- Email: [email protected]
Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1β- and cAMP-induced group II phospholipase A2 expression in rat renal mesangial cells
Biochimica et Biophysica Acta 1391 Ž1998. 213–222
Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1 b- and cAMP-induced group II phospholipase A 2 expression in rat renal mesangial cells Gaby Walker
a,1
, Dieter Kunz
a,1
, Werner Pignat b, Josef Pfeilschifter
c,)
a
c
Department of Pharmacology, Biozentrum, UniÕersity of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland b Research Department, Pharmaceuticals DiÕision, NoÕartis Pharma, CH-4002 Basel, Switzerland Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-UniÕersitat, ¨ Theodor-Stern-Kai-7, D-60590 Frankfurt am Main, Germany Received 22 August 1997; revised 21 November 1997; accepted 27 November 1997
Abstract Expression of group II phospholipase A 2 ŽPLA 2 ; EC 3.1.1.4. in rat renal mesangial cells is triggered in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin 1 b ŽIL-1 b . or tumor necrosis factor a and agents that elevate cellular levels of cyclic AMP ŽcAMP. such as forskolin, an activator of adenylate cyclase. Treatment of mesangial cells with IL-1 b or forskolin for 24 h induces group II PLA 2 activity secreted into cell culture supernatants by about 15-fold and 11-fold, respectively. Platelet-derived growth factor ŽPDGF.-BB potently inhibits secretion of IL-1 b- and forskolin-induced group II PLA 2 activity. By Western and Northern blot analyses, we demonstrate that this is due to a reduction of PLA 2 protein levels and the corresponding PLA 2 mRNA steady-state levels. Basic fibroblast growth factor ŽbFGF. virtually does not inhibit IL-1 b-stimulated group II PLA 2 activity, but markedly inhibits forskolin-induced expression of group II PLA 2 activity. These effects are caused by changes in the corresponding PLA 2 protein and PLA 2 mRNA steady-state levels. Inhibition of protein kinase C ŽPKC. by the potent and selective PKC inhibitor calphostin C converted the inhibitory action of PDGF into a bFGF-type of response thus suggesting that PKC is a major effector in PDGF-induced inhibition of IL-1 b-stimulated group II sPLA 2 expression. In summary, our data suggest that PDGF and bFGF differentially modulate in a stimulus-specific manner the expression of group II PLA 2 in mesangial cells. q 1998 Elsevier Science B.V. Keywords: Phospholipase A 2 , secretory; Gene expression; Interleukin 1 b ; Cyclic AMP; Platelet-derived growth factor; Fibroblast growth factor
1. Introduction Phospholipase A 2 Ž PLA 2 . comprises a group of lipolytic enzymes that specifically release fatty acids, ) 1
Corresponding author. Fax: q49-69-6301-7942. Both authors contributed equally to this study.
often arachidonic acid, from the sn-2 position of membrane phospholipids for production of important lipid mediators such as eicosanoids and plateletactivating factor w1,2x. Arachidonic acid and its numerous metabolites act as intracellular and intercellular messengers contributing to normal cellular physiology by modifying the activity of intracellular en-
0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 2 0 4 - X
214
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
zymes and ion channels. Moreover, PLA 2 and its products function as substrates for the generation of inflammatory lipid mediators that play an important role in the pathogenesis of inflammatory diseases. In most tissues and cells, the synthesis of eicosanoids is limited by the availability of intracellular free arachidonic acid. The major mechanism considered to control the level of intracellular free arachidonic acid is the activation of PLA 2 and the direct release of the fatty acid from membrane glycerophospholipids. In recent years, four distinct types of phospholipases have been identified: Ž1. secretory PLA 2 ŽsPLA 2 ., a disulfide cross-linked, low molecular weight protein of 13–15 kDa which is secreted by cells and requires mM Ca2q for optimal activity; Ž 2. cytoplasmic PLA 2 ŽcPLA 2 . , a protein with a molecular weight of 70–80 kDa with a different requirement for Ca2q Ž m M rather than mM Ca2q .; Ž3. intracellular Ca2q-independent PLA 2 ŽiPLA 2 . with a molecular weight of 80 kDa; and Ž4. platelet-activating factor acetylhydrolase ŽPAF-AH or PAF-PLA 2 ., a group of several isozymes with molecular weights ranging from 29 kDa to 45 kDa which, most prominently release acetyl groups Ž for review, see Ref. w3x.. Based on their primary structure, the sPLA 2 s are further classified into two groups w4x. Mammalian group I PLA 2 comprises the pancreatic-type of PLA 2 and mammalian group II PLA 2 is synthesized and secreted from many cell types and is believed to play a role in the initiation and propagation of inflammatory processes Ž for review, see Ref. w5x.. Two potent pro-inflammatory cytokines, IL-1 b and TNFa , have been shown to induce the synthesis and secretion of group II PLA 2 in mesangial cells w6,7x and in a variety of other cell types Ž for review, see Ref. w8x.. The cytokine effect is blocked by actinomycin D and cycloheximide, thus demonstrating a requirement for both transcription and de novo protein synthesis. This is confirmed by the observation that IL-1 b and TNFa increase the level of mRNA for group II PLA 2 in these cells w9,10x. We also demonstrated an increased secretion of group II PLA 2 in rat vascular smooth muscle cells w11x and rat mesangial cells w12,13x in response to cAMP-elevating agents. Furthermore, forskolin, an activator of adenylate cyclase, synergistically interacts with IL-1 b to increase group II PLA 2 mRNA levels in mesangial cells w14x.
The aim of this study was to evaluate whether growth factors such as PDGF and FGF, known to play an important role in the regulation of the inflammatory response and wound healing, would modulate induction of group II PLA 2 in rat renal mesangial cells. We show that PDGF-BB suppresses IL-1 b- and cAMP-induced group II PLA 2 expression, whereas bFGF virtually does not affect IL-1 b-, but inhibits cAMP-stimulated group II PLA 2 expression. 2. Materials and methods 2.1. Materials Recombinant human IL-1 b was generously supplied by Dr. Christiane Rordorf, Novartis Pharma, Basel, Switzerland. The cDNA-clone pEX6, coding for human b-actin was a gift from Dr. U. Aebi, Basel, Switzerland. w1- 14 CxOleic acid and 32 P-dATP Žspecific activity 3000 Cirmmol. were from Amersham, Dubendorf, Switzerland. Nylon membranes ¨ ŽGene Screen. were purchased from DuPont de Nemours International, Regensdort, Switzerland; PDGF was donated by Dr. Michael Pech, F. Hoffmann-La Roche, Basel, Switzerland; Žser 70,88 . bFGF was generously supplied by Dr. Michael Fox, Amgen, Thousand Oaks, CA, USA; PDGF-BB was a kind gift of Dr. Michael Pech, F. Hoffmann-La Roche, Basel, Switzerland; Calphostin C was from Calbiochem, Zurich, Switzerland. Cell culture media and ¨ nutrients were from Gibco, Basel, Switzerland and all other chemicals used were either from Merck, Darmstadt, Germany or Fluka, Basel, Switzerland. 2.2. Cell culture Rat mesangial cells were cultured as described previously w15x. In a second step, single cells were cloned by limited dilution using 96-micro well plates. Clones with apparent mesangial cell morphology were used for further processing. The cells exhibited the typical stellate morphology. Moreover, there was positive staining for the intermediate filaments desmin and vimentin, which are considered to be specific for myogenic cells, positive staining for Thy 1.1 antigen, negative staining for the factor VIII-related antigen and cytokeratin, excluding endothelial and epithelial
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
215
Table 1 Effect of PDGF-BB and bFGF on IL-1 b-stimulated PLA 2 activity secreted from mesangial cells Addition
PLA 2 secretion Žc.p.m.r100 m l.
Fold induction
Control IL-1 b Ž2 nM. IL-1 b Ž2 nM. q PDGF-BB Ž1 ngrml. IL-1 b Ž2 nM. q PDGF-BB Ž10 ngrml. IL-1 b Ž2 nM. q PDGF-BB Ž100 ngrml. Control IL-1 b Ž2 nM. IL-1 b Ž2 nM. q bFGF Ž1 ngrml. IL-1 b Ž2 nM. q bFGF Ž10 ngrml. IL-1 b Ž2 nM. q bFGF Ž100 ngrml.
823 " 53 12 456 " 489 11 278 " 556 5791 " 195 1381 " 92 958 " 78 14 375 " 342 15 281 " 624 14 456 " 549 15 289 " 498
y 15.1 13.7 7.0 1.7 y 15.0 15.9 15.0 15.9
Confluent mesangial cells were incubated with the indicated concentrations for 24 h. Thereafter, the medium was withdrawn and PLA 2 activity determined as described in Section 2. Results are means" SE Ž n s 4..
cell contamination, respectively. The generation of inositol trisphosphate upon activation of the angiotensin II AT1 receptor was used as a functional criterion for characterizing the cloned cell line. The cells were grown in RPMI 1640 supplemented with
10% fetal calf serum, penicillin Ž100 unitsrml., streptomycin Ž100 m grml. and bovine insulin at 0.66 unitsrml ŽSigma.. For the experiments, passages 6–16 of mesangial cell were used.
