Glucocorticoids inhibit TNFα-induced cytosolic phospholipase A2 activity

Bioch#nica et Biophyskra Acta, 1127 (1992) 163-167 © 1992 Elsevier Science Publishers B.V. All rights reserved 11005-2760/92/$05.00

163

BBALIP 53973

Glucocorticoids inhibit TNFa-induced cytosolic phospholipase A 2 activity Margarete Goppelt-Struebe and Wolfgang Rehfeldt Institute of Moh,cular Pharmacology, Medical School Hamzoz,er, Hannot'er (Germany) (Received 2 February 1992)

Key words: Cytosolic phospholipase A.~; Glucocorticoid; Tumor necrosis fiictor; Phospholipase A

The cytosolic phospholipase A 2 (PLA 2) was characterized in the human epithelial carcinoma cell lille HEp-2 by its apparent molecular mass (about 8{) kDa); its in vitro activation by micromolar concentrations of calcium; and its calcium-dependent associatien with cellular membranes. The activity of this enzyme was induced by an overnight incubation with tumor necrosis factor a (TNFa). Glucocorticoids only moderately reduced PLA. aci'dvity in c,.m:rol cells, but completely inhibited the TNF¢~-induced increase in the activity of the high-molecular-weight cytosolic PLA2.

Introduction

Inflammatory reactions are characterized by complex interactions between various types of cell. Cytokines and arachidonic acid metabolites have been shown to play an essential role in mediating cell--cell interactions. Inflammatory cytokines, interleukin 1 and 6, and tumor necrosis factor alpha (TNFa), induce the synthesis of eicosanoids. Prostaglandins, on the other hand, act back on the synthesis of cytokines, thus building one of the multiple metabolic autocrine or paracrine loops. The primary reaction in the biosynthesis of eicosanoids is the release of arachidonic acid from the membrane phospholipids, which in most cells is mediated by phospholipases A 2 (PLA2). Besides the pancreatic-type enzymes, two types of PLA 2 have been characterized. The low-molecular-weight PLA2s (type 11) are membrane-bound enzymes, which are secreted and function extracellularly [1,2]. These enzymes are found in inflammatory lesions, e.g. the synovia of inflamed joints [3]. Their synthesis is induced by cytokines [4-8] and they have been shown to act pro-inflammatory when given exogenously to cells [9]. However, these PLA2s have no substrate specificity for arachidonic acid or other polyunsaturated fatty acids, Correspondence to: M. Goppelt-Struebe, Medizinische Klinik IV, Universifiit Erlangen-Niirnberg, Loschgestr. 8 1/2, D-8520 Erlangen, Germany. Abbreviations: TNFa, tumor necrosis factor; PLA 2, phospholipase A 2•

i.e., no specificity for the fatty acids, which are the precursors of eicosanoid synthesis. The second type of PLA 2, a high-molecular-weight PLA 2, has been characterized very recently in platelets, mesangial cells and monocytic cells, macrophages and corresponding precursor cells (Refs. 10-12 and citations therein). The enzyme is located intraceilularly and preferentially cleaves araehidonic acid or other polyunsaturated fatty acids, which suggests its importance in eicosanoid biosynthesis. In mesangial cells it has been shown to be regulated by arginine vasopressin and epidermal growth factor [13,14]. The effect of cytokines on the synthesis or activity of this type of PLA2 has not yet been characterized. Tumor necrosis factora (TNFa) has been shown to be an inducer of eicosanoid synthesis in many cell types. It also induces the release of arachidonic acid, indicating an effect on phospholipases. An activation of the secreted PLA 2 has been demonstrated and rece',~tly been confirmed on the molecular level, i.e., mRNA and protein synthesis [5-8]. An effect of TNFa on the high molecular weight cytosolic PLA 2, however, has not yet been shown, partly due to the fact that in some earlier papers the activity measured was not attributed to one or the other form of PLA 2. Glucocorticoids, which are potent anti-inflammatory drugs, have long been discussed as inducers of lipocortins, which were suggested to interact with PLA 2 and thereby should mediate the effect of glucocorticolds on eicosanoid synthesis. This concept has been questioned by the results of many different investigators [15]. Very few data are available on the effect of

