM-32 and RNA transcription kits were obtained from Promega Biotech, Madison, WI. Cycloheximide, actinomycin D, paraquat, and other chemicals were purchased from Sigma Chemical, St. Louis, MO. Cell culture methods. NIH H820 human pulmonary adenocarcinoma cells were isolated at the National Cancer Institute, Bethesda, MD, and kindly provided by Dr. A. Gazdar. These cells have biochemical and histological properties similar to type II pulmonary epithelial cells with junctional complexes, apical microvilli, and multilamellar inclusion bodies. Cells were incubated at 37°C in 95% air-5% COa, in RPMI-1640 (GIBCO) supplemented with hormones and growth factors as previously described (16). Medium was changed every 4 days, and cells routinely passed once a week. Before all experiments, cells were suspended in 5 ml of fresh medium. Gene transcription was inhibited by treating cells with actinomycin D (10 pg/ml). Transcriptional analysis demonstrated that 10 pg/ml actinomycin
CC 1991 the American
Physiological
Society
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TLJMOR
NECROSIS
FACTOR
AND
D blocked transcription of the surfactant-associated proteins. Protein synthesis was inhibited by treating cells with 10 PM cycloheximide, which inhibited [“Slmethionine incorporation by >90% in this cell line. Cells were harvested, washed vigorously, suspended in 1 ml of 5 mM K,HPO, (pH 7.8), and sonicated on ice for 45 s. SOD activity assay. SOD activity in cell lysates was measured by inhibition of nitro blue tetrazolium reduction by 0,. (22). Mn-SOD activity is discerned from CuZn-SOD by its resistance to 5 mM potassium cyanide. The protein and DNA content of cell lysate was measured as previously described (3, 13). Mn-SOD activity was expressed per milligram protein and per nanogram DNA. RNA extraction for hybridization experiments. To isolate RNA, H820 cells were sonicated in 4 M guanidine thiocyanate and centrifuged through 5.7 M cesium chloride. The RNA was dissolved in water, phenol extracted, and precipitated with ethanol (26). The amount of RNA in aqueous solution was determined by absorbance at 260 nM. For Northern blot analysis, equal amounts of total cellular RNA were electrophoresed through a 1.2% agarose-7% formaldehyde gel and transferred to Nytran (Schleicher and Schuell, Keene, NH) filters. The filters were probed with ““P-labeled antisense RNA transcribed from PGEM-3Z (Promega) into which the Mn-SOD cDNA was subcloned (26). The filters were hybridized at 60°C for 18 h, washed stringently at 60°C and exposed to XAR film at -80°C. Rehybridization with another probe was performed after washing the membrane for 2 h at 65°C in 5 mM tris(hydroxymethyl)aminomethane chloride (pH 8.0), 0.2 mM EDTA, 0.5% sodium pyrophosphate, and 0.1~ Denhart’s solution. h/In-SOD was detected as a l.O-kb message, with additional less intense bands observed at 2.0, 2.5, and 4.0 kb. Surfactant protein A (SP-A) and ,& ac t in mRNAs were detected as 2.2- and 1.8-kb species, respectively. Relative message intensity was quantitated by scanning densitometry of the autoradiograms (Helena Laboratories). Resistance to paraquat. Equal numbers of H820 cells were incubated for 48 h in the presence or absence of 25 rig/ml of TNF -cy. Paraquat was then added in concentrations varying from 2 to 20 PM. After 48 h in paraquat, 400 nM neutral red was added directly to the cultures. As described by Finter (6), only viable cells retain the dye. Two hours after addition of dye, cells were washed three times in phosphate-buffered saline (pH 7.4), centrifuged, and lysed with 1:195% ethanol-100 mM sodium citrate (pH 4.2) and absorbance at 540 nm was measured. Statistics. Comparisons between groups were made with the Student’s t test and one-way analysis of variance. RESULTS
