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CYSTATIN C SECRETION BY RAT GLOMERULAR MESANGIAL CELLS: AUTOCRINE LOOP FOR in vitro GROWTH-PROMOTING ACTIVITY C. TavCra I, J. Leung-Tack l, D. Prevot I, M.C. Gensac l, J. Martinez *, P. Fulcmnd *, and A. Colle l* 1 INSERM U 133, Faculte deMtdecine, 133route de Narbonne,31062 Toulouse,France 2 CCIPE, Faculti de Pharmacie,avenueCharlesFlahaut, 34060Montpellier, France Received
December
16,
1991
Cystatin C, the major inhibitor of the cysteineproteinasesfound in humanandmt body fluids, is particularly abundantin seminalplasmaand cerebrospinalfluid. In a precedentreport, we have evidenced noteworthy levels of cystatin C in tat kidney cortex. In the presentstudy, we show that rat mesa&al glomerular cells produce cystatin C. Immunoprecipitation of extracts of metabolically labeledcells and culture mediashowedthat the synthesizedcystatin C is a 15.5 + 0.5 kDa protein. The protein was releasedinto the culture supematant(1.6 + 0.26 ug/ 106 cells / 24h). Urinary rat cystatin C and PPPR synthetic peptide (5-8 N-terminal sequenceof rat cystatin C) increasedmesangialcell proliferation. Affinity chromatography on Ultrogel-avidinbiotin-PPPR of extracts of metabolically labeledcells indicate the existenceof a PPPR binding protein of 46 kDa. The resultsdescribedin this work suggest,for glomerular rat mesa&al cells in vitro, an autocrine regulationof proliferation by cystatin C. 0 1992 Academic Press, Inc.
Proteins of the cysteine proteinasefamily participate in numerous cellular processes including protein catabolism (l), proteolytic processingof prohormones(2), penetration of normal tissuesby malignant cells (3). Cystatin C is a major cysteine proteinaseinhibitor (4,5) belonging to family 2 of the cystatin superfamily (6). Human cystatin C, previously known as gamma-traceor post-gammaglobulin, is an alkaline low molecular weight protein (13 kDa) described for the first time in 1961, in cerebrospinal fluid (7,8), and isolated from urine of patients with tubular disorders (9, IO). It was shown to be a 120 amino-acid polypeptide containing 2 disullide bridges. Under normal physiological conditions, cystatin C is presentin all testedhuman biological fluids ( 11,12) and is particularly abundantin seminalplasma(13) and cerebrospinal fluid (14). Immunohistochemical studies of human cystatin C have also shownthat this protein is particularly expressedin neuroendocrinecells( 15,16). Rat cystatin C is an alkaline low molecularmassprotein, it hasbeenisolatedfrom urine after induction of a tubular dysfunction with sodium chromate, and from seminal vesicles (17,18). The primary structure of rat cystatin C was determinedby nucleotide and amino-acid sequencing (19,20). In rat, all tissuesand biological fluids already examined contained measurablelevels of cystatin C. Seminalvesiclesanddifferent compoundsof the central nervous * To whom correspondenceshouldbe addressed. 0006-291X/92 Copyright All rights
$1.50
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
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system contained the highest amount of cystatin C. Except these compartments, the kidney cortex area demonstrated noteworthy levels of cystatin C (21). Secretion of cystatin C by monocytes or macrophages (22) and the specific interaction between cystatin C and the fourth component of complement may be of particular interest at tissue sites of inflammation (23). Cystatins C and the 5-8 N-terminal synthetic tetrapeptides (KPPR in human and PPPR in rat), modulate leukocyte chemotaxis and phagocytosis-associated respiratory burst and were thus suggested to play a regulatory role in inflammatory processes (24, 25, 26). Treatment of mouse 3T3 tibroblasts with chicken cystatin was able to induce a stimulation of cell proliferation in culture (27). Lastly, an increase in cystatin C mRNA levels was observed in serum-free mouse embryo cells, after treatment with picomolar concentrations of TGFB (28). In this work, we have investigated the synthesis and secretion of cystatin C by rat glomerular mesa&al cells. The effects of rat cystatin C and synthetic peptide PPPR have been studied on the growth of rat mesangial cells. Finally we have investigated the possible presence of specific binding site(s) for PPPR. MATERIALS