164 glucocorticoids on the activity of intracellular phospholipases, membrane-bound or cytosc~!ic. We have shown previously that glucocorticoids inhibit membrane-bound PLA 2 activity in bone-marrow-derived macrophages [16] and human monocytic cells [17], without being able at that time to further characterize the type of PLA2 involved. We now present data, showing the induction of the high.molecular-weight cytosolic PLA 2 by TNFa and the inhibition of the enzyme activity by glucocordcoids. Materials and Methods Cultivation of cells. The human epidermoid larynx carcinoma cell line HEp-2 was obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured in MEM medium (Gibco), supplemented with 5% FCS. In activation experiments, HEp-2 cells were grown to confluence in 40-ram.diameter petri dishes. They were pre-incubated with or without dexamethasone phosphate (ga-fluoro16t~-methyl-prednisolone phosphate, Decadron ~, MSD Merck, Sharp & Dohme, Miinchen, Germany) for 3 h and subsequently incubated with rh T N F a (BASF/Knoll, Ludwigshafen, Germany) for 14 h (concentrations as indicated). At the end of the incubation, the cells were washed twice with phosphate-buffered saline (137 mM NaCl/2.7 mM KCI/8 mM Na2HPO4/ 1.5 mM KH2PO 4) and removed with a rubber policeman. Cell fractionation. The cells were disrupted by sonication in 20 mM Tris-HCI (plt 7.4)/340 mM sucrose/5 raM 2-mercaptoethanol/I mM EDTA. In some experiments, CaCi, (1 mM or 0.5 mM) was added instead of EDTA. Cellular organelles were removed by centrifugation at 6500 x g for 20 rain. Cytosol and membranes were separ:lted [~y c¢ptrifugation at 100000 × for 45 rain in a B,'ckman TLI00 ultracentrifuge. Membranes were resuspended in the same buffer as described above. Protein was determined by a micro-titer Bradford assay ~,,,ith bovine serum albumin as standard

[181, Characterization of PLA,. The cytosol of HEp-2 cellswas prepared as described above and fractionated on an FPLC su~e~ose 12 column (Pharmacia LKB) equilibrated with buffer containing I0 m M Tris-HCl (pH 7.6)/150 m M NaCI/10% glycerol (v/v)/5 m M 2-mercaptoethanol/0.1 m M phenylmethanesulphonyl fluoride/l /zg/ml leupeptin). The column was calibrated with blue dextran (2 MDa), catalase (230 kDa), •},-globulin(169 kDa), bovine serum albumin (67 kDa), carbonic anhydrase (29 kDa) and tt-lactalbumin (14 kDa) as molecular mass standards. Determination of PLA 2 activity. The activity was determined with t~-l-palmitoyl-2-[1-~4C]arachidonyipbosphatidylcholine (New England Nuclear, Boston, MA, USA) as substrate as described previously [16]. If

not indicated otherwise the assay mixture contained 1 /~M phospholipid, corresponding to about 30 000 cpm, 5 mg bovine serum album[n/ml, 0.3/z M dioleylglycerol and 5 mM CaCi 2 in 100 mM Tris-HC! (pH 9.5). The micromolar calcium concentrations were calculated by a computer program, using EGTA/CaCI a mixtures in 20 mM Tris/HCi (pH 7.6). Reaction time was 15 to 30 min at 37°C. The liberated fatty acid was separated from substrate that had not reacted by heptane extraction, and the radioactivity was determined by liquidscintillation counting. Results and Discussion