Fig. 1. Inhibition of IL-1 b-stimulated group II PLA 2 protein expression by PDGF-BB. Mesangial cells were incubated for 12 h with vehicle ŽControl., IL-1 b Ž2 nM. or IL-1 b Ž2 nM. plus the indicated concentrations of PDGF-BB in the presence of w 35 Sxmethionine. Thereafter, group II PLA 2 protein was immunoprecipitated and samples were analyzed as described in Section 2.
Fig. 2. Effect of bFGF on the formation of IL-1 b-stimulated group II PLA 2 protein expression. Mesangial cells were incubated for 12 h with vehicle ŽControl., IL-1 b Ž2 nM. or IL-1 b Ž2 nM. plus the indicated concentrations of bFGF in the presence of w 35 Sxmethionine. Thereafter, group II PLA 2 protein was immunoprecipitated and samples were analyzed as described in Section 2.
216
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
lowed by 2 ml of heptane and 3 ml of water. After vigorous shaking and phase separation, a portion Ž 2.5 ml. of the upper phase was passed over a column of silicic acid Ž 3.5 cm = 0.5 cm.. Free w1- 14 Cxoleic acid was quantitatively eluted with 1 ml of ethylacetate. Radioactivity was determined in a scintillation counter. Phospholipase A 2 activity is expressed as w1- 14 Cxoleate radioactivity released by 100 m l of cell culture supernatant. 2.4. Metabolic labeling and immunoprecipitation
Fig. 3. Inhibition of IL-1 b-stimulated induction of group II PLA 2 mRNA in mesangial cells by PDGF-BB. Mesangial cells were incubated for 24 h with vehicle ŽControl., IL-1 b Ž2 nM. or IL-1 b Ž2 nM. plus the indicated concentrations of PDGF-BB. Total cellular RNA Ž20 m g. was successively hybridized to 32 P-labeled group II PLA 2 and b-actin cDNA probes as described in Section 2.
Mesangial cells were stimulated with agents for 6 h in DMEM without methionine. Newly synthesized proteins were labeled by addition of 80 m Ci of 35 L-w Sxmethionine Ž 1175 Cirmmol; DuPont NEN. to 2 ml of methionine-free culture medium and incubations continued for further 6 to 8 h. Subsequently, immunoprecipitation was performed by using 2 ml of culture supernatant containing proteinase inhibitors at
2.3. Phospholipase A 2 assay Phospholipase A 2 activity was determined with w1- 14 Cxoleate-labelled Escherichia coli as substrate as described previously w16x. The substrate was prepared by growing E. coli in the presence of w114 x C oleate, followed by autoclaving to inactivate endogenous phospholipases. Over 95% of the label incorporated by E. coli was in phospholipid and, as demonstrated by hydrolysis with snake venom Ž Crotalus adamanteus., more than 95% of the w114 x C oleate was in the sn-2 position of the phospholipids. Assay mixtures Ž1.0 ml. contained 100 mM Tris–HCl buffer ŽpH 7.0., 1 mM CaCl 2 , 2.5 = 10 8 w1- 14 Cxoleate-labeled E. coli Ž5 nmol of phospholipids, 5000–8000 c.p.m.. and the enzyme to be tested at a dilution producing approximately 5% substrate hydrolysis. Reaction mixture were incubated for 1 h at 378C in a shaking water bath. The reaction was stopped by the addition of 5 ml of propane-2olrn-heptaner1 M H 2 SO4 Ž40:10:1, by volume. fol-
Fig. 4. Effect of bFGF on the formation of IL-1 b-stimulated group II PLA 2 mRNA. Mesangial cells were incubated for 24 h with vehicle ŽControl., IL-1 b Ž2 nM. or IL-1 b Ž2 nM. plus the indicated concentrations of bFGF. Total cellular RNA Ž20 m g. was successively hybridized to 32 P-labeled group II PLA 2 and b-actin cDNA probes as described in Section 2.
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
a final concentration of 1 mM phenylmethylsulfonyl fluoride, 10 m M leupeptin and 20 m M pepstatin. A total of 100 m l of a 1:1 slurry of a specific monoclonal anti-PLA 2 antibody w17,18x coupled to protein G-Sepharose were added and incubated overnight at 48C. The proteins were solubilized from the beads by heating for 5 min at 958C in 0.1 ml of SDS–PAGE sample buffer Ž3.6% Ž wrv. SDS, 15% Žwrv. glycerol, 120 mM Tris–HCl, pH 6.8, 0.125 M dithiothreitol. and subjected to SDS–PAGE Ž 15% Ž wrv. acrylamide gel..
217
insert from clone pEX6 coding for human b-actin. DNA-probes Ž 0.5 y 1 = 10 6 dpmrml. were radioactively labelled with 32 P-dATP by random priming ŽBoehringer Mannheim.. Hybridisation reactions were performed in 50% Ž vrv. formamide, 5 = SSC, 5 = Denhardt’s solution, 1% Ž wrv. SDS, 10% Žwrv. dextran sulfate and 100 m grml salmon sperm DNA. Filters were washed 3 times in 2 = SSCr0.1% SDS at room temperature for 15 min and then 2 times in 0.2 = SSCr1% SDS at 658C for 30 min. Filters were exposed for 24–48 h to Kodak X-Omat XAR-film using intensifying screens.
2.5. Northern blot analysis Confluent mesangial cells were washed twice with PBS and incubated in DMEM, supplemented with 0.1 mgrml of fatty acid-free bovine serum albumin, with or without agents for the indicated time periods. Cells were washed twice with PBS and harvested using a rubber policeman. Total cellular RNA was extracted from the cell pellets using the guanidinium thiocyanatercesium chloride method w19x. Samples of 20 m g RNA were separated on 1% agarose gels containing 0.66 M formaldehyde prior to the transfer to gene screen membranes ŽNew England Nuclear. . After UV-crosslinking and prehybridization for 2 h, the filters were hybridized for 16–18 h to a 32 P-labelled EcoRIrHindIII-cDNA insert from p139-1 coding for rat group II PLA 2 w20x. To correct for variation in RNA amount, the PLA 2 probe was stripped with boiling 0.1 = SSCr0.1% SDS and the blots were rehybridized to the 32 P-labeled BamHIrSalI cDNA
3. Results PLA 2 activity secreted from mesangial cells was measured by hydrolysis of w 14 Cxoleate-labeled E. coli phospholipid used as a substrate and expressed as w1- 14 Cxoleate radioactivity released by 100 m l of cell culture supernatant. As shown in Table 1, stimulation of mesangial cells for 24 h with IL-1 b increases PLA 2 activity measured in culture supernatants by approximately 15-fold compared to unstimulated cells. Coincubation of the cells with IL-1 b and PDGF-BB potently inhibits PLA 2 activity ŽTable 1.. In contrast, treatment of the cells with IL-1 b and bFGF does not affect the secretion of PLA 2 activity into cell culture supernatants as shown in Table 1. The experiments described here have been performed with Ž ser 70,88 . bFGF in which cysteine residues at positions 70 and 88, which are not conserved among
Table 2 Effect of PDGF-BB and bFGF on forskolin-stimulated PLA 2 activity secreted from mesangial cells Addition
PLA 2 secretion Žc.p.m.r100 m l.
Fold induction
Control Forskolin Ž10 Forskolin Ž10 Forskolin Ž10 Forskolin Ž10 Control Forskolin Ž10 Forskolin Ž10 Forskolin Ž10 Forskolin Ž10
730 " 37 8146 " 320 8046 " 714 7641 " 103 3478 " 127 743 " 40 8148 " 280 9208 " 191 7649 " 300 5176 " 244
y 11.1 11.0 10.5 4.7 y 10.9 12.4 10.3 7.0
m M. m M. q PDGF-BB Ž1 ngrml. m M. q PDGF-BB Ž10 ngrml. m M. q PDGF-BB Ž100 ngrml. m M. m M. q bFGF Ž1 ngrml. m M. q bFGF Ž10 ngrml. m M. q bFGF Ž100 ngrml.
Confluent mesangial cells were incubated with the indicated concentrations of forskolin, PDGF-BB and bFGF for 24 h. Thereafter, the medium was withdrawn and PLA 2 activity determined as described in Section 2. Results are means" SE Ž n s 4..