Effect of TNF-a on SOD actiuity. TNF-a significantly increased Mn-SOD activity in human pulmonary adenocarcinoma cells (Table 1). Mn-SOD activity was increased 24 h after addition of TNF-cu, with a further increase after 48 h. TNF-cu increased Mn-SOD activity at the lowest dose of 0.1 rig/ml, an effect sustained through 100 rig/ml. In contrast to the stimulatory effect
MN-S1_JPER,OXIDE
TABLE TNF-tu, ng/rnl
Control 0.1
1. Effect of TNF-cu on SOD activity 24 h
48 h
n
Mn-SOD
CuZn-SOD
n
Mn-SOD
7
18k6
40t21
15 3
16t5 :‘32&17” 62t37* 41t25* 44t13” 118t61*
1.0 10 25 100
L297
DISMUTASE
3 4 3
32t 10* 76t7*
30t9 20t9
6 12 7
CuZn-SOI)
50535 175t7 32kll
49t23 51t42 37k24
Values are means t SD expressed as U activit.y/mg protein; n, no. of separate experiments for each concentration of’ TNF-tu. Cells were incubated for 24 or 48 h with 0.1-100 r-g/ml TNF-tu. SOD activity was assayed by the nitro blue tetrazolium method. * Significantly diff’erent from control, 1’ < 0.05.
of TNF-a on Mn-SOD, TNF-a had no effect on CuZnSOD activity. Normalization of Mn-SOD activity to DNA content rather than protein did not alter activity results. Cellular protein content was not affected by TNF-cu (data not shown). The effect of TNF -cy on Mn-SOD activity was not unique to H820 cells. TNF-ti also increased Mn-SOD activity in the human pulmonary adenocarcinoma cell line A549. Control cells contained 161 t 9 U Mn-SOD activity/mg protein, while cells treated with TNF-tu (25 rig/ml) for 48 h contained 401 t 132 U/mg protein (n =
3, P < 0.05). Anti-TNF-a antibodies. To confirm that the increase in Mn-SOD activity was due specifically to TNF-cu, H820 cells were incubated with TNF-cu (25 rig/ml) for 48 h in the presence or absence of monoclonal antibodies to human TNF-a (Boehringer-Mannheim, Indianapolis, IN). Anti-TNF-cw antibodies completely blocked the increase in Mn-SOD activity: control cells had 20 U MnSOD activity/mg protein, 70 U/mg protein after TNF-tu, and 19 U/mg protein after TNF-cu antibodies with TNFcy(n = 2). Effect of TNF-cu on A4n-SOD mRNA. Northern blots of total cellular mRNA from H820 cells (Figs. 1A and 3A) demonstrated a major RNA species hybridizing with the Mn-SOD cRNA. This l.O-kb message encodes for the NHz-terminal leader sequence, the active protein, and the 3’-untranslated sequence including the polyadenylation sequence (26). Other mRNA species which are specific for Mn-SOD were also observed, including a 4.0kb message which is distinct from ribosomal mRNA. These larger Mn-SOD mRNA species are present in mammalian tissue and are felt to arise from differential splicing of the primary transcript (9, 26). TNF-cu increases both the l.O- and 4.0-kb message in H820 cells. To examine the time course of Mn-SOD mRNA induction by TNF- cy,H820 cells were incubated with TNFti for 0, 6, 12, or 48 h. Total cellular RNA was analyzed for Mn-SOD and ,&actin mRNAs. Figure 1A is a representative autoradiogram, and Fig. 1B the densitometric analysis of this autoradiogram. Both the l.O- and 4.0-kb Mn-SOD mRNAs are increased at 6, 12, and 48 h. ,@ Actin mRNA is unchanged. To ensure the reproducibility of these findings H820 cells were treated with TNF-cu (25 rig/ml) for 24 or 48 h in four separate experiments, and the l.O-kb Mn-SOD mRNA quantified by densitometric analysis (Fig. 2). There was a significant increase
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L298
TUMOR
A
NECROSIS
FACTOR
AND
MN-SUPEROXIDE
DISMUTASE
n
Mn-SOD
Mn-SOD El-Actm
-4.0
0 0
24 Time
FIG. 2. In 4 separate experiments H820 cells were incubated in 25 rig/ml TNF-cu for 24 or 48 h. Total cellular RNA was recovered, electrophoresed, and transferred to Nytran. After hybridization, l.O-kb Mn-SOD and 1.8-kb p-actin mRNAs were quantitated by densitometric analysis. Results are expressed as absorption relative to control, and histograms illustrate means + SD. Mn-SOD mRNA was significantly increased over control at both 24 and 48 h (P < 0.05), and /3-actin mRNA was unchanged.
