JOURNAL OF CELLULAR PHYSIOLOGY 153491-497 (1992)

Regulation of Cu,Zn-Superoxide Dismutase in Bovine Pulmonary Artery Endothelial Cells X U E - J U N KONG AND BARRY L. FANBURG*

Depdrlment of Medione, Pulmonary Division, New England Medrcd Center, Boston, Mdsbdchusett, 02 7 I 1 To evaluate the regulation of endothelial cell Cu,Zn-SOD, we have exposed bovine pulmonary artery endothelial cells in culture to hyperoxia and hypoxia,

second messengers or related agonists, hormones, free radical generating systems, endotoxin, and cytokinesand have measured Cu,Zn-SODprotein of thew cells by an ELISA developed in our laboratory. Control preconfluent and confluent cells in room air contained 196 ? 18 ng Cu,Zn-SOD/lO' cells. A231 87 (0.33 pM), forskolin (10 pMj, isobutylmethylxanthine (0.1 mM), dexamcthasonc (1 pM), triiodothyronine (1 kM) and retinoic acid (1 pM) failed to alter this level of Cu,ZnSOD. Exposure to anoxia and hyperoxia both elevated the level -1.5-2.0-fold over 20% oxygen-exposed controls at 48-72 hr. Similarly, exposures to glucose oxidase (0.0075 units/ml), menadione (1 2.5 p M ) , xanthine-xanthine oxidase (10 pM, 0.03 unitsirnlj and H,O, (0.0005%)increased the level u p to two-threefold over controls at 2 4 4 8 h r . Lipopolysaccharide, TCF,,, TNF,, and 11-1 also increased levels of cellular Cu,Zn-SOU, but only in proliferating cells. 11-2, 11-4, interferon-y, and CM-CSF had n o effect o n Cu,Zn-SOD. All treatments that elevated SOD resulted in inhibition of cellular growth, but decreased growth of cells at confluence alone was not associated with increased Cu,Zn-SOU. We propose from these studies that Cu,Zn-SOD of endothelial cells is not under conventional second messenger or hormonal regulation, but that up-regulation of the enzyme is associated with (and perhaps stimulated by) free-radical or oxidant production that also may be influenced by availability of certain cytokines under replicating conditions. 6 1992 WiIcy-Liss, Inc. Superoxide dismutase (SOD) is a n important cellular enzyme for limiting tissue injury caused by enhanced 02-production. It accelerates the conversion of 0,- to H,O, and 0, by a dismutation reaction. The ubiquitous nature of SOD in all aerobic species has given rise to the hypothesis that this enzyme is essential for organisms that metabolize molecular oxygen (McCord and Fridovich, 1969; McCord et al., 1971). Unlike prokaryocytes, eukaryotic cells contain Cu,Zn-SOD and Mn-SOD, but no Fe-SOD (Fridovich, 1978). Yeast (Gregory et al., 1974) and endothelial cells (Housset and Junod, 1981) in vitro and rat lung (Crapo and Tierney, 1974; Stevens and Autor, 1977) in vivo have been found to possess elevated levels of SOD when exposed to hyperoxia. Administration of endotoxin to oxygen-exposed rats has also been shown to elevate the activity and content of SOD (Frank et al., 1980; Hass et al., 1982). Utilizing a radioimmunoassay for Cu,ZnSOD, Shiki et al. (1987) found that endotoxin alone had little effect in 24 h r on Cu,Zn-SOD content of bovine pulmonary artery endothelial cells in culture, whereas i t elevated that of Mn-SOD several-fold within this interval. Assayama et al. (1985) reported a similar effect of endotoxin on human monocytes in culture. More recently, Wong and Goeddel (1988) have described selective induction of mRNA of Mn-SOD, but not of Cu,ZnSOD, in a variety of human, mouse, and r a t cell lines in response to TNFa, TGFB1,and 11-1.Shaffer et al. (19901, 8.1