AND
METHODS
Cystatin C measurement.Purification and identification of rat cystatin C have been described in details (17). Briefly, urine from sodium chromate intoxicated rats was chromatographied on an affinity column of Sepharose coupled to carboxymethyl-papain (Cm-papain). The second purification step consisted of an anion-exchange chromatography column equilibrated in 0.02M Tris-HCl, pH 9.2, as a starting buffer. Gradient elution was performed using the same buffer containing 0 to 0.3M NaCl. Antisera against purified rat cystatin C were raised in rabbits. Rat cystatin C levels were determined using the radioimmunoassay previously described (29). Fetal calf serum (FCS) was shown not to react in this rat cystatin C quantification method. Peptide synthesis. Peptides PPPR and biotinylated PPPR were synthesized according to a technique already described for tuftsin and postin (KPPR). Their purification was undertaken by a preparative HPLC system (PREP LC 500, Waters) on a 500 Cl8 column, and controlled by analytical HPLC (Waters 440) in a Cl8 column eluted with 0.1% TFA, 20% acetonitril and 80% H,O, at a flow rate of 1mVmin (25,30). Cell culture. -Rat mesa&al cells were obtained from cultures of glomeruli isolated by graded sieving of kidney cortex suspension obtained after collagenase digestion (0.35 U/l) (31). For primary cultures, about 400 isolated glomeruli were plated into 25 cm2 flask (NUNC) in a humidified atmosphere of 95% air-5% CO, and overlayed with complete medium consisting of RPM1 1640 supplemented with 10% fetal calf serum (FCS), 50 U/ml penicillin, 5Opg/ml streptomycin and 2mM glutamine. After 3 weeks of culture, mesangial cells were identified by morphological and functional criteria and were shown to constitute 95% of the total cells (32,33). -Protein synthesis : After washing, subconfluent cultures ( lo6 cells per 25 cm2 flask) were incubated for 24h at 37°C in leucine-free medium added with 100 pCi of X-leucine (140 Ci/ mmol, Amersham). Conditionned media were harvested and cells were solubilized by sonication (2 x 15 s). These protein solutions were used for protein characterization. -For cystatin C characterization, proteins were chromatographied (Cm-papain and anion exchange columns) or selectively immunoprecipitated as follows: lml of Ultrogel-Protein A (IBF, France) was coupled to 5 ml of anti-rat cystatin C rabbit antiserum according to the manufacturer’s recommendations; 50 ~1 of Ultrogel-Protein A-IgG were incubated overnight at 4°C with cystatin C containing samples ( 3H-leucine mdiolabeled protein solutions); 100 1.11 of Sephacryl S-300 were added and the samples were centrifuged at 2,000g for 10 min. The pellets were washed until supematant radioactivity approached zero. The radiolabeled proteins, specifically bound in the pellet, were dissolved into 100 ~1 of electrophoresis buffer containing 0.06M Tris-HCl, pH 6.7, 2% SDS, 8% glycerol, 2% B-mercaptoethanol and 0.005% bromophenol blue. Samples were then boiled for 3 min at 90°C. Proteins were analyzed by electrophoresis on 4.5-12% (wb) discontinuous SDS - polyacrylamide slab gels. After protein fucation, the gels were dried and exposed to Hyperfilm-MP (Amersham). 1083
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-For binding site partial characterization, the solution of solubilized cells was chromatographied on an affinity column consisting of Ultrogel-avidin (IBF, France) coupled to biotin-PPPR according to the manufacturer’s recommendations. Proteins presenting afftnity for PPPR were specifically retained on the column and eluted with 0.2M Glycine buffer, pH 2.8 containing 0.5M NaCl. The eluted proteins were analyzed by SDS-polyacrylamide gel electrophoresis (4.5 10%) and autoradiographied. Specificity of binding sites for PPPR was demonstrated by eluting radiolabeled proteins bound on the afftnity column with lo-6M PPPR in PBS. Mesangial cell growth assays. 