DACTIN
0
6
12
48
(hrs)
46
Hours
-.m.w. *.-‘-’ --- *--
is the densitometric analysis of this autoradiogram. Both the LOand 4.0-kb messages are increased at 0.1 ng Mn-SOD 1Kb TNF-a/ml, with progressive increases through 100 ng/ Mn-SOD 4Kb ml. Examination of the densitometric analysis demonB-Actin strates a slight increase in /?-actin mRNA over the dose range used. However, the increase in P-actin mRNA is minor compared with the increase in Mn-SOD mRNA. In contrast to the induction of Mn-SOD in H820 cells, TNF-(Y inhibited expression of SP-A, a major surfactant protein in H820 cells (Fig. 4). Effect of cycloheximide or actinomycin D on induction of Mn-SOD by TNF-CY. Cycloheximide and actinomycin
..c .. ---
0
_-- *-------------------------J( Y,‘,YYY.,.,
6
12
18
24
30
36
42
48
Time (hrs) FIG. 1. A: time course for TNF-cu induction of Mn-SOD mRNA. Cells were incubated in 25 rig/ml TNF-cu for 6, 12, or 48 h. Total cellular RNA was isolated and 40 fig RNA/sample was electrophoresed, transferred to Nytran, and hybridized with 32P-labeled Mn-SOD cRNA. After washing, membranes were reprobed for p-actin using a ‘“P-labeled cDNA. B: densitometric analysis of above autoradiogram demonstrates relative increase of 1.0 and 4.0-kb Mn-SOD mRNAs over time. P-Actin mRNA is unchanged.
in the l.O-kb Mn-SOD message at both 24 and 48 h, with no change in P-actin mRNA. The dose-response relationship for TNF-a! and Mn-SOD mRNA is shown in Fig. 3. Figure 3A is a representative autoradiogram, and Fig. 3B
D were used to evaluate the role of new protein synthesis and gene transcription in the induction of Mn-SOD. Figure 5A is a representative autoradiogram, and Fig. 5B is a histogram summarizing the results of three separate experiments. Cycloheximide did not prevent the increase in Mn-SOD mRNA, indicating that new protein synthesis was not required for induction of Mn-SOD mRNA by TNF-cu. Cycloheximide alone appears to increase MnSOD mRNA. Actinomycin D completely blocked the increase in Mn-SOD mRNA, indicating that gene transcription was required for induction of Mn-SOD mRNA by TNF-a. Cellular protection from paraquat. The herbicide paraquat (l,l’-dimethyl-4,4’-bipyridinium) exerts its cytotoxic effects by increasing production of 020 (5). If, as proposed, Mn-SOD has a protective role against oxygen injury by scavenging 020, then cells with increased MnSOD activity should be protected against paraquat. To test this hypothesis, H820 cells were pretreated with TNF-(U and resistance to paraquat toxicity was assessed. TNF-a (25 rig/ml) protected H-820 cells from paraquat killing, with significantly improved survival at each paraquat concentration (Fig. 6). At the LD50 (6 PM) for
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TUMOR
NECROSIS
FACTOR
AND MN-SUPEROXIDE
L299
DISMUTASE
Mn-SOD
A
SP -A
DACTIN
0.1 10 TNF Ing/mll
0
25
FIG. 4. Effect of TNF-cu on mRNA for surfactant protein A and pactin. H820 cells were incubated with TNF-ol (0.1-25 rig/ml) for 48 h. Total cellular RNA was isolated and 40 rg RNA/sample was electrophoresed, transferred to Nytran, and hybridized sequentially with 3zPlabeled surfactant protein A cDNA and p-actin cDNA.