218
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
IL-1 b in the presence of bFGF does not inhibit but rather increases expression of PLA 2 protein as shown in Fig. 2. Northern blot analyses were performed to assess whether growth factors affect the corresponding PLA 2 mRNA steady-state levels. Mesangial cells were incubated with IL-1 b and PDGF-BB for 24 h. Total cellular RNA was isolated and Northern blots were probed with a radioactively labeled cDNA coding for group II PLA 2 . Whereas in unstimulated cells no PLA 2 mRNA is detectable, IL-1 b strongly induces the formation of PLA 2 mRNA Ž Figs. 3 and 4, respectively. . PDGF-BB inhibits the formation of PLA 2 mRNA upon induction by IL-1 b ŽFig. 3.. As shown in Fig. 4, coincubation of the cells with IL-1 b and bFGF does not significantly change PLA 2 mRNA levels. Incubation of mesangial cells with forskolin, an activator of the catalytic moiety of adenylate cyclase,
Fig. 5. Inhibition of forskolin-stimulated expression of group II PLA 2 protein by PDGF-BB. Mesangial cells were incubated for 12 h with vehicle ŽControl., forskolin Ž10 m M. or forskolin Ž10 m M. plus the indicated concentrations of PDGF-BB in the presence of w 35Sxmethionine. Thereafter, group II PLA 2 protein was immunoprecipitated and samples were analyzed as described in Section 2.
the FGF family members, have been replaced with serines in order to reduce intermolecular dimer formation at high protein concentration. This molecule has biological activity identical to that of native bFGF w21x. Selected experiments have been performed with native bFGF and gave identical results. In order to determine whether the modulation of PLA 2 activity by PDGF-BB and bFGF is due to changes in the corresponding PLA 2 protein levels, we performed immunoprecipitations of radioactively labeled PLA 2 protein from mesangial cell culture supernatants using a monoclonal anti-PLA 2 antibody. As shown in Figs. 1 and 2, IL-1 b induces the de novo synthesis of PLA 2 protein which is secreted into cell culture supernatants. Coincubation with IL1 b and different amounts of PDGF-BB inhibits cytokine-stimulated secretion of PLA 2 protein. Maximal inhibition is observed at a concentration of 100 ngrml of PDGF-BB ŽFig. 1. . Incubation of cells with
Fig. 6. Effect of bFGF on the formation of forskolin-stimulated group II PLA 2 protein expression. Mesangial cells were incubated for 12 h with vehicle ŽControl., forskolin Ž10 m M. or forskolin Ž10 m M. plus the indicated concentrations of bFGF in the presence of w 35 Sxmethionine. Thereafter, group II PLA 2 protein was immunoprecipitated and samples were analyzed as described in Section 2.
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
Fig. 7. Inhibition of forskolin-stimulated induction of group II PLA 2 mRNA by PDGF-BB. Mesangial cells were incubated for 24 h with vehicle ŽControl., forskolin Ž10 m M. or forskolin Ž10 m M. plus the indicated concentrations of PDGF-BB. Total cellular RNA Ž20 m g. was successively hybridized to 32 P-labeled group II PLA 2 and b-actin cDNA probes as described in Section 2.
increases PLA 2 activity secreted into cell culture supernatants by about 11-fold compared to unstimulated cells as shown in Table 2. Coincubation of cells with forskolin and PDGF-BB markedly inhibits PLA 2 activity secreted from the cells Ž Table 2. . However, PDGF inhibition was less pronounced in forskolinstimulated cells as compared to IL-1 b-stimulated mesangial cells Ž Table 1. . The use of higher concentrations of PDGF-BB Ž) 100 ngrml. does not reveal stronger inhibition Ždata not shown. . Similarly, bFGF reduces forskolin-induced PLA 2 activity to about 7-fold ŽTable 2.. As for PDGF-BB, higher concentrations Ž) 100 ngrml. of bFGF do not result in a more potent inhibition Ž data not shown. . Using immunoprecipitations of radioactively labeled PLA 2 protein we show that forskolin strongly induces the de novo synthesis of PLA 2 protein Ž Figs. 5 and 6, respectively. . Coincubation with forskolin and different amounts of PDGF-BB concentration, dependently inhibits stimulated secretion of PLA 2
219
protein Ž Fig. 5.. Incubation of the cells with forskolin in the presence of bFGF gives a moderate inhibition of PLA 2 protein expression only at a maximal concentration of 100 ngrml bFGF ŽFig. 6. . Northern blot analyses were performed to detect whether growth factors affect corresponding PLA 2 mRNA steady-state levels. Mesangial cells were incubated with forskolin and PDGF-BB for 24 h and Northern blots of total cellular RNA were probed with a radioactively labeled cDNA coding for group II PLA 2 . Whereas in unstimulated cells no PLA 2 mRNA is detectable, forskolin strongly induces the formation of PLA 2 mRNA ŽFigs. 7 and 8, respectively. . PDGF-BB inhibits the formation of PLA 2 mRNA upon induction by forskolin at a maximal concentration of 100 ngrml ŽFig. 7.. As shown in Fig. 8, coincubation of the cells with forskolin Ž 10 m M. and bFGF slightly reduces PLA 2 mRNA levels at a maximal concentration of 100 ngrml. A possible explanation for the divergent effects of PDGF and bFGF on group II sPLA 2 expression is the
Fig. 8. Effect of bFGF on the forskolin-stimulated induction of group II PLA 2 mRNA. Mesangial cells were incubated for 24 h with vehicle ŽControl., forskolin Ž10 m M. or forskolin Ž10 m M. plus the indicated concentrations of bFGF. Total cellular RNA Ž20 m g. was successively hybridized to 32 P-labeled group II PLA 2 and b-actin cDNA probes as described in Section 2.
220
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
Table 3 Effect of calphostin C on PDGF-BB-dependent suppression of PLA 2 activity secreted from mesangial cells stimulated by IL-1 b Addition
PLA 2 secretion Žc.p.m.r100 m l.
Fold induction
Control Calphostin C Ž100 nM. IL-1 b Ž2 nM. IL-1 b Ž2 nM. q PDGF-BB Ž10 ngrml. IL-1 b Ž2 nM. q PDGF-BB Ž10 ngrml. q Calphostin C Ž100 nM.
354 " 10 803 " 24 5519 " 349 2070 " 83 4233 " 374
y 2.3 16.0 5.8 12.0
Confluent mesangial cells were incubated for 24 h with vehicle Žcontrol. or the indicated concentrations of compounds. Thereafter, the medium was withdrawn and PLA 2 activity determined as described in Section 2. Results are means" SE Ž n s 4..
different degree of activation of protein kinase C ŽPKC.. In order to determine whether the inhibitory effect of PDGF-BB on PLA 2 expression is due to its pronounced capability to activate protein kinase C, we measured PLA 2 activity secreted into cell culture supernatants from cells that were stimulated in the presence of calphostin C, a potent and selective inhibitor of protein kinase C w22x. Mesangial cells were incubated for 24 h with IL-1 b , IL-1 b plus PDGF-BB and a combination of IL-1 b plus PDGFBB and calphostin C and subsequently PLA 2 activity was determined. As shown in Table 3, the inhibitory effect of PDGF-BB on IL-1 b-stimulated PLA 2 expression can almost completely be reversed under the action of calphostin C.