1992 WILEY-LISS, INC

Visner et al. (1990, 1991), and Kuni-ichi et al. (1989) obtained a similar induction of Mn-SOD mRNA in brain vessel endothelial, porcine pulmonary artery endothelial, rat pulmonary epithelial, and human breast cancer cells within several hours, but there was no effect on mRNA of Cu,Zn-SOD of these cells during this period of time. We undertook the present study to systematically compare the relative effects of a variety of agents, including hormones, second messengers, free radical generating systems, cytokines, hyperoxia, and hypoxia on Cu,Zn-SOD protein, measured by a sensitive ELISA, of pulmonary artery endothelial cells in culture. This is the first such study to our knowledge where a full spectrum of comparisons has been made directly on Cu,ZnSOD protein of the same endothelial cells. The results show that Cu,Zn-SOD of these cells is not influenced by hormones or changes in LCa+,l or CAMPbut is elevated reproduceably by agents that generate oxygen-based free radicals, H,O,, exposure to hyperoxia or hypoxia, lipopolysaccharide and certain cytokines, including TNF,,, TGF,,, and 11-1. In contrast, 11-2, 11-4, interferon-?, and GM-CSF had no effect on cellular Cu,Zn-

Received April 10,1992; accepted July 7,1992.

"To whom reprint requestslcorrespondence should be addressed.

492

KONG AND FANBURG

SOD. Elevations of Cu,Zn-SOD were usually associated with inhibition of cellular proliferation and occurred relatively late (after 24 hr) during the exposure periods, which may explain differences of our studies from previously reported data where measurements were only done within 24 h r (Assayama et al., 1985; Wong and Goeddel, 1988; Kuni-ichi et al., 1989; Shaffer et al., 1990; Visner et al., 1990, 1991).

rabbit antibovine Cu,Zn-SOD antiserum containing 4% human albumin that was prepared in our laboratory and incubated for 2 hr at 37°C. Plates were washed with TBS-T, filled with alkaline phosphatase-conjugated goat antirabbit 1gG (1:2,000 diluted in TBS-T containing 4% human plasma) and incubated again for 2 h r a t 37°C. Following this, the wells were washed again and incubated with 150 pl (per well) of substrate solution (one tablet of p-nitrophenyl phosphate in 20 ml 50 mM MATERIALS AND METHODS diethanolamine buffer) for 1 h r at room temperature. Glucose oxidase, menadione, xanthine, xanthine The reaction was stopped by the addition of 50 pl of 2N oxidase, TGF,,, TNF,, IL-1,2,4, IFN-)I, GM-CSF, NaOH. Purified bovine erythrocyte Cu,Zn-SOD lipopolysaccharide (E. coli, 026:B6), 3-isobutyl-l-meth- (Sigma) was utilized for preparation of standard curves ylxanthine (IBMX), dexamethasone, L-triiodothyro- that were linear in the 0.1-1.0 ng range, allowing meanine, isoproterenol, and forskolin were all from Sigma surements for as few a s 250 endothelial cells. Plates Chemical (St. Louis, MO). A23187 was from Calbio- were read in a Dynatech MR 5000 microplate reader. chem, Behring Diagnostics (La Jolla, CA). Hydrogen Dilutions were made when the linear range of measureperoxide was from the Fisher Scientific Co. (Pitts- ments was exceeded. burgh, PA). All chemicals were of the highest purity Oxygen exposures commercially available. Culture dishes were placed in sealed plastic chamCell culture bers (Billups Rothenberg, Delmar, CA) and gassed with Experiments were performed using 3rd-5th pas- different concentrations of oxygen, 5% CO,, and balsaged bovine pulmonary artery endothelial cells ob- ance nitrogen. We have previously reported that these tained as previously described (Shimada et al., 1981). cells tolerate exposures to anoxia quite well as long as Cells were cultured in RPMI 1640 (Gibco Lab, Grand substrate for glycolysis is present (Lee and Fanburg, Island, NY) supplemented with 10%)fetal bovine serum 1987). Control cells were maintained in these chambers (Sigma) and penicillin, 100 unitsiml (Marsam Pharma- with 20% O,, 5% COz, and balance N2. ceuticals, Cherry Hill, NJ), streptomycin, 100 pgiml (Eli Lilly, Indianapolis, IN), and amphotericin B, 1.25 Statistical analyses pgiml (Gibco Lab, Grand Island, NY). The cell seeding Each agent or exposure was tested in triplicate and density was approximately 4 x lo4 cells per 35 mm assays were done in duplicate. Statistical significance culture dish in 2 ml medium, which was replaced every 48 hr. The cells were cultured a t 37°C in humidified air was determined by Anova and represents P < 0.05. containing 5 6 COz until they reached various stages of RESULTS preconfluency or confluency, which was usually Effects of hormones and agents that elevate achieved a t 5-6 days. Then, test agents were added o r intracellular [Ca ’ 2] and CAMPon Cu,Zn-SOD cells were exposed to hypoxia or hyperoxia. Various agents were tested t o evaluate the influence Cell harvesting and counting and preparation of of hormones and second messengers on cellular Cu,Zncell-free extracts SOD. The Ca+2 ionophore A23187 and forskolin were At the end of exposure periods, culture media was used a t concentrations previously found to elevate enremoved by suctioning and cell monolayers were rinsed dothelial cellular ICa-”l and CAMP,respectively, of botwice with warm phosphate-buffered saline (PBS), vine pulmonary artery endothelial cells in culture pH = 7.4. Then, 2 ml per dish of 0.1% trypsin was added (Dasarathy and Fanburg, 1988,1989). IBMX was used and after a 2-min incubation, cells were dispersed by a t a concentration that increases levels of another progentle pipetting and a n aliquot was diluted in Isoton tein of the endothelial cells (i.e,,angiotensin converting (Fisher Scientific) for counting in a Coulter Counter, enzyme) (Dasarathy and Fanburg, 1988). DexamethaModel ZM. The remaining trypsinized cells were centri- sone, triiodothyronine, and retinoic acid, known agofuged and the pellet was suspended in 1 ml PBS, nists for the steroid receptor superfamily (Umesono pH = 7.4, and sonicated to prepare cell free extracts, et al., 1988; O’Malley, 1990) were similarly tested for which were stored a t -70°C prior to performing the their effects on cellular SOD. These experiments showed no stimulation of Cu,Zn-SOD by any of these ELISA. agents for intervals up to 72 h r of exposure (Table 1). ELISA for Cu,Zn-SOD There was also no influence on cell counts by these Cu,Zn-SOD measurements were carried out accord- agents. ing to a n ELISA developed in our laboratory (Das and by exposures of cells to Fanburg, 1992). In brief, 100 pl of Cu,Zn-SOD or cell Elevation of Cu,Zn-SOD anoxia and hyperoxia free extracts in 0.01 N Na,CO,, NaHCO, buffer, The next series of experiments evaluated the influpH = 9.6, were placed into wells of a 96-well microtiter plate (Dynatech Laboratories, Alexandria, VA) and in- ences of exposures to anoxia or hyperoxia (95% 0,) on cubated a t 65°C for 3 hr. The wells were then washed levels of endothelial cellular Cu,Zn-SOD. Both anoxia four times with Tris-buffered saline containing 0.05%) and hyperoxia blocked the proliferation of these cells Tween 20 (TBS-T). The wells were filled with 100 ~1 of (Fig. l a ) ,but stimulated a n elevation of cellular Cu,Zn-