8 ACTIN
-1.8 0
0.1
1.0
10
25
100
TNF(ng/ml) B
1 Kb Mn-SOD 4 Kb Mn-SOD ~~~~~~~~~~~~~~~~~ D-actin ----
)( ---.
20 -
CON
30
$ a, t s
I3 ACTIN
.
15-
TNF
CHX
TNF+ CHX
ACT-D
TNF+
ACT-D
B
1
T
n
Mn-SOD B-Actin
lo.-: 2 u oe
?
0 .? 3 8%
FIG. 3. A: dose response for TNF-(U induction of Mn-SOD mRNA. Cells were incubated for 48 h with 0.1-100 rig/ml of TNF-a. Total cellular RNA was isolated and 40 fig RNA/sample was separated by electrophoresis, transferred to Nytran, and hybridized with “P-labeled Mn-SOD cRNA. After washing, membranes were reprobed for /3-actin using a “P-labeled cDNA. B: densitometric analysis of above autoradiogram demonstrates relative increase of l.O-kb Mn-SOD and 4.0-kb Mn-SOD mRNAs and &actin mRNA.
control cells, 95% of TNF-a-treated cells survived, 85% at the LDT5 (10 PM), and 77% at the LDgo (14 PM). DISCUSSION
TNF-(U increased Mn-SOD activity and mRNA in a time- and dose-dependent manner in a human pulmo-
10
0 control
TNF
CHX
CHX+
TNF
ActD
ActD
+ TNF
5. A: effect of cycloheximide and actinomycin D on induction of Mn-SOD mRNA by TNF-ol. Cells were incubated for 24 h with medium (Con), 25 rig/ml TNF-(Y (TNF), 10 PM cycloheximide (Chx), cycloheximide plus TNF-cu (TNF + Chx), 10 rg/ml actinomycin D (Act D), or actinomycin D plus TNF-cu (TNF + Act D). Total cellular RNA was isolated, separated by electrophoresis, and transferred to Nytran. Blots were hybridized with [“*P]Mn-SOD cRNA and then /3-[“‘Plactin cDNA, with resulting representative autoradiogram. B: densitometric analysis of l.O-kb Mn-SOD and 1.8-kb P-actin mRNAs was done on autoradiograms from 3 separate experiments. Histogram values represent means f SD of these 3 experiments. Results are expressed as absorbance relative to control. Mn-SOD mRNA was increased in cells treated with TNF-cu, cycloheximide, and TNF-a plus cycloheximide (P < 0.05). fl-Actin was unchanged. FIG.
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TIJMOR
NECROSIS
FACTOR
AND
control tnf 100
20
Kh 20
10 Paraquat
30
(uM)
FI(:. 6. Effect of TNF-tu on parayuat killing. Cells were preincuhat.ed for 48 h in presence or absence of’ 25 rig/ml TNF-tu. Paraquat (2-20 FM) was then added for 48 h. Cell viability was measured by neutral red uptake and retention. Values represent means t SD. TNF-tu improved survival (P < 0.05) at each paraquat concentrat,ion.