4. Discussion Glomerular mesangial cells are a major determinant in the regulation of the glomerular filtration rate. These cells are an abundant source of vasoactive substances, such as prostaglandins or platelet-activating factor which act as intracellular or intercellular messengers, playing an important role in the crosscommunication between glomerular cells Ž for review, see Refs. w8,23x.. Thus, mesangial cells may help to preserve structure and function of the glomerulus. Under pathological conditions, mesangial cells may be activated by factors released from immune cells such as macrophages or neutrophils invading the glomerulus or by serum factors. Such activated mesangial cells have the potential to produce a myriad of inflammatory mediators including eicosanoids, nitric oxide and other reactive oxygen species as well as growth factors. The aberrant production of such
mediators may sustain connective tissue accumulation, resulting in irreversible alteration of glomerular structure and function and finally ending up in glomerulosclerosis. Cytokine-triggered synthesis and secretion of group II PLA 2 by rat mesangial cells contributes to the synthesis of eicosanoids such as prostaglandin E 2 that occurs in parallel to the enzyme secretion w24x. Coincubation with a monoclonal antibody that specifically binds and neutralizes group II PLA 2 or a specific inhibitor of group II PLA 2 attenuates IL-1 b- and tumour necrosis factor a-stimulated prostaglandin E 2 production by approximately 50% to 70%. Moreover, extracellular PLA 2 secreted by mesangial cells is able to stimulate in a paracrine fashion arachidonic acid release and prostaglandin E 2 production by the adjacent glomerular endothelial and epithelial cells w24x. In recent years, it has become evident that besides inflammatory cytokines like IL-1 b or tumour necrosis factor a , growth factors, notably PDGF, FGF and TGF-b are crucially involved in the development and progress of glomerular inflammation and subsequent tissue repair Ž for review, see Refs. w25,26x.. These effects are due to their actions on glomerular cells causing proliferation, production of extracellular matrix and regulation of enzymes producing inflammatory mediators. The aim of this study was to evaluate whether growth factors such as PDGF or FGF would modulate group II PLA 2 expression. We report here that PDGF-BB, which binds to and activates a tyrosine kinase receptor in mesangial cells w26–28x potently inhibits IL-1 b- and, less pronounced, forskolinstimulated expression of group II PLA 2 activity secreted into mesangial cell culture supernatants thus
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
confirming previous observations by us and others w9,29x. It is evident from our studies that suppression occurs at multiple levels of group II PLA 2 expression. The loss of PLA 2 activity is due to reduction of PLA 2 protein and corresponding PLA 2 mRNA steady-state levels. Whether the decrease in formation of PLA 2 mRNA levels is due to decreased gene transcription or changes in mRNA half-life remains to be demonstrated. Insulin-like growth factors are another type of tyrosine kinase activating growth factors that have been shown to counteract IL-1 bstimulated group II PLA 2 expression in rabbit articular chondrocytes by a post-transcriptional mechanism w30,31x. Surprising effects were noted for bFGF which also binds to a tyrosine kinase receptor. For IL-1 b-induced expression of group II PLA 2 bFGF does, if anything, slightly stimulate whereas forskolin-stimulated PLA 2 activity is considerably reduced by bFGF. A potentiating effect on IL-1 b-induced group II sPLA 2 expression has also been reported for another tyrosine kinase receptor ligand, i.e., epidermal growth factor w27x. A possible explanation for the disparate effects of the two growth factors on IL-1 b-stimulated PLA 2 expression is the different degree of activation of protein kinase C. Whereas PDGF-BB potently stimulates phosphoinositide turnover and subsequent activation of protein kinase C w28,32x, bFGF is a poor activator of this signaling pathway in mesangial cells w33x. Our results clearly show that coincubation of mesangial cells with PDGF-BB and calphostin C, a potent and selective inhibitor of protein kinase C w22x, reverses the inhibitory effect of PDGF. This is very interesting in the context that the ´-isozyme of protein kinase C tonically suppresses inducible nitric oxide synthase expression in mesangial cells w34x, an enzyme that is also induced by IL-1 b and cAMP w35,36x and differentially modulated by PDGF and bFGF w33x. Recently the identification of novel secretory PLA 2 s Žnow called group IIc and V, respectively. has been reported w37x. Several investigators that had not rigorously identified the secreted PLA 2 s had to reevaluate their findings. Mast cells and the macrophage-like P388D1 cells are good examples thereof w37x. We used not only immunological techniques in the identification w7x but also purified and
221
sequenced the secreted enzyme w38x. A total of 36 out of 40 amino acids could be identified with certainty. Since these were completely identical to those of rat group II PLA 2 , we concluded that the enzyme secreted by mesangial cells was Žin the nomenclature of those days. a group II PLA 2 . Of course, one can never exclude the possibility that a few percent of the secreted sPLA 2 was in fact another type than type IIa and that we did not get the signals from these enzymes in the sequenator runs. However, of the 36 amino acids identified with certainty, 15 and 19 are different from rat group V and group IIc, respectively. Thus, the vast majority of the enzyme secreted by mesangial cells remains a group IIa PLA 2 , no matter what has been rectified for other cells. In summary, our data provide novel aspects of the regulation of IL-1 b and forskolin-stimulated group II PLA 2 expression in renal mesangial cells and highlight the complexity of cross-talk between signaling pathways involved in the induction and modulation of inflammatory enzymes.
Acknowledgements This work was supported by Swiss National Science Foundation Grant 31-43090.95 and by a grant from the Commission of the European Communities ŽBiomed 2 PL 950979.. The supply of recombinant IL-1 b , PDGF-BB and bFGF by Christiane Rordorf, Michael Pech and Michael Fox, respectively, is gratefully acknowledged.
References w1x H. van den Bosch, Biochim. Biophys. Acta 604 Ž1980. 161–246. w2x K.B. Glaser, D. Mobilio, J.Y. Chang, N. Senko, Trends Pharmacol. Sci. 14 Ž1993. 92–98. w3x M. Roberts, FASEB J. 10 Ž1996. 1159–1172. w4x R.L. Heinrikson, E.T. Krueger, P.S. Keim, J. Biol. Chem. 252 Ž1977. 4913–4921. w5x W. Pruzanski, P. Vadas, Immunol. Today 12 Ž1991. 143– 146. w6x J. Pfeilschifter, W. Pignat, K. Vosbeck, F. Marki, ¨ Biochem. Biophys. Res. Commun. 159 Ž1989. 385–394. w7x C. Schalkwijk, J. Pfeilschifter, F. Marki, ¨ H. van den Bosch, Biochem. Biophys. Res. Commun. 174 Ž1991. 268–275.
222
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
w8x J. Pfeilschifter, Phospholipase A 2 in clinical inflammation, in: K.B. Glaser, P. Vadas ŽEds.., Molecular Approaches to Pathophysiology, CRC Press, New York, 1995, pp. 25–51. w9x H. Muhl, ¨ T. Geiger, W. Pignat, F. Marki, ¨ H. van den Bosch, K. Vosbeck, J. Pfeilschifter, FEBS Lett. 291 Ž1991. 249– 252. w10x Y. Nakazato, M.S. Simonson, W.H. Herman, M. Konieczowski, M. Sedor, J.R. Sedor, J. Biol. Chem. 266 Ž1991. 14119–14127. w11x J. Pfeilschifter, W. Pignat, F. Marki, I. Wiesenberg, Eur. J. ¨ Biochem. 181 Ž1989. 237–242. w12x J. Pfeilschifter, J. Leighton, W. Pignat, F. Marki, K. Vos¨ beck, Fresenius, J. Anal. Chem. 337 Ž1990. 139–140. w13x J. Pfeilschifter, J. Leighton, W. Pignat, F. Marki, K. Vos¨ beck, Biochem. J. 273 Ž1991. 199–204. w14x H. Muhl, ¨ T. Geiger, W. Pignat, F. Marki, ¨ H. van den Bosch, N. Cerletti, D. Cox, G. McMaster, K. Vosbeck, J. Pfeilschifter, FEBS Lett. 301 Ž1992. 190–194. w15x J. Pfeilschifter, K. Vosbeck, Biochem. Biophys. Res. Commun. 175 Ž1991. 372–379. w16x F. Marki, R. Franson, Biochim. Biophys. Acta 879 Ž1986. ¨ 149–156. w17x A.J. Aarsman, J.G.N. De Jong, E. Arnoldussen, F.W. Neys, P.D. van Wassenaar, H. van den Bosch, J. Biol. Chem. 264 Ž1989. 10008–10014. w18x J.G.N. De Jong, H. Amesz, A.J. Aarsman, H.B.M. Lenting, H. van den Bosch, Eur. J. Biochem. 164 Ž1987. 129–135. w19x J. Sambrock, J. Fritsch, T. Maniatis, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1989. w20x R.H.N. van Schaik, N.M. Verhoeven, F.W. Neijs, A.J. Aarsman, H. van den Bosch, Biochim. Biophys. Acta 1169 Ž1993. 1–15.
w21x G.M. Fox, S.G. Schiffer, M.F. Rohde, L.B. Tsai, A.R. Banks, T. Arakawa, J. Biol. Chem. 263 Ž1988. 18452– 18458. w22x E. Kobayashi, H. Nakano, M. Morimoto, T. Tamaoki, Biochem. Biophys. Res. Commun. 156 Ž1989. 548–553. w23x J. Pfeilschifter, News Physiol. Sci. 9 Ž1994. 271–276. w24x J. Pfeilschifter, C. Schalkwijk, V.A. Briner, H. van den Bosch, J. Clin. Invest. 92 Ž1993. 2516–2522. w25x N. Wardle, Nephron 73 Ž1996. 1–7. w26x H.E. Abboud, Kidney Int. 43 Ž1993. 252–267. w27x M.J.B.M. Vervoordeldonk, C.G. Schalkwijk, J. Pfeilschifter, H. van den Bosch, Biochem. J. 315 Ž1996. 435–441. w28x J. Pfeilschifter, M. Hosang, Cell Signal. 3 Ž1991. 413–424. w29x P. Vadas, E. Stefanski, M. Wloch, B. Grouix, H. van den Bosch, B. Kennedy, Eur. J. Biochem. 235 Ž1996. 557–563. w30x F. Berenbaum, G. Thomas, S. Poiraudeau, G. Bereziat, M.T. ´´ Corvol, J. Masliah, FEBS Lett. 340 Ž1994. 51–55. w31x C. Jacques, G. Bereziat, L. Humbert, J.-L. Olivier, M.-T. ´´ Corvol, J. Masliah, F. Berenbaum, J. Clin. Invest. 99 Ž1997. 1864–1872. w32x J. Pfeilschifter, A. Huwiler, J. Auton. Pharmacol. 16 Ž1996. 315–318. w33x D. Kunz, G. Walker, W. Eberhardt, U.K. Messmer, A. Huwiler, J. Pfeilschifter, J. Clin. Invest. 100 Ž1997. 2800– 2809. w34x H. Muhl, ¨ J. Pfeilschifter, Biochem. J. 303 Ž1994. 607–612. w35x H. Muhl, ¨ D. Kunz, J. Pfeilschifter, Br. J. Pharmacol. 112 Ž1994. 1–8. w36x D. Kunz, H. Muhl, ¨ G. Walker, J. Pfeilschifter, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 5387–5391. w37x J.A. Tischfield, J. Biol. Chem. 272 Ž1997. 17247–17250. w38x C. Schalkwijk, J. Pfeilschifter, F. Marki, ¨ H. van den Bosch, J. Biol. Chem. 267 Ž1992. 8846–8851.
Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1 b- and cAMP-induced group II phospholipase A 2 expression in rat renal mesangial cells Gaby Walker
a,1
, Dieter Kunz
a,1
, Werner Pignat b, Josef Pfeilschifter
c,)
a
c
Department of Pharmacology, Biozentrum, UniÕersity of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland b Research Department, Pharmaceuticals DiÕision, NoÕartis Pharma, CH-4002 Basel, Switzerland Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-UniÕersitat, ¨ Theodor-Stern-Kai-7, D-60590 Frankfurt am Main, Germany Received 22 August 1997; revised 21 November 1997; accepted 27 November 1997
Abstract Expression of group II phospholipase A 2 ŽPLA 2 ; EC 3.1.1.4. in rat renal mesangial cells is triggered in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin 1 b ŽIL-1 b . or tumor necrosis factor a and agents that elevate cellular levels of cyclic AMP ŽcAMP. such as forskolin, an activator of adenylate cyclase. Treatment of mesangial cells with IL-1 b or forskolin for 24 h induces group II PLA 2 activity secreted into cell culture supernatants by about 15-fold and 11-fold, respectively. Platelet-derived growth factor ŽPDGF.-BB potently inhibits secretion of IL-1 b- and forskolin-induced group II PLA 2 activity. By Western and Northern blot analyses, we demonstrate that this is due to a reduction of PLA 2 protein levels and the corresponding PLA 2 mRNA steady-state levels. Basic fibroblast growth factor ŽbFGF. virtually does not inhibit IL-1 b-stimulated group II PLA 2 activity, but markedly inhibits forskolin-induced expression of group II PLA 2 activity. These effects are caused by changes in the corresponding PLA 2 protein and PLA 2 mRNA steady-state levels. Inhibition of protein kinase C ŽPKC. by the potent and selective PKC inhibitor calphostin C converted the inhibitory action of PDGF into a bFGF-type of response thus suggesting that PKC is a major effector in PDGF-induced inhibition of IL-1 b-stimulated group II sPLA 2 expression. In summary, our data suggest that PDGF and bFGF differentially modulate in a stimulus-specific manner the expression of group II PLA 2 in mesangial cells. q 1998 Elsevier Science B.V. Keywords: Phospholipase A 2 , secretory; Gene expression; Interleukin 1 b ; Cyclic AMP; Platelet-derived growth factor; Fibroblast growth factor
1. Introduction Phospholipase A 2 Ž PLA 2 . comprises a group of lipolytic enzymes that specifically release fatty acids, ) 1
Corresponding author. Fax: q49-69-6301-7942. Both authors contributed equally to this study.
often arachidonic acid, from the sn-2 position of membrane phospholipids for production of important lipid mediators such as eicosanoids and plateletactivating factor w1,2x. Arachidonic acid and its numerous metabolites act as intracellular and intercellular messengers contributing to normal cellular physiology by modifying the activity of intracellular en-
0005-2760r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 2 0 4 - X
214
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
zymes and ion channels. Moreover, PLA 2 and its products function as substrates for the generation of inflammatory lipid mediators that play an important role in the pathogenesis of inflammatory diseases. In most tissues and cells, the synthesis of eicosanoids is limited by the availability of intracellular free arachidonic acid. The major mechanism considered to control the level of intracellular free arachidonic acid is the activation of PLA 2 and the direct release of the fatty acid from membrane glycerophospholipids. In recent years, four distinct types of phospholipases have been identified: Ž1. secretory PLA 2 ŽsPLA 2 ., a disulfide cross-linked, low molecular weight protein of 13–15 kDa which is secreted by cells and requires mM Ca2q for optimal activity; Ž 2. cytoplasmic PLA 2 ŽcPLA 2 . , a protein with a molecular weight of 70–80 kDa with a different requirement for Ca2q Ž m M rather than mM Ca2q .; Ž3. intracellular Ca2q-independent PLA 2 ŽiPLA 2 . with a molecular weight of 80 kDa; and Ž4. platelet-activating factor acetylhydrolase ŽPAF-AH or PAF-PLA 2 ., a group of several isozymes with molecular weights ranging from 29 kDa to 45 kDa which, most prominently release acetyl groups Ž for review, see Ref. w3x.. Based on their primary structure, the sPLA 2 s are further classified into two groups w4x. Mammalian group I PLA 2 comprises the pancreatic-type of PLA 2 and mammalian group II PLA 2 is synthesized and secreted from many cell types and is believed to play a role in the initiation and propagation of inflammatory processes Ž for review, see Ref. w5x.. Two potent pro-inflammatory cytokines, IL-1 b and TNFa , have been shown to induce the synthesis and secretion of group II PLA 2 in mesangial cells w6,7x and in a variety of other cell types Ž for review, see Ref. w8x.. The cytokine effect is blocked by actinomycin D and cycloheximide, thus demonstrating a requirement for both transcription and de novo protein synthesis. This is confirmed by the observation that IL-1 b and TNFa increase the level of mRNA for group II PLA 2 in these cells w9,10x. We also demonstrated an increased secretion of group II PLA 2 in rat vascular smooth muscle cells w11x and rat mesangial cells w12,13x in response to cAMP-elevating agents. Furthermore, forskolin, an activator of adenylate cyclase, synergistically interacts with IL-1 b to increase group II PLA 2 mRNA levels in mesangial cells w14x.
The aim of this study was to evaluate whether growth factors such as PDGF and FGF, known to play an important role in the regulation of the inflammatory response and wound healing, would modulate induction of group II PLA 2 in rat renal mesangial cells. We show that PDGF-BB suppresses IL-1 b- and cAMP-induced group II PLA 2 expression, whereas bFGF virtually does not affect IL-1 b-, but inhibits cAMP-stimulated group II PLA 2 expression. 2. Materials and methods 2.1. Materials Recombinant human IL-1 b was generously supplied by Dr. Christiane Rordorf, Novartis Pharma, Basel, Switzerland. The cDNA-clone pEX6, coding for human b-actin was a gift from Dr. U. Aebi, Basel, Switzerland. w1- 14 CxOleic acid and 32 P-dATP Žspecific activity 3000 Cirmmol. were from Amersham, Dubendorf, Switzerland. Nylon membranes ¨ ŽGene Screen. were purchased from DuPont de Nemours International, Regensdort, Switzerland; PDGF was donated by Dr. Michael Pech, F. Hoffmann-La Roche, Basel, Switzerland; Žser 70,88 . bFGF was generously supplied by Dr. Michael Fox, Amgen, Thousand Oaks, CA, USA; PDGF-BB was a kind gift of Dr. Michael Pech, F. Hoffmann-La Roche, Basel, Switzerland; Calphostin C was from Calbiochem, Zurich, Switzerland. Cell culture media and ¨ nutrients were from Gibco, Basel, Switzerland and all other chemicals used were either from Merck, Darmstadt, Germany or Fluka, Basel, Switzerland. 2.2. Cell culture Rat mesangial cells were cultured as described previously w15x. In a second step, single cells were cloned by limited dilution using 96-micro well plates. Clones with apparent mesangial cell morphology were used for further processing. The cells exhibited the typical stellate morphology. Moreover, there was positive staining for the intermediate filaments desmin and vimentin, which are considered to be specific for myogenic cells, positive staining for Thy 1.1 antigen, negative staining for the factor VIII-related antigen and cytokeratin, excluding endothelial and epithelial
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
215
Table 1 Effect of PDGF-BB and bFGF on IL-1 b-stimulated PLA 2 activity secreted from mesangial cells Addition
PLA 2 secretion Žc.p.m.r100 m l.
Fold induction
Control IL-1 b Ž2 nM. IL-1 b Ž2 nM. q PDGF-BB Ž1 ngrml. IL-1 b Ž2 nM. q PDGF-BB Ž10 ngrml. IL-1 b Ž2 nM. q PDGF-BB Ž100 ngrml. Control IL-1 b Ž2 nM. IL-1 b Ž2 nM. q bFGF Ž1 ngrml. IL-1 b Ž2 nM. q bFGF Ž10 ngrml. IL-1 b Ž2 nM. q bFGF Ž100 ngrml.
823 " 53 12 456 " 489 11 278 " 556 5791 " 195 1381 " 92 958 " 78 14 375 " 342 15 281 " 624 14 456 " 549 15 289 " 498
y 15.1 13.7 7.0 1.7 y 15.0 15.9 15.0 15.9
Confluent mesangial cells were incubated with the indicated concentrations for 24 h. Thereafter, the medium was withdrawn and PLA 2 activity determined as described in Section 2. Results are means" SE Ž n s 4..
cell contamination, respectively. The generation of inositol trisphosphate upon activation of the angiotensin II AT1 receptor was used as a functional criterion for characterizing the cloned cell line. The cells were grown in RPMI 1640 supplemented with
10% fetal calf serum, penicillin Ž100 unitsrml., streptomycin Ž100 m grml. and bovine insulin at 0.66 unitsrml ŽSigma.. For the experiments, passages 6–16 of mesangial cell were used.