493

REGULATION OF ENDOTHELIAL CELL Cu,Zn-SOD TABLE 1. Effect of hoimiones and second messengers on cellular proliferation and Cu, Zn-SOD'

Agent tested Control

24 h r

Cu.Zn-SOD. ng/1O6 cells (Cell number, x 10Gcells) 48 hr

72 hr

228 ? 19 (0.97 I 0.08)

204 I 17 (1.36 ? 0.14)

189 20 (1.68 t 0.14)

216 ? (0.95 ? 224 ? (0.98

0.09) 20 0.1)

196 I 15 (1.39_t 0.11) 211 2 21 (1.41 i 0.13)

166 ? 18 (1.70 t 0.16:) 197 I 12 (1.65 ? 0.15)

190 L 29 (1.02 i 0.1) 215 t- 12 (0.96 5 0.09)

225 i 25 (1.36 2 0.121 213 -t 26 17.42 ? 0.13)

175 2 14 (1.73 20.141 198 21 (1.7 i 0.15)

196 -C 15 (0.98 i 0.06) 241 i 28 (1.05 i 0.111

205 2 19 (1.32 f 0.12, 226: 2 11 (1.42 i 0.111

195 t 15 11.71 i 0.16) 214 t- 20 11.75 5 0.13)

204 2 15 (1.15i 0.09) 220 i 20 (1.05 i 0.1)

219 15 11.52 0.14) 217 i 14 (1.47 ? 0.12)

217 2 1 6 (1.02 ? O . l i 217 i- 20 (1.14 2 0.08)

223 2 16 (1.56 20.11) 228 t- 30 (1.62 2 0.12)

209 i 9 (1.03 t 0.08) 219 i 11 (1.13 0.09)

224 t 16 (1.54 0.11) 239 ? 25 (1.48 -t 0.13)