Characterization of the PI~!, in HEp-2 cells The intracellular PLA 2 is defined by its molecular weight, calcium-dependent activity, its calcium-dependent association with cellular membranes and its substrate specificity. This enzyme was characterized in the human epithelial carcinoma cell line HEp-2. In this cell line TNFa has been shown previously to induce prostaglandin synthesis [ 19]. The cells were disrupted by sonication and the cytosolic fraction was prepared in the presence of 1 mM EDTA, to shift the enzyme activity h,to the cytoso! (see below). The buffer contained 2-mercaptoethanol to inhibit type ii PLA 2, which possibly might also exist in these cells. Therefore, no enzyme activity was determined, when the cytosol or the membrane fraction were assayed with oleic-acid-labelled phospholipids as substrate (data not shown). The size of the enzyme was determined by gel filtration. The column was run with a buffer containing 150 mM NaCl, in order to avoid enzyme aggregation. The enzyme activity eluted as a single peak (Fig. la). Comparison with standard proteins indicated a molecular mass of 70 to 80 kDa (inset in Fig. l a). This molecular mass is in agreement with that deduced from the amino-acid composition of the enzyme from U937 cells [10,11]. No activity peak or shoulder was detected in fractions 13 to 14, which would correspond to a low-molecular-mass enzyme ( < 20 kDa). Using arachidonylphosphatidylcholine as substrate, the enzyme activity was optimal at alkaline pH. Addition of diolein to the lipid/bovine serum albumin substrate was not mandatory but enhanced the enzyme activity as has been shown for other PLA 2s of this type as well [20]. The enzyme was active at micromolar concentrations of calcium in the in vitro assay The activity was further enhanced, when millimolar concentrations of calcium were used (Fig. lb). The distribution of the enzyme between the cellular fractions was dependent on the calcium concentration in the buffer used for cell fractionation: The membrane-bound fraction of the PLA 2 was enhanced when EDTA was omitted from the buffer (Fig. lc). The calcium-depen-

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dent association with the membrane, the in vivo substrate of the enzyme, is discussed as one mechanism of activation of this enzyme [21-23]. We could thus show that the enzyme activity, determined in the membranes and the cytosolic fractions by the assay described above, is attributed to the cytosolic high-molecular-weight type of PLA 2.

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Figure 2. Induction of the PI,A 2 activity by TNFa. HEp-2 cells were incubated with TNFa for 14 h in the concentn~tions indicated. PLA2 activity was determined in the cytosol in the presence of 5 mM CaCI 2. Data points with error bars indicate means :t: S.E. of 3-5 independent experiments.

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Fig. 1. Characterization of the cytosolic PEA 2 in HEp-2 cells: a) I,)¢termination of the molecular weight by chromatography on a superose 12 column, b) Calcium-dependency of the cytosolic PLA2 in the in vitro assay. Data are means+S.D, of triplicates, c) Calcium-dependent distribution of the PLA 2 between membranes and cytosol. The cells were fractionated in the presence of (A) ! mM EDTA, (D) 0.5 mM EDTA, (C) no additives, (D) 0.5 mM CaCI 2, (E) ] mM CaCI2. The enzyme activity was determined in the membrane (hatched bars) and cytosolic fraction (open bars) in the presence of 5 mM CaCi2.

Induction of the high molecular weight PLA 2 by TNFa TNFa interacts with cells by binding to surface receptors, which have also been characterized on HEp2 cells [24,25]. Functionally TNFa induces antiviral activity in these cells [26], mediated by the induction of interferon fl 1 [27]. As one of the multiple cellular events following receptor activation, the release of arachidonic acid due to PLA 2 activation has been described. In contrast to the rapid activation of type 11 PLA2s by TNFa reported in the literature (e.g., Refs. 8, 28), there was no effect of TNFa on the activity of the high-molecular-weight enzyme detectable in HEp-2 cells within minutes or hours (incubation time up to 4 h; data not shown). Incubation of HEp-2 cells with TNFa for 14 h induced a concentration-dependent increase in PLAz activity, determined in the cytosolic fraction (Fig. 2). The enhanced activity was measurable in the in vitro assay at micromolar as well as millimolar calcium concentrations (Table I). Incubation of the cells with cycloheximide, an inhibitor of protein synthesis, prevented the induction (data not shown). The time course of induction and the sensitivity to cycioheximide suggest that the higher activity in TNFa-treated cells was due to an increase in de novo protein synthesis rather than an activation by enzyme modification. The molecular