Fig. 1. Inhibition of IL-1 b-stimulated group II PLA 2 protein expression by PDGF-BB. Mesangial cells were incubated for 12 h with vehicle ŽControl., IL-1 b Ž2 nM. or IL-1 b Ž2 nM. plus the indicated concentrations of PDGF-BB in the presence of w 35 Sxmethionine. Thereafter, group II PLA 2 protein was immunoprecipitated and samples were analyzed as described in Section 2.
Fig. 2. Effect of bFGF on the formation of IL-1 b-stimulated group II PLA 2 protein expression. Mesangial cells were incubated for 12 h with vehicle ŽControl., IL-1 b Ž2 nM. or IL-1 b Ž2 nM. plus the indicated concentrations of bFGF in the presence of w 35 Sxmethionine. Thereafter, group II PLA 2 protein was immunoprecipitated and samples were analyzed as described in Section 2.
216
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
lowed by 2 ml of heptane and 3 ml of water. After vigorous shaking and phase separation, a portion Ž 2.5 ml. of the upper phase was passed over a column of silicic acid Ž 3.5 cm = 0.5 cm.. Free w1- 14 Cxoleic acid was quantitatively eluted with 1 ml of ethylacetate. Radioactivity was determined in a scintillation counter. Phospholipase A 2 activity is expressed as w1- 14 Cxoleate radioactivity released by 100 m l of cell culture supernatant. 2.4. Metabolic labeling and immunoprecipitation
Fig. 3. Inhibition of IL-1 b-stimulated induction of group II PLA 2 mRNA in mesangial cells by PDGF-BB. Mesangial cells were incubated for 24 h with vehicle ŽControl., IL-1 b Ž2 nM. or IL-1 b Ž2 nM. plus the indicated concentrations of PDGF-BB. Total cellular RNA Ž20 m g. was successively hybridized to 32 P-labeled group II PLA 2 and b-actin cDNA probes as described in Section 2.
Mesangial cells were stimulated with agents for 6 h in DMEM without methionine. Newly synthesized proteins were labeled by addition of 80 m Ci of 35 L-w Sxmethionine Ž 1175 Cirmmol; DuPont NEN. to 2 ml of methionine-free culture medium and incubations continued for further 6 to 8 h. Subsequently, immunoprecipitation was performed by using 2 ml of culture supernatant containing proteinase inhibitors at
2.3. Phospholipase A 2 assay Phospholipase A 2 activity was determined with w1- 14 Cxoleate-labelled Escherichia coli as substrate as described previously w16x. The substrate was prepared by growing E. coli in the presence of w114 x C oleate, followed by autoclaving to inactivate endogenous phospholipases. Over 95% of the label incorporated by E. coli was in phospholipid and, as demonstrated by hydrolysis with snake venom Ž Crotalus adamanteus., more than 95% of the w114 x C oleate was in the sn-2 position of the phospholipids. Assay mixtures Ž1.0 ml. contained 100 mM Tris–HCl buffer ŽpH 7.0., 1 mM CaCl 2 , 2.5 = 10 8 w1- 14 Cxoleate-labeled E. coli Ž5 nmol of phospholipids, 5000–8000 c.p.m.. and the enzyme to be tested at a dilution producing approximately 5% substrate hydrolysis. Reaction mixture were incubated for 1 h at 378C in a shaking water bath. The reaction was stopped by the addition of 5 ml of propane-2olrn-heptaner1 M H 2 SO4 Ž40:10:1, by volume. fol-
Fig. 4. Effect of bFGF on the formation of IL-1 b-stimulated group II PLA 2 mRNA. Mesangial cells were incubated for 24 h with vehicle ŽControl., IL-1 b Ž2 nM. or IL-1 b Ž2 nM. plus the indicated concentrations of bFGF. Total cellular RNA Ž20 m g. was successively hybridized to 32 P-labeled group II PLA 2 and b-actin cDNA probes as described in Section 2.
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
a final concentration of 1 mM phenylmethylsulfonyl fluoride, 10 m M leupeptin and 20 m M pepstatin. A total of 100 m l of a 1:1 slurry of a specific monoclonal anti-PLA 2 antibody w17,18x coupled to protein G-Sepharose were added and incubated overnight at 48C. The proteins were solubilized from the beads by heating for 5 min at 958C in 0.1 ml of SDS–PAGE sample buffer Ž3.6% Ž wrv. SDS, 15% Žwrv. glycerol, 120 mM Tris–HCl, pH 6.8, 0.125 M dithiothreitol. and subjected to SDS–PAGE Ž 15% Ž wrv. acrylamide gel..
217
insert from clone pEX6 coding for human b-actin. DNA-probes Ž 0.5 y 1 = 10 6 dpmrml. were radioactively labelled with 32 P-dATP by random priming ŽBoehringer Mannheim.. Hybridisation reactions were performed in 50% Ž vrv. formamide, 5 = SSC, 5 = Denhardt’s solution, 1% Ž wrv. SDS, 10% Žwrv. dextran sulfate and 100 m grml salmon sperm DNA. Filters were washed 3 times in 2 = SSCr0.1% SDS at room temperature for 15 min and then 2 times in 0.2 = SSCr1% SDS at 658C for 30 min. Filters were exposed for 24–48 h to Kodak X-Omat XAR-film using intensifying screens.
2.5. Northern blot analysis Confluent mesangial cells were washed twice with PBS and incubated in DMEM, supplemented with 0.1 mgrml of fatty acid-free bovine serum albumin, with or without agents for the indicated time periods. Cells were washed twice with PBS and harvested using a rubber policeman. Total cellular RNA was extracted from the cell pellets using the guanidinium thiocyanatercesium chloride method w19x. Samples of 20 m g RNA were separated on 1% agarose gels containing 0.66 M formaldehyde prior to the transfer to gene screen membranes ŽNew England Nuclear. . After UV-crosslinking and prehybridization for 2 h, the filters were hybridized for 16–18 h to a 32 P-labelled EcoRIrHindIII-cDNA insert from p139-1 coding for rat group II PLA 2 w20x. To correct for variation in RNA amount, the PLA 2 probe was stripped with boiling 0.1 = SSCr0.1% SDS and the blots were rehybridized to the 32 P-labeled BamHIrSalI cDNA
3. Results PLA 2 activity secreted from mesangial cells was measured by hydrolysis of w 14 Cxoleate-labeled E. coli phospholipid used as a substrate and expressed as w1- 14 Cxoleate radioactivity released by 100 m l of cell culture supernatant. As shown in Table 1, stimulation of mesangial cells for 24 h with IL-1 b increases PLA 2 activity measured in culture supernatants by approximately 15-fold compared to unstimulated cells. Coincubation of the cells with IL-1 b and PDGF-BB potently inhibits PLA 2 activity ŽTable 1.. In contrast, treatment of the cells with IL-1 b and bFGF does not affect the secretion of PLA 2 activity into cell culture supernatants as shown in Table 1. The experiments described here have been performed with Ž ser 70,88 . bFGF in which cysteine residues at positions 70 and 88, which are not conserved among
Table 2 Effect of PDGF-BB and bFGF on forskolin-stimulated PLA 2 activity secreted from mesangial cells Addition
PLA 2 secretion Žc.p.m.r100 m l.
Fold induction
Control Forskolin Ž10 Forskolin Ž10 Forskolin Ž10 Forskolin Ž10 Control Forskolin Ž10 Forskolin Ž10 Forskolin Ž10 Forskolin Ž10
730 " 37 8146 " 320 8046 " 714 7641 " 103 3478 " 127 743 " 40 8148 " 280 9208 " 191 7649 " 300 5176 " 244
y 11.1 11.0 10.5 4.7 y 10.9 12.4 10.3 7.0
m M. m M. q PDGF-BB Ž1 ngrml. m M. q PDGF-BB Ž10 ngrml. m M. q PDGF-BB Ž100 ngrml. m M. m M. q bFGF Ž1 ngrml. m M. q bFGF Ž10 ngrml. m M. q bFGF Ž100 ngrml.
Confluent mesangial cells were incubated with the indicated concentrations of forskolin, PDGF-BB and bFGF for 24 h. Thereafter, the medium was withdrawn and PLA 2 activity determined as described in Section 2. Results are means" SE Ž n s 4..