*

Dexamethasone 1 PM

1 nM

+

Triiodothylanine 1 PM 1nM

Retinoic acid 1 PM 1 nM

A23187 0.33 p,M 0.033 PM

Forskolin i n &M 1 PM

IBMX' 0.1 mM 0.01 rnM 'Mean t SD In 'PIUS

13

+

-

210 i- 21 (1.84 0.15) 211 2 19 (1.90 i 0.161 +

+

*

208 i 27 (1.88 2 0.14)

204 t- 15 (1.91 t- 0.17) 216 i 1 X (1.86 t- 0.19) 219 i 16 (1.85 i 0.16)

41.None of the differences were sta1isticall.v significant isoprotereno1110 'MM) =

T

T

5

zoo

0

too

48

,z

Exposure time (hours)

24

48

72

Exposure time (hours)

Fig. 1.(a) Effect of anoxia and hyperoxia on cell proliferation. Symbo1s:B 207i oxygen; D 0% oxygen; ISI 95% oxygen. Cells were exposed to anoxia or 95% 0, for intervals noted on abscissa. *P < .05 vs. 20% 0,. (n = 4).(b) Effect of anoxia and hyperoxia on cellular Cu,Zn-SOD. Exposures wcre t h e same as for (a).

SOD at 72 h r exposure (Fig. lb). Cellular morphological changes (enlargement, vacuolization) only occurred with exposure to hyperoxia for 72 h r or more. There were no detectable morphological changes with exposure to anoxia at any time during the study, consistent with our previous data (Lee and Fanburg, 1987).

Effect of oxygen-based free radicals on cellular proliferation and Cu,Zn-SOD A variety of methods to produce intra- or extracellular oxygen-based free radicals and H,O, were tested for their effects on Cu,Zn-SOD production by cells. All of

494

KONG AND PANBURG TABLE 2. Effects of oxidants on cellular proliferation and Cu, Zn-SOD'

Agent tested Control Glucose oxidase (0.005 U/ml)

24 hr

Cu, Zn-SOD, ng/106 cells (Cell number, x 10" cells) 48 hr

220 2 12 ( 1 0 s ? 0 12)

(1 51 t 0 09)

196 18 (1.97 ? 0.1)

28* 0 07) 710 i 41" (0 76 0 07)

370 i 22" 0 06) 542 + 43* (0 67 ? 0 06)

232 i 24 (0.81 f 0.1) 376 t 32" (0.65 -t 0.04)

276 -t 23* (106 0 11) 393 -t 32* ( 0 83 I 0 09)

314 + 12* ( 1 2 1 ? 0 1) 540 ? 43* (0 65 -t 0 0.5)

294 t 16" (1.27 t 0.14) 479 t 50" (0.53 0.05)

226 ? 19 (1 12 t 0 13) 233 ? 20 (0 9 s 0 08)

313 I 2 4 "

(0 96 I 0 09) 620 t 53* (0 82 2 0 06)

394 + 16* (0.9 -t 0.07) 670 -t 43* (0.75 t 0.08)

435

I0 84

(0.0075 U h l !

Menadione (10 pMj

+

+

+

(12.5 bM!

Xanthine (5pM) + xanthine oxidase (0.03 U/mll

+ xanthine oxidasc (0.06 Uiml) Hydrogcn peroxide 0.0005% 0.00104

Paraquat 150 p M )

*

470 2 23* (0 86 ? 0 05) 630 i 36* (071 2 0 0 5 ) 360 i 32" (0 84 2 0 06)

193 t 21

(0 8

+

510 t 41* (0 89 i 0 07) 672 -t 53" ( 0 76 i 0 08) 420 -t 39* (0 92 I 0 07)

72 hr

*

+

612 ? 48* (0.85 5 0.04) 789 t 52" (0.74 t 0.06) 300 2 28" (0.73 t 0.05)

'Significances regarding cell number diff'crmces are not Included. Mean z SD (11 - 41. -: .05, compared with control

*I'

these exposures, including those to glucose oxidase, menadione, xanthineixanthine oxidase, hydrogen peroxide, and paraquat, inhibited cellular proliferation and produced cellular injury and detachment at sufficiently high concentrations or prolonged exposures. All of the agents also elevated Cu,Zn-SOD, despite variations in time courses of this effect. Concentrations of the agents that elevated Cu,Zn-SOD while producing minimal or no cellular morphological changes are noted in Table 2. Enhancements of Cu,Zn-SOD occurred for most agents at 24 hr. However, the effect of xanthineixanthine oxidase was seen only at and after 48 hr. All of the exposures blocked cellular proliferation.