166 TABLE ! ,c:

Induction of the cytosolic phospholipase A: by TNFa Phospholipase A2 activity was determined in n = 5 independent preparations (* P < 0.02 * *P < 0.001, paired t-test). Induction rate: ratio of the activities in TNFa-induced cells to control cells. Phospholipase A 2 activity (pmol/mg protein per rain) Assay condition:

1 ~ M CaCI 2 (pH %0)

5 mM CaCi 2 (pH 9.5)

Control TNFa5ng/ml

14.2 :!: 6.0 23.1 +11.2 *

647 1008

lnductionrate

!.58± 0.15

E1200 r=

-~ ¢= 1000 ,_> ,,b,,, C~

±123 4-189"*

!.56±

800

60O

--J

"

400

0.11

mechanism of PLAz-induction by TNFa, however, remains to be investigated.

Interference of glucocorticoids with PLA : activity HEp-2 cells were pre.incubated with dexamethasone for 3 h and then incubated with or without TNFa for 14 h to induce PLA 2 activity. A concentration-dependent inhibition of PLA 2 activity was observed (Fig. 3). There was a modest reduction of the specific PLA 2 activity in the untreated cells. The TNFa-induced induction of PLA 2 activity, however, was almost completely impaired by dexamethasone at 10 -6 or 10 -7 M, but not reduced below the activity determined in untreated cells (Table ll). The effect was independent of the in vitro assay conditions. A similar effect of glucocorticoids has been observed before: in U937 cells, glucocorticoids had no effect on the basal PLA 2 activity, but completely abolished the phorbolester-induced increase in enzyme activity [17]. This implicates that glucocorticoids wilt interfere most effectively with the activated enzyme, e.g., in inflamed tissues. We could thus show for the first time that the high-molecular-weight ¢ytosolic phospholipase A 2 activity is induced by TNFa and that the intiuction is impaired by incubation with dexamethasone. This system will thus permit further investigation of the molec-

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were incubated with (e) or without ( v ) TNFa (5 ng/ml) and the concentrations of dexamethasone as indicated. PLA: activity (means ± S,D. of triplicates) was determined in the cytosol in the presence of 5 mM CaC! 2.

ular mechanisms underlying the changes in enzyme activity. Acknowledgements The technical assistance of Mrs. S. Eickemeier is gratefully acknowledged. This work was supported by a grant from the Stiftung Niedersachsen (467-013/91). References ! Dennis, E.A., Rhee, S.G., Billah, M.M. and Hannun, Y.A. (19ql) FASEB J. 5, 2068-2077. 2 Pruzanski, W. and Vadas, P. (1991) lmmunol. Today, 12, 143-146.

3 Seilhamer, JJ., Plant, S,, Pruzanski, W., Schilling, J., Stefanski, E., Vadas, P. and Johnson, L.K. (1989) J. Biochem. 106, 38-42. 4 Suf~s P., Van Roy, F. and Fiers, W. (1988) FEBS Left. 232, 24-28. 5 Nakalto, T.00hara, O., Teraoka, H. and Arita, H. (1990) FEBS Left. 261, 171-174. 6 0 k a , S. and Arita, H. (1991) J. Biol. Chem. 266, 9956-9960.

TABLE !!

EJyec¢ o[ dexamethasone on the actirity of the cyrosolic phospholii~se A: Phmpholipa~ A : activity was determined as indicated. To compare experiments with different absolute activities, the act!vitv or ccatrol cells was taken as 100%. Data are means ± S.D. of a experiments; * P < 0.005 compared to TNFa-treated cells. Phospholipase A 2 activity (relative to control) [Dexam~thu~one]:

0

10 -~ M

10 -~' vl

A,v,wy ~th ! ~,M C,O: Ca = 5) Control TNFa 5 ng/ml

100 i58.4 ± 16.0

65.1 ± 10.0 94.4-*- 15.4 *

65.5+ 14.4 93.1 ± 19.3 *

Assay w~h 5 mM CaCl: ea -- J) Control TNFa 5 ng/ml

100 162 ± 6

92 ±13 111 ± 6 "

99 ± 9 99 ± 1 4 "

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