218
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
IL-1 b in the presence of bFGF does not inhibit but rather increases expression of PLA 2 protein as shown in Fig. 2. Northern blot analyses were performed to assess whether growth factors affect the corresponding PLA 2 mRNA steady-state levels. Mesangial cells were incubated with IL-1 b and PDGF-BB for 24 h. Total cellular RNA was isolated and Northern blots were probed with a radioactively labeled cDNA coding for group II PLA 2 . Whereas in unstimulated cells no PLA 2 mRNA is detectable, IL-1 b strongly induces the formation of PLA 2 mRNA Ž Figs. 3 and 4, respectively. . PDGF-BB inhibits the formation of PLA 2 mRNA upon induction by IL-1 b ŽFig. 3.. As shown in Fig. 4, coincubation of the cells with IL-1 b and bFGF does not significantly change PLA 2 mRNA levels. Incubation of mesangial cells with forskolin, an activator of the catalytic moiety of adenylate cyclase,
Fig. 5. Inhibition of forskolin-stimulated expression of group II PLA 2 protein by PDGF-BB. Mesangial cells were incubated for 12 h with vehicle ŽControl., forskolin Ž10 m M. or forskolin Ž10 m M. plus the indicated concentrations of PDGF-BB in the presence of w 35Sxmethionine. Thereafter, group II PLA 2 protein was immunoprecipitated and samples were analyzed as described in Section 2.
the FGF family members, have been replaced with serines in order to reduce intermolecular dimer formation at high protein concentration. This molecule has biological activity identical to that of native bFGF w21x. Selected experiments have been performed with native bFGF and gave identical results. In order to determine whether the modulation of PLA 2 activity by PDGF-BB and bFGF is due to changes in the corresponding PLA 2 protein levels, we performed immunoprecipitations of radioactively labeled PLA 2 protein from mesangial cell culture supernatants using a monoclonal anti-PLA 2 antibody. As shown in Figs. 1 and 2, IL-1 b induces the de novo synthesis of PLA 2 protein which is secreted into cell culture supernatants. Coincubation with IL1 b and different amounts of PDGF-BB inhibits cytokine-stimulated secretion of PLA 2 protein. Maximal inhibition is observed at a concentration of 100 ngrml of PDGF-BB ŽFig. 1. . Incubation of cells with
Fig. 6. Effect of bFGF on the formation of forskolin-stimulated group II PLA 2 protein expression. Mesangial cells were incubated for 12 h with vehicle ŽControl., forskolin Ž10 m M. or forskolin Ž10 m M. plus the indicated concentrations of bFGF in the presence of w 35 Sxmethionine. Thereafter, group II PLA 2 protein was immunoprecipitated and samples were analyzed as described in Section 2.
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
Fig. 7. Inhibition of forskolin-stimulated induction of group II PLA 2 mRNA by PDGF-BB. Mesangial cells were incubated for 24 h with vehicle ŽControl., forskolin Ž10 m M. or forskolin Ž10 m M. plus the indicated concentrations of PDGF-BB. Total cellular RNA Ž20 m g. was successively hybridized to 32 P-labeled group II PLA 2 and b-actin cDNA probes as described in Section 2.
increases PLA 2 activity secreted into cell culture supernatants by about 11-fold compared to unstimulated cells as shown in Table 2. Coincubation of cells with forskolin and PDGF-BB markedly inhibits PLA 2 activity secreted from the cells Ž Table 2. . However, PDGF inhibition was less pronounced in forskolinstimulated cells as compared to IL-1 b-stimulated mesangial cells Ž Table 1. . The use of higher concentrations of PDGF-BB Ž) 100 ngrml. does not reveal stronger inhibition Ždata not shown. . Similarly, bFGF reduces forskolin-induced PLA 2 activity to about 7-fold ŽTable 2.. As for PDGF-BB, higher concentrations Ž) 100 ngrml. of bFGF do not result in a more potent inhibition Ž data not shown. . Using immunoprecipitations of radioactively labeled PLA 2 protein we show that forskolin strongly induces the de novo synthesis of PLA 2 protein Ž Figs. 5 and 6, respectively. . Coincubation with forskolin and different amounts of PDGF-BB concentration, dependently inhibits stimulated secretion of PLA 2
219
protein Ž Fig. 5.. Incubation of the cells with forskolin in the presence of bFGF gives a moderate inhibition of PLA 2 protein expression only at a maximal concentration of 100 ngrml bFGF ŽFig. 6. . Northern blot analyses were performed to detect whether growth factors affect corresponding PLA 2 mRNA steady-state levels. Mesangial cells were incubated with forskolin and PDGF-BB for 24 h and Northern blots of total cellular RNA were probed with a radioactively labeled cDNA coding for group II PLA 2 . Whereas in unstimulated cells no PLA 2 mRNA is detectable, forskolin strongly induces the formation of PLA 2 mRNA ŽFigs. 7 and 8, respectively. . PDGF-BB inhibits the formation of PLA 2 mRNA upon induction by forskolin at a maximal concentration of 100 ngrml ŽFig. 7.. As shown in Fig. 8, coincubation of the cells with forskolin Ž 10 m M. and bFGF slightly reduces PLA 2 mRNA levels at a maximal concentration of 100 ngrml. A possible explanation for the divergent effects of PDGF and bFGF on group II sPLA 2 expression is the
Fig. 8. Effect of bFGF on the forskolin-stimulated induction of group II PLA 2 mRNA. Mesangial cells were incubated for 24 h with vehicle ŽControl., forskolin Ž10 m M. or forskolin Ž10 m M. plus the indicated concentrations of bFGF. Total cellular RNA Ž20 m g. was successively hybridized to 32 P-labeled group II PLA 2 and b-actin cDNA probes as described in Section 2.
220
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
Table 3 Effect of calphostin C on PDGF-BB-dependent suppression of PLA 2 activity secreted from mesangial cells stimulated by IL-1 b Addition
PLA 2 secretion Žc.p.m.r100 m l.
Fold induction
Control Calphostin C Ž100 nM. IL-1 b Ž2 nM. IL-1 b Ž2 nM. q PDGF-BB Ž10 ngrml. IL-1 b Ž2 nM. q PDGF-BB Ž10 ngrml. q Calphostin C Ž100 nM.
354 " 10 803 " 24 5519 " 349 2070 " 83 4233 " 374
y 2.3 16.0 5.8 12.0
Confluent mesangial cells were incubated for 24 h with vehicle Žcontrol. or the indicated concentrations of compounds. Thereafter, the medium was withdrawn and PLA 2 activity determined as described in Section 2. Results are means" SE Ž n s 4..
different degree of activation of protein kinase C ŽPKC.. In order to determine whether the inhibitory effect of PDGF-BB on PLA 2 expression is due to its pronounced capability to activate protein kinase C, we measured PLA 2 activity secreted into cell culture supernatants from cells that were stimulated in the presence of calphostin C, a potent and selective inhibitor of protein kinase C w22x. Mesangial cells were incubated for 24 h with IL-1 b , IL-1 b plus PDGF-BB and a combination of IL-1 b plus PDGFBB and calphostin C and subsequently PLA 2 activity was determined. As shown in Table 3, the inhibitory effect of PDGF-BB on IL-1 b-stimulated PLA 2 expression can almost completely be reversed under the action of calphostin C.