Effects of lipopolysaccharide and cytokines on Cu,Zn-SOD Lipopolysaccharide showed a profound effect on inhibition of cellular proliferation (Fig. 2a) and a t concentrations > 100 ngiml produced marked alteration in cellular morphology. Use of a lower concentration (10 nginil) resulted in more moderate inhibition of cellular proliferation and caused elevation of Cu,Zn-SOD a t and after 48 h r in culture (Fig. 2b). A variety of cytokines were tested for their effects on Cu,Zn-SOD (Table 3). TGF,, (2 ngiml), TGF (2 ngi ml) + FeC1, (40 pm), TNFa (10 ngiml), and Pi-1 (500 unitsiml) showed no effect on confluent cells (data not shown), but these concentrations of cytokines reduced proliferation of cells and elevated Cu,Zn-SOD when applied at lower cellular densities and times of more active cellular proliferation. The combination of TGFPl + FeC1, produced a striking elevation of Cu,ZnSOD, consistent with our previous observations of enhancement of "pro-oxidant" effects of TGFPl in the presence of iron (Das and Fanburg, 1991). Several other

cytokines including 11-2 (500 Uiml), 11-4 (1ngiml) interferon-y (100 Uiml), and GM-CSF (100 Uiml) had no effect on Cu,Zn-SOD when tested on preconfluent cells.

Effect of cellular proliferation on stimulation of Cu,Zn-SOD We considered the possibility that inhibition of proliferation might nonspecifically account for the stimulatory changes of Cu,Zn-SOD that we have observed in these studies. To test the effects of cellular proliferation on Cu,Zn-SOD we performed two sets of experiments. In the first set we tried to growth arrest the cells by performing cultures at low concentrations of FBS, or in the absence of FBS. These experiments were unsuccessful since the endothelial cells were either not growtharrested or injured by this condition of culture, probably due to the formation of endogenous mitogen(s1 by endothelial cells. Second, we plated cells a t low density in a large number of dishes and removed a set of dishes daily for measurement of cell counts and Cu,Zn-SOD. This study showed that SOD levels per cell were no different during rapid stages of proliferation from those at confluence (days 8-12) when proliferation had ceased (Fig. 3). DISCUSSION Cu,Zn-SOD regulation has received considerable attention during the last decade because of its protective effects against oxidant injury and oxygen toxicity. Early studies showed that endotoxin administered t o rats subsequently exposed to hyperoxia elevated total lung SOD activity (Frank and Roberts, 1979; Frank et al., 1980; Hass et al., 1982). The stimulation of SOD activity by hyperoxia and endotoxin (Salmonella typhimurium) was subsequently confirmed in porcine pulmonary artery endothelial cells (Block, 1983). Cu,Zn-

495

REGULATION OF ENDOTHELIAL CELL Cu,Zn-SOD

a

w,

$

600

=. g

500

P

-

400

fi 0

2 7v

300

200

100

0 24

48

72

96

72

24

Incubation time (hours)

Exposure Time (hours)

Fig. 2. Lipopolysaccharide effect on cellular growth (a, and Cu-Zn-SOD (b). Experiments were carried out as described in Materials and Methods. n = 4. Symbols: LPS (10 ng/ml); LPS (20 control; ng/ml). * P < .05 compared with control.

TABLE 3. Effects of cytokines on cellular proliferation and Cu, Zn-SOD’

Aeent tested Control

TGF,,

(2.0 ngiml) +FcCl, (40 pM)

TNF,, 10 ngiml 20 ngiml Control 11-1 (500 Uiml)

11-2 (500 Uiml) 11-4 (1ng/ml!