nary adenocarcinoma cell line, NIH H820. These results confirm work of other investigators using different cell lines (1, 24, 28). The enhancement of Mn-SOD in H820 cells was not due to a generalized stimulatory effect of TNF-cr on cellular proteins; SP-A mRNA was decreased, whereas P-actin and CuZn-SOD were unchanged. The increase in Mn-SOD was specific to TNF-a, anti-TNFcy antibodies blocked the increase in Mn-SOD activity. Induction of h/In-SOD by TNF-cu appears to be pretranslational. Actinomycin D blocked the increase in Mn-SOD mRNA by TNF- cy, indicating that gene transcription is required. TNF-cu regulates transcription of other proteins including collagenase, jun (2), the HLA complex (19), and macrophage-specific colony-stimulating factor (21). Cycloheximide did not block the induction of Mn-SOD mRNA by TNF-cu, suggesting that synthesis of new protein intermediates is not required. Cycloheximide alone increased Mn-SOD mRNA, an effect reported for other messages. Cycloheximide increases m,yc (12), c-fos (20), and the EGF receptor (10) mRNAs by increasing message half-life. In combination with TNF-a, cycloheximide increased Mn-SOD mRNA more than either alone. This apparent synergy may be due to separate mechanisms. For example, TNF-cu may increase Mn-SOD mRNA transcription, whereas cycloheximide stabilizes the cytoplasmic message. Cellular events involved in the regulation of Mn-SOD gene expression are unclear. In prokaryotic cells, MnSOD is induced by oxygen, redox-active compounds such as viologen or quinones, and heat shock, all oxidative stresses (8, 19). In eukaryotic cells, oxygen also may regulate Mn-SOD expression. Rats exposed to hyperoxia have increased Mn-SOD activity in whole lung (4) and alveolar type II cells (7). Mn-SOD activity is also increased in mammalian cell lines after exposure to paraquat (11) and X-irradiation (16). One common pathway of oxygen, paraquat, and X-irradiation is the production
MN-SUPEROXIDE
DISMUTASE
of reactive oxygen metabolites, supporting the hypothesis that the cellular oxidative state regulates Mn-SOD expression. TNF-a increases production of oxygen metabolites in several models. In cell culture, TNF-a increases 0,. (15, 23) and malondialdehyde (14), a free radical lipid peroxidation product. Anaerobic conditions or exogenous antioxidants (14, 29) significantly decrease TNF-c-w cytotoxicity, again suggesting that cellular effects of TNF-cu are mediated by oxygen metabolism. Whether the induction of Mn-SOD by TNF-cw involves reactive oxygen metabolites remains to be clarified. Under hyperoxic conditions, there is increased production of reactive oxygen metabolites including 0,. , HzOz and hydroxyl radical. Although oxygen radicals arise from multiple subcellular sources, mitochondria contribute at least 25% of whole lung oxygen radical production (27). This observation is consistent with the histological response of mitochondrial enlargement and inner mitochondrial membrane swelling seen early in pulmonary oxygen injury. Mn-SOD, strategically located in the mitochondrial matrix, may provide an important initial defense against 0, . To examine the protective effect of Mn-SOD against oxidant injury, H820 cells were pretreated with TNF-cw to increase Mn-SOD and then exposed to paraquat. Paraquat, a dipyridylium herbicide, increases mitochondrial production of 0~0. In our study, TNF-cu pretreatment significantly protected human pulmonary adenocarcinoma cells against paraquat-induced cell death. These results confirm the findings of Krall et al. (ll), who demonstrated that Mn-SOD protected HeLa cells against paraquat cytotoxicity. Generalizations from these findings must be made with caution. only in the mitochondria, Paraquat produces 0, whereas hyperoxia increases production in a number of subcellular organelles. Thus cellular injury associated with hyperoxia is likely to be more complex than with paraquat. Future experiments will directly determine whether increased Mn-SOD protects against oxygen injury in vitro and in vivo. TNF-a may also be protecting against paraquat through mechanisms unrelated to MnSOD. Wong et al. (28) reported that catalase and glutathione system enzymes were unchanged by TNF-a. However, TNF-a affects many cell processes, some of which could alter the cytotoxicity of paraquat. NIH H820 are similar to type II alveolar epithelial cells. The type II cell is important in oxygen injury because it contains a major portion of pulmonary SOD (7) and serves as a progenitor cell for repopulating the damaged alveolar epithelial lining. The current findings raise the possibility that inflammatory mediators may directly interact with respiratory epithelial cells to affect important protective and functional components of the lung. l
l
The authors wish to thank discussions and critical review This work was supported Blood Institute Grant HL-39605 Research Foundation. Address reprint requests to Received
5 February,
Jeffrey A. Whitsett for his many helpful of this paper. in part by National Heart, Lung, and to J. R. Wispi! and Children’s Hospital J. R. Wisp&
1990; accepted
in final
form
5 September
1990.