105mesangialcells were seededon 60mmdiameterculture dishesin 2 ml of RPM1 medium supplementedwith 10% FCS. After 48h of incubation, the cultureswere serum-depletedduring the following 48h. The mediawere removed and replaced by 2 ml of serum-freemedium, supplementedor not with the various moleculesto be tested.To estimate cell proliferation, cells were either trypsinized and counted in a Coulter Counter (Coultronics France) or submitted to 3H-thymidine uptake as follows : 5 uCi methyl- 3Hthymidine (42 Wmmol, Amersham)were addedto eachwell; after 24h, cells were washedand 5 ml of 5% TCA were addedfor 30 min.; the preparationswere rlnced with ethanol, lysed with 1 ml of 1% SDS, O.lM NaOH, 2% Na,C03 and their l3radioactivity, incorporatedinto DNA, wascounted(19OOADLiquid Scintillation Analyzer; Packard). Protein concentrationswere determinedaccordingto the Bradford method(34). Data were expressedasmeans+ SEM. RESULTS In isolatedtat glometuli cultures,the highestlevel of secretedrat cystatin C wasobtained after a 20 day period of culture, when mesangialcellscomposed95% of the cultured cells (data not shown). In conventional mesangialcell cultures i.e. in presenceof 10% FCS, a cystatin C secretionwas detectedin the conditionedmedium,and wasshownto be time-dependentvarying from 0.21 f 0.09 ug / 106 cells at 6h to 2.2 -+0.3 pg/ 106 cells at 48h (fig.lA).
In the same
conditions,the intracellular cystatin C levels remainedstablearound 0.10 f 0.04 ug/ 106cells. The radiolabeledcystatin C secretedby rat mesar@alcellsduring a 24h incubationperiod with 3H-leucinepresentedidentical characteristicsto thoseof the rat urine cystatin C we already characterized (17). Both the secretedand the cellular radiolabeled cystatin C lysate had an apparent molecular mass of 15.5 f 0.5 kDa, as determined by SDS-PAGE after immunoprecipitation (fig. 1B). This molecule was retained on a Cm-papain affinity column, whereasits major part was not retained on an anion exchangechromatography at pH 9.2. As shown by RIA, cystatin C was eluted in the peaks 1 and 2 (fig.lC), the peaks 3, 4 and 5 representedother cysteineproteinaseinhibitors. The effects of native urinary rat cystatin C (PI: 10.2) and of synthetic peptide PPPR were testedon the proliferation of rat mesangialcells cultured in the absenceof FCS. The two compounds(l&M)
stimulatedthe cell proliferation, this effect wassimilar to that obtainedwith
IOM~MEGF (fig.;lA). The increasein cell proliferation, estimatedby two different methods(cell counting and 3H-thymidine incorporation) was dose-dependent.The stimulation of DNA synthesisis shownin figure 2 B. Affity chromatography,wasperformed on Ultrogel-avidine coupled to biotin-PPPR It retained radiolabeledproteins from lysates of mesangialcells cultured with 3H-leucine, thus indicating the existenceof binding proteins. Thesemoleculescould be eluted by low pH buffer (0.2M Glycine, pH 2.8, 0.5M NaCl) or more specifically by 10m6MPPPR (Bg.3A). No radiolabeledproteins unspecifically bound the affinity column. One major protein was eluted 1084
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-33-w
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Fkure 1. Synthesis andsecretion of cystatinC by ratmesangial cellsin culture. A. Rat cystatinC levelwasmeasured by RIA methodin theculturemediumof rat mesangial cellsafter different incubationtimes.For eachpoint, the numberof cellswasdetermined. CystatinC secretionwastimedependent. B. Radiolabeledproteinsfrom culture medium(lane 1) or cellular lysate(lane 2) were analyzed by SDS-polyacrylamide gel electrophoresisand autoradiography after immunoprecipitation by anti-ratcystatinC antisera.CystatinC synthesized by rat mesangial cellsshoweda molecularmassof 15.5* 0.5 kDa. C. Anion exchangechromatography: characterization of cystatinC from themediumof rat mesangial cellsculturedwith 3H-leucine. The culturemediawerechromatographied on Cmpapainaffinity column.Cysteineproteinase inhibitors,retainedon the affinity columnand thenelutedby O.OlMNaOHsolution,wereloadedonanionexchangechromatography. The two first peaksweredetectableby bothradioactivitycountandRIA method(shaded areas) indicatingthat theycontaincystatinC synthesized by mesangial cells.Peaks3,4 and5 were constituted by cysteineproteinase inhibitorsdifferentfromcystatinC.
from the affinity column by low pH buffer and was detected as a 46 kDa protein following analysisof the radiolabeledmaterial, by 10%SDS-PAGE and autotadiography (fig.3B).
DISCUSSION Our resultsdemonstratedthat conventional culturesof tat glomerular meaangialcells(in presenceof 10% FCS) were able to secreteconsiderableamountsof cystatin C (1.6 f 0.3 pg/ 106 cells / 24h). The maximum of secretion arose between 6h and 24h of culture. By comparison,much lower amountsof cystatin C (26.5 + 6.8 ng/ 106 cells/ 24h) were shown to be secretedby alveolar macrophages(22). 1085
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0%) 6 0 500 2 P 8 400 e8; *z 3 300
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Time
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-1 M)
Figure 2. Effect of tat cystatin C and of synthetic peptide PPPR on the induction of rat mesangial cell proliferation. A. Rat mesangial cells, cultured without FCS, were counted after various periods of treatment with purified rat cystatin C (IO-‘M) or synthetic peptide PPPR (lo-‘M). 10e9M EGF (Sigma) was used as a positive control. The two molecules stimulated mesangial cell proliferation after 48h of treatment. B. Dose dependent effect of rat cystatin C and PPPR on mesangial cell proliferation determined by 3H-thymidine incorporation. Molecules are added to culture medium at various concentrations ( 10mgMto lo-‘M ). Results are expressed in percent of control. 10 -‘M of rat cystatin C and PPPR induced a maximum incorporation of 426% and 349% respectively and 10e9M EGF stimulated the mesangial cell proliferation by 480%.
The use of 3H-leucineallowed us to show that cystatin C was de nova synthesizedby mesangialcells in vitro. Moreover, the synthesizedcystatin C exhibited the samecharacteristics to those of rat urinary cystatin C, i.e. chromatographic behaviour on Cm-papain and anion
B PPPR
0 0
I 50 Elution
(IO -‘+A)
I 100 volume
1 150 (ml)
3. Binding site for PPPR on mesangial cellular lysates. A. Affinity chromatography on Ultrogel-avidine-biotine-PPPR retained radiolabeled proteins from mesangial cellular lysates. These proteins were eluted by 10 dM PPPR indicating specificity of the binding proteins for PPPR. B. The material eluted from the affinity column by low pH buffer (0.2M Glycine, pH 2.8, 0.5M NaCl) then analyzed by 10% SDS-PAGE and autoradiography consists in one major protein of 46 kDa. Lane 1: sample in loading buffer. Lane 2: sample reduced with 2mM dithiothreitol in 50mM sodium phosphate pH 7, 2% (w/v) SDS at 100°C for Sm, and alkylated by iodoacetamide (IOOmM fmal).
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exchange chromatography (peak 1: native form with pIB9.2; peak 2: truncated form with pIC9.2) (17), apparentmolecularmass(15.5 f 0.5 kDa) andimmunological characteristics. Cell growth assaysshowedthat 10%l and lo-7M of native cystatin C (17) induced an increaseof rat mesangialcell proliferation. The maximum of stimulation wasobtainedwith 10-7 M rat cystatin C. Besides,eggwhite cystatin, anothermemberof the secondfamily of cystatins, hasalready been shownto have a proliferative effect on mouse3T3 fibroblasts (27), but much higher concentrations( 10m5M)of cystatin were required. Interestingly, differencesbetweenegg white cystatin or human and rat cystatin C especially concerned the N-terminal parts of the molecules: the egg white cystatin doesnot contain the particular KPPR sequencepresent in human cystatin C (35) or the PPPR present in rat cystatin C ( 17,18). Otherwise, we have already suggestedthat thesetwo tetrapeptidescould exert a possibleimmunomodulatingactivity, similar to that of the native cystatins C (24,25,26). In the present study, we evidenced proliferative effect of PPPR ( 10-7M), i.e.the synthetic peptide corresponding to the 5-8 Nterminal sequenceof rat cystatin C. This effect wasquite identical to that of the native molecule. Lastly, affinity chromatography on Ultrogel-avidin-biotin-PPPR allowed us to detect specific PPPR binding site of 46 kDa in mesangialcell lysates. The resultsdescribedhere, namely: a significant secretionof cystatin C, a proliferative effect of both cystatin C and PPPR and lastly the existenceon mesangialcell lysatesof PPPR binding proteins, suggesta possible autocrine regulation by cystatin C of rat glomerular mesangialcell proliferation. Moreover, the 5-8 N-terminal sequenceof rat cystatin C (PPPR) could play a major role in this cell proliferation. Recent studiesdocumentedthe presenceof the cysteine proteinases,cathepsinesB, H and L in glomeruli and demonstratedthe ability of endogenousglomerular cysteine proteinases to degradeintact glomerular basementmembranes(36). Thus, the local secretionof cystatin C may be an important factor in glomerular diseasescharacterized by matrix expansion and mesangialcell proliferation. Analogous effects have already beendescribedfor other substances andparticularly for growth factors (37).
Acknowledgments We wish to thank Dr. Jean-Marie DARBON for reading the paper and for valuable suggestions.We are grateful to Dr. Nelly BLAES for the critical review of the manuscript. We thank Jean-ClaudeLEPERT andChristianMORA for assistancewith photography. REFERENCES 1.
Barrett, A.J., and Kirschke, H. (1981) in Methods in Enzymology (L. Lorand, ed.) Vol 80, pp. 535-561, Academic Press,New York. 2. Marks, N., Berg, M.J., and Benuck, B. (1986) Arch. Biochem. Biophys. 249,489-499. 3. Maciewicz, R.A., Wardale R.J., Etherington, D.J., and Paraskeva,C. (1989) Int. J. Cant. 43,478-484. 4. Bnin, J., Popovic, T., Turck, V., Borchart, U., and Machleidt, W. (1984) Biochem. Biophys. Res. Commun. 118, 103-109. 5. Barrett, A.J., Davies, M.E., and Grubb, A. (1984) B&hem. Biophys. Res. Commun. 120, 631-636. 6. Barrett, A.J., Fritz, H., Grubb, A., Isemura, S., Jarvinen., M., Katunuma, N., Machleidt, W., Muller Esterl, W., Sasaki,M., and Turk, V. (1986) B&hem. J. 236, 311-312. 7. Clausen,J. (1961) Proc. Sot. Exp. Biol. (N.Y.) 107, 170-172. 1087
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8. Mac Pherson, C.F.C., and Cosgrove, J.B.R. (1961) Can. J. Biochem. 39, 1567-1574. 9. Cejka, J., and Fleischmann, L.E. (1973) Arch. B&hem. Biophys. 157, 168-176. 10. Manuel, Y., Leclercq, M., Colli, A., and Tonnelle, C. (1973) in Protides of the biological fluids, Proceedings of the Twenty First Colloquium Brugge (H. Peetem, ed.) pp. 505-5 11. 11. Hochwald, G.M., and Thorbecke, G.E. (1962) Proc. Sot. Exp. Biol. Med. 109,91-95. 12. Abrahamson, M., Barrett, A.J., and Salvesen, G. (1986) J. Biol. Chem. 261, 1128211289. 13. Collt, A., Guinet, R., Leclercq, M., and Manuel, Y (1976) Clin. Chim. Acta 67, 93-97. Liifberg, H., and Grubb, A. (1979) Stand. J. Clin. Lab. Invest. 39, 619-626. :t Grubb, A., and Liitberg, H. (1982) Proc. Sot. Acad. Sci. USA 79,3024-3027. 16: MGller, C.A., LGlberg, H., Grubb, A., Olsson, S.O., Davies, M