IFN--y (100 U/ml) GM-CSF (100 Uiml)

24 h r

Cu, Zn-SOD, ng/lOGcells (Cell number, x 10‘ cells) 48 hr 72 hr

180 2 11 (0.21 -t 0.01) 220 ? 21x (0.20 % 0.01) 294 % 25* (0.2 -t 0.02) 210 I8.0“ (0.21 ? 0.01) 230 f 21* (0.22 0.02)

*

175 19 (0 65 0 021 310 r 22+ (0 43 -t 0 021 360 L 18* (0 39 2 0 02) 220 % 13* (0 49 r 0 03) 254 i 31* (0 45 0 04)

192 i 17 (0.2 i 0.02) 231 2 21 (0.2 % 0.01) 221 i 30 (0.2 * 0.02) 200 + 22 (0.2 ? 0.02) 195 2 18 (0.2 5 0.01) 208 22 (0.2 0.01)

210 2 20 (0 7 i 0051 310 -t 29* (0 56 2 0 041 205 I 21 (0 65 i 0 06) 212 i- 13 (0 71 i 0 04) 210 21 (0 68 i 0 03) 225 -t 25 (0 66 i 0 041

* *

+

+

*

*

96 h r

200 * 15 (101 I 0 05) 446 I 5 0 * ( 0 56 t 0 04) 1206 2 89* (0 37 2 0 02) 412 2 34* (0 55 2 0 04) 426 2 39* (0 52 t 0 04)

145 i 12 (1.22 ? 0.09) 360 ? 25* (0.67 i 0.06) 450 i 25” (0.62 i 0.051 270 ? 19” (0.81 T 0.07) 316 -t 23” (0.74 i 0.041

225 t 25

185 i 16 (1.32 i 0.1) 380 i 29“ (0.8 2 0.04) 175 % 17 (1.28 -t 0.091 180 i 17 (1.28 i 0.011 197 i 20 (1.3 ? 0.07) 160 ? 13 (1.33 ? 0.08)

(11 t 0 07)

400 t 35* (0 78 2 0 05) 190 % 17 (1 09 0 11 200 2 20 (1 05 % 0 1) 233 22 (1.11-t 0.061 225 -t 21 (1.08 2 0.05)

*

+

‘Mcan + SD In - 41. *ps: .05, compared with contrui

SOD mRNA along with Cu,Zn-SOD protein measured by radioimmunoassay were then reported to be elevated in rat lung by the administration of 500 pgikg endotoxin (Iqbal et al., 1989). Thus, many studies have indicated that the lung and at least one of its constituent cells (the endothelial cell) respond to hyperoxia and endotoxin by a n elevation of the level of Cu,Zn-SOD. In contrast, more recent data have presented possibly conflicting results by demonstrating that mRNA for Mn-SOD, and not that for Cu,Zn-SOD, is stimulated in a large variety of cell culture systems exposed to cytokines and lipopolysaccharide that may produce free radical intermediates (Wong and Goeddel, 1988). Mn-

SOD expression and enzymatic activity are elevated in human adenocarcinoma cells in response to TNFa (Warner et al., 1991). Mn-SOD mRNA elevation has also been reported t o be a n early response of lungs of the rat subjected t o hyperoxia in vivo, but Mn-SOD enzymatic activity was not elevated concurrently with the increase in mRNA (Chiang et al., 1990; Ho et al., 1990). We have made use of a highly sensitive ELISA to evaluate possible modes of regulation of Cu,Zn-SOD in endothelial cells. Although the elevation of second messengers and treatment of cells with hormones have been demonstrated to stimulate the synthesis of some

496

KONG AND FANRURG

lung homogenates from rats exposed to hypoxia, and Frank (1982) has demonstrated protective effects against oxidant injury of rat lung by pre-exposures to hypoxia prior to hyperoxia. It is also possible that a stimulat,ion of free radical generation results from exposure to relative hyperoxic conditions after removal of cells from the hypoxic environment prior to carrying out assays. Thus, all exposures of bovine pulmonary artery endothelial cells to factors or agents that enhance formation of free radicals or their byproducts elevated cellular Cu,Zn-SOD. Coupled with our data that hormones and second messengers have no effect on cellular Cu,Zn-SOD, the results suggest a direct action of free . I . I . I . I . 100 0 2 4 6 8 1 0 1 2 radicals or their byproducts on a n intracellular site(s) Culture time ( days) to stimulate processes for Cu,Zn-SOD production. Why there is a delay in Cu,Zn-SOD production in response to Fig. 3. Effect of cellular proliferation on Cu,Zn-SOD. Cells were the stimulus is not known. Although we have not yet seeded at - 4 X lo4 cellsidish in a series of culture dishes (time 0)and evaluated the influence of the agents tested on genetic at each subsequent day 4 dishes were removed for cell counts and expression of Cu,Zn-SOD in our cells, Jornot and Junod measurement of Cu,Zn-SOD. (n = 4). (1991) have shown that exposures to one oxidant (hyperoxia) actually decrease general protein synthesis of human umbilical vein endothelial cells a t a translaproteins in cells in culture, the production of Cu,Zn- tional level and have provided data indicating that the SOD by these cellular events, to our knowledge, has site of stimulation of Cu,Zn-SOD, at least in response to never been assessed. We have found that agents that hyperoxia, is a t the cellular level of transcription. Enwe previously demonstrated to elevate [Ca2+J and hanced expression of Cu,Zn-SOD mRNA with exposure CAMPof endothelial cells had no effect on Cu,Zn-SOD to hyperoxia took 3-5 days to occur in their experimenof these cells. These results differ from a 46-fold eleva- tal system (Jornot and Junod, 1992). We have considered the possibility that enhancement tion of Cu,Zn-SOD activity by calcium reported during sporulation of Physarum Polycephalum mold (Nations of levels of cellular Cu,Zn-SOD may be simply reguet al., 1987). Exposures to a variety of intra- and extra- lated by the growth status of the cells since all treatcellular free radical generating systems, H,O,, hyper- ments that elevated Cu,Zn-SOD also decreased cellular oxia, hypoxia, LPS, TNFa, TGFP1, and 11-1 all in- proliferation. Since it was difficult to totally growth creased cellular levels of Cu,Zn-SOD. Pronounced arrest endothelial cells by deletion of serum from the among the responses was the elevation caused by culture medium, we assessed the effect of growth reguTGFPl in the presence of added FeCI,. We have previ- lation in Cu,Zn-SOD by measuring Cu,Zn-SOD in cells ously reported that the combination of TGFPl and a t different stages of proliferation. The Cu,Zn-SOD conFeC1, has a strong “pro-oxidant” effect on endothelial tent of cells a t confluence (nonproliferating) was no cells and have attributed it to the facilitation of the different from that of highly proliferating cells, sugHaber-Weiss reaction where iron is required for the gesting that the extent of cellular proliferation did not generation of hydroxyl radical (Das and Fanburg, influence the level of Cu,Zn-SOD. Growth restriction of 1991). A requirement for the presence of proliferating cells in the presence of 0.01% serum and subsequent cells and optimization of the cytokine/cellular ratio is synchronized release indicated that Cu,Zn-SOD mRNA also consistent with our previous data showing inhibi- expression occurred primarily in the S-phase of cell tion of proliferation of endothelial cells by TGFPl cycling (unpublished data). The precise associations of lipopolysaccharide and cytokines with free radical for(White et al., 1992). Lipopolysaccharide has also been reported to produce mation and mechanisms of stimulation of gene exprescellular actions through generation of free radicals sion for Cu,Zn-SOD by free radical formation remain to (Shiki e t al., 1987; Visner et al., 1990) and its effect on be determined. Cu,Zn-SOD of endothelial cells is consistent with the ACKNOWLEDGMENTS effects of better recognized systems for free radical genThis research was supported by HL42376 from the eration (e.g., xanthinelxanthine oxidase, menadione (Hjordis e t al., 1982) and paraquat (Hassan and Frido- NHLBI, National Institutes of Health. We are indebted vich, 1979; Lee and Hassen, 1985; Matters and Scandal- to Ms. Deborah La Perche for preparing the manuscript. ios, 1986; Krall e t al., 1988). LITERATURE CITED Exposure t o hyperoxia is generally recognized to enhance formation of 02-based free radicals and their pro- Assayama, K., Janco, R.L., and Burr, I.M. (1985)Selective inductionof tective enzymes, but it is less well established that exMn-SOD in human monocytes. Am. J. Physiol., 249:C393-C397. posures to hypoxia may produce a similar effect, Block, E.R. (1983) Endotoxin protects against hyperoxic alterations in lung endothelial cell metabolism. J . Appl. Physiol. Respirat. Enviperhaps through generation of increased NADH reducran. Exercise. Physiol., 54f‘I/:24-30. ing equivalents via lactose metabolism and enhanced Chiang, A.A., Crapo, J.D., and Ho, Y.S. (1990) Expression of antioxiformation of 0, . Sjostroni and Crapo (1983) have redant enzymes during hyperoxia in neonatal rat lungs. Am. Rev. Repir. Dis., 141 (suppZi:A 819. ported enhanced cyanide insensitive oxygen uptake in 4

-ry-

Cellnumber

--c

CuZn-SOD

380

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