4. Discussion Glomerular mesangial cells are a major determinant in the regulation of the glomerular filtration rate. These cells are an abundant source of vasoactive substances, such as prostaglandins or platelet-activating factor which act as intracellular or intercellular messengers, playing an important role in the crosscommunication between glomerular cells Ž for review, see Refs. w8,23x.. Thus, mesangial cells may help to preserve structure and function of the glomerulus. Under pathological conditions, mesangial cells may be activated by factors released from immune cells such as macrophages or neutrophils invading the glomerulus or by serum factors. Such activated mesangial cells have the potential to produce a myriad of inflammatory mediators including eicosanoids, nitric oxide and other reactive oxygen species as well as growth factors. The aberrant production of such
mediators may sustain connective tissue accumulation, resulting in irreversible alteration of glomerular structure and function and finally ending up in glomerulosclerosis. Cytokine-triggered synthesis and secretion of group II PLA 2 by rat mesangial cells contributes to the synthesis of eicosanoids such as prostaglandin E 2 that occurs in parallel to the enzyme secretion w24x. Coincubation with a monoclonal antibody that specifically binds and neutralizes group II PLA 2 or a specific inhibitor of group II PLA 2 attenuates IL-1 b- and tumour necrosis factor a-stimulated prostaglandin E 2 production by approximately 50% to 70%. Moreover, extracellular PLA 2 secreted by mesangial cells is able to stimulate in a paracrine fashion arachidonic acid release and prostaglandin E 2 production by the adjacent glomerular endothelial and epithelial cells w24x. In recent years, it has become evident that besides inflammatory cytokines like IL-1 b or tumour necrosis factor a , growth factors, notably PDGF, FGF and TGF-b are crucially involved in the development and progress of glomerular inflammation and subsequent tissue repair Ž for review, see Refs. w25,26x.. These effects are due to their actions on glomerular cells causing proliferation, production of extracellular matrix and regulation of enzymes producing inflammatory mediators. The aim of this study was to evaluate whether growth factors such as PDGF or FGF would modulate group II PLA 2 expression. We report here that PDGF-BB, which binds to and activates a tyrosine kinase receptor in mesangial cells w26–28x potently inhibits IL-1 b- and, less pronounced, forskolinstimulated expression of group II PLA 2 activity secreted into mesangial cell culture supernatants thus
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
confirming previous observations by us and others w9,29x. It is evident from our studies that suppression occurs at multiple levels of group II PLA 2 expression. The loss of PLA 2 activity is due to reduction of PLA 2 protein and corresponding PLA 2 mRNA steady-state levels. Whether the decrease in formation of PLA 2 mRNA levels is due to decreased gene transcription or changes in mRNA half-life remains to be demonstrated. Insulin-like growth factors are another type of tyrosine kinase activating growth factors that have been shown to counteract IL-1 bstimulated group II PLA 2 expression in rabbit articular chondrocytes by a post-transcriptional mechanism w30,31x. Surprising effects were noted for bFGF which also binds to a tyrosine kinase receptor. For IL-1 b-induced expression of group II PLA 2 bFGF does, if anything, slightly stimulate whereas forskolin-stimulated PLA 2 activity is considerably reduced by bFGF. A potentiating effect on IL-1 b-induced group II sPLA 2 expression has also been reported for another tyrosine kinase receptor ligand, i.e., epidermal growth factor w27x. A possible explanation for the disparate effects of the two growth factors on IL-1 b-stimulated PLA 2 expression is the different degree of activation of protein kinase C. Whereas PDGF-BB potently stimulates phosphoinositide turnover and subsequent activation of protein kinase C w28,32x, bFGF is a poor activator of this signaling pathway in mesangial cells w33x. Our results clearly show that coincubation of mesangial cells with PDGF-BB and calphostin C, a potent and selective inhibitor of protein kinase C w22x, reverses the inhibitory effect of PDGF. This is very interesting in the context that the ´-isozyme of protein kinase C tonically suppresses inducible nitric oxide synthase expression in mesangial cells w34x, an enzyme that is also induced by IL-1 b and cAMP w35,36x and differentially modulated by PDGF and bFGF w33x. Recently the identification of novel secretory PLA 2 s Žnow called group IIc and V, respectively. has been reported w37x. Several investigators that had not rigorously identified the secreted PLA 2 s had to reevaluate their findings. Mast cells and the macrophage-like P388D1 cells are good examples thereof w37x. We used not only immunological techniques in the identification w7x but also purified and
221
sequenced the secreted enzyme w38x. A total of 36 out of 40 amino acids could be identified with certainty. Since these were completely identical to those of rat group II PLA 2 , we concluded that the enzyme secreted by mesangial cells was Žin the nomenclature of those days. a group II PLA 2 . Of course, one can never exclude the possibility that a few percent of the secreted sPLA 2 was in fact another type than type IIa and that we did not get the signals from these enzymes in the sequenator runs. However, of the 36 amino acids identified with certainty, 15 and 19 are different from rat group V and group IIc, respectively. Thus, the vast majority of the enzyme secreted by mesangial cells remains a group IIa PLA 2 , no matter what has been rectified for other cells. In summary, our data provide novel aspects of the regulation of IL-1 b and forskolin-stimulated group II PLA 2 expression in renal mesangial cells and highlight the complexity of cross-talk between signaling pathways involved in the induction and modulation of inflammatory enzymes.
Acknowledgements This work was supported by Swiss National Science Foundation Grant 31-43090.95 and by a grant from the Commission of the European Communities ŽBiomed 2 PL 950979.. The supply of recombinant IL-1 b , PDGF-BB and bFGF by Christiane Rordorf, Michael Pech and Michael Fox, respectively, is gratefully acknowledged.
References w1x H. van den Bosch, Biochim. Biophys. Acta 604 Ž1980. 161–246. w2x K.B. Glaser, D. Mobilio, J.Y. Chang, N. Senko, Trends Pharmacol. Sci. 14 Ž1993. 92–98. w3x M. Roberts, FASEB J. 10 Ž1996. 1159–1172. w4x R.L. Heinrikson, E.T. Krueger, P.S. Keim, J. Biol. Chem. 252 Ž1977. 4913–4921. w5x W. Pruzanski, P. Vadas, Immunol. Today 12 Ž1991. 143– 146. w6x J. Pfeilschifter, W. Pignat, K. Vosbeck, F. Marki, ¨ Biochem. Biophys. Res. Commun. 159 Ž1989. 385–394. w7x C. Schalkwijk, J. Pfeilschifter, F. Marki, ¨ H. van den Bosch, Biochem. Biophys. Res. Commun. 174 Ž1991. 268–275.
222
G. Walker et al.r Biochimica et Biophysica Acta 1391 (1998) 213–222
w8x J. Pfeilschifter, Phospholipase A 2 in clinical inflammation, in: K.B. Glaser, P. Vadas ŽEds.., Molecular Approaches to Pathophysiology, CRC Press, New York, 1995, pp. 25–51. w9x H. Muhl, ¨ T. Geiger, W. Pignat, F. Marki, ¨ H. van den Bosch, K. Vosbeck, J. Pfeilschifter, FEBS Lett. 291 Ž1991. 249– 252. w10x Y. Nakazato, M.S. Simonson, W.H. Herman, M. Konieczowski, M. Sedor, J.R. Sedor, J. Biol. Chem. 266 Ž1991. 14119–14127. w11x J. Pfeilschifter, W. Pignat, F. Marki, I. Wiesenberg, Eur. J. ¨ Biochem. 181 Ž1989. 237–242. w12x J. Pfeilschifter, J. Leighton, W. Pignat, F. Marki, K. Vos¨ beck, Fresenius, J. Anal. Chem. 337 Ž1990. 139–140. w13x J. Pfeilschifter, J. Leighton, W. Pignat, F. Marki, K. Vos¨ beck, Biochem. J. 273 Ž1991. 199–204. w14x H. Muhl, ¨ T. Geiger, W. Pignat, F. Marki, ¨ H. van den Bosch, N. Cerletti, D. Cox, G. McMaster, K. Vosbeck, J. Pfeilschifter, FEBS Lett. 301 Ž1992. 190–194. w15x J. Pfeilschifter, K. Vosbeck, Biochem. Biophys. Res. Commun. 175 Ž1991. 372–379. w16x F. Marki, R. Franson, Biochim. Biophys. Acta 879 Ž1986. ¨ 149–156. w17x A.J. Aarsman, J.G.N. De Jong, E. Arnoldussen, F.W. Neys, P.D. van Wassenaar, H. van den Bosch, J. Biol. Chem. 264 Ž1989. 10008–10014. w18x J.G.N. De Jong, H. Amesz, A.J. Aarsman, H.B.M. Lenting, H. van den Bosch, Eur. J. Biochem. 164 Ž1987. 129–135. w19x J. Sambrock, J. Fritsch, T. Maniatis, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1989. w20x R.H.N. van Schaik, N.M. Verhoeven, F.W. Neijs, A.J. Aarsman, H. van den Bosch, Biochim. Biophys. Acta 1169 Ž1993. 1–15.
w21x G.M. Fox, S.G. Schiffer, M.F. Rohde, L.B. Tsai, A.R. Banks, T. Arakawa, J. Biol. Chem. 263 Ž1988. 18452– 18458. w22x E. Kobayashi, H. Nakano, M. Morimoto, T. Tamaoki, Biochem. Biophys. Res. Commun. 156 Ž1989. 548–553. w23x J. Pfeilschifter, News Physiol. Sci. 9 Ž1994. 271–276. w24x J. Pfeilschifter, C. Schalkwijk, V.A. Briner, H. van den Bosch, J. Clin. Invest. 92 Ž1993. 2516–2522. w25x N. Wardle, Nephron 73 Ž1996. 1–7. w26x H.E. Abboud, Kidney Int. 43 Ž1993. 252–267. w27x M.J.B.M. Vervoordeldonk, C.G. Schalkwijk, J. Pfeilschifter, H. van den Bosch, Biochem. J. 315 Ž1996. 435–441. w28x J. Pfeilschifter, M. Hosang, Cell Signal. 3 Ž1991. 413–424. w29x P. Vadas, E. Stefanski, M. Wloch, B. Grouix, H. van den Bosch, B. Kennedy, Eur. J. Biochem. 235 Ž1996. 557–563. w30x F. Berenbaum, G. Thomas, S. Poiraudeau, G. Bereziat, M.T. ´´ Corvol, J. Masliah, FEBS Lett. 340 Ž1994. 51–55. w31x C. Jacques, G. Bereziat, L. Humbert, J.-L. Olivier, M.-T. ´´ Corvol, J. Masliah, F. Berenbaum, J. Clin. Invest. 99 Ž1997. 1864–1872. w32x J. Pfeilschifter, A. Huwiler, J. Auton. Pharmacol. 16 Ž1996. 315–318. w33x D. Kunz, G. Walker, W. Eberhardt, U.K. Messmer, A. Huwiler, J. Pfeilschifter, J. Clin. Invest. 100 Ž1997. 2800– 2809. w34x H. Muhl, ¨ J. Pfeilschifter, Biochem. J. 303 Ž1994. 607–612. w35x H. Muhl, ¨ D. Kunz, J. Pfeilschifter, Br. J. Pharmacol. 112 Ž1994. 1–8. w36x D. Kunz, H. Muhl, ¨ G. Walker, J. Pfeilschifter, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 5387–5391. w37x J.A. Tischfield, J. Biol. Chem. 272 Ž1997. 17247–17250. w38x C. Schalkwijk, J. Pfeilschifter, F. Marki, ¨ H. van den Bosch, J. Biol. Chem. 267 Ž1992. 8846–8851.