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TUMOR
NECROSIS
FACTOR
AND
MN-SUPEROXIDE
REFERENCES 1. ASOH, K., Y. WATANABE, H. MIZOGUCHI, M. MAWATAI, M. ONO, K. KOHNO, AND M. KUWANO. Induction of manganese superoxide dismutase by tumor necrosis factor in human breast cancer MCF7 cell line and its TNF-resistant variant. Biochim. Biophys. Acta 162: 794-801, 1989. 2. BRENNER, D. A., M. OHARA, P. ANGEL, M. CHOJKIER, AND M. KARIN. Prolonged activation ofjun and collagenase genes by tumor necrosis factor N. Nature Lond. 337: 661-663, 1989. 3. BRUNK, C. F., K. C. JONES, AND T. W. JAMES. Assay for nanogram quantities of DNA in cellular homogenates. Anal Biochem. 92: 497-500, 1979. 4. CRAPO, J. D., B. C. BARRYS, J. A. FOSCUE, AND J. SHELBURNE. Structural and biochemical changes in rat lungs occurring during exposure to lethal and adaptive doses of oxygen. Am. Reu. Respir. Dis. 122: 123-143, 1980. 5. FARRINGTON, J. A., J. EBERT, E. J. LAND, AND K. FLETCHER. Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implication for the mode of action of bipyridyl herbicides. Biochim. Biophys. Acta 314: 372-381, 1973. 6. FINTER, N. B. Dye uptake methods for assessing viral cytopathogenicity and their application to interferon assays. J. Gen. ViroL. 5: 419-427, 1969. 7. FORMAN, H. J., AND A. B. FISHER. Antioxidant enzymes of rat granular pneumocytes. Constitutive levels and effect of hyperoxia. Lab. Invest. 45: l-6, 1981. a. HASSAN, H. Biosynthesis and regulation of superoxide dismutases. Free Radical Biol. Med. 5: 377-385, 1988. 9. HO, Y., AND J. D. CRAPO. Isolation and characterization of cornplementary DNAs encoding human manganese-containing superoxide dismutase. FEBS Lett. 229: 256-260, 1988. 10. KESAVAN, P., P. DAS, J. KERN, AND M. DAS. Regulation of stability and synthesis of EGF-receptor mRNAs encoding for intact and truncated receptor forms. Oncogene 5: 483-488, 1990. 11. KRALL, J., A. C. BAGLEY, G. T. MULLENBACH, R. A. HALLEWELL, AND R. E. LYNCH. Superoxide mediates the toxicity of paraquat for cultured mammalian cells. J. Biol. Chem. 263: 1910-1914, 1988. 12. KRONKE, M., C. SCHLUTER, AND K. PFIZENMAIER. Tumor necrosis factor inhibits myc expression in HL-60 cells at the level of mRNA transcription. Proc. Natl. Acad. Sci. USA 84: 469-473, 1987. 13. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. Protein measurement with the Folin phenol reagent. J. BioZ. Chem. 193: 265-275, 1951. 14. MATTHEWS, N., M. L. NEALE, S. K. JACKSON, AND J. M. STARK. Tumor cell killing by tumor necrosis factor: inhibition by anaerobic conditions, free radical scavengers and inhibitors of arachidonic metabolism. Immunology 62: 153-155, 1987. 15. MEIER, B., H. H. RADEKE, S. SELLS, M. YOUNES, H. SIES, K. RESCH, AND G. G. HABERNEHL. Human fibroblasts release reactive
16 ’ 17
18 * 1g *
20*
21*
22.
23.
24.
25.
26.
27. 28.
29.
DISMUTASE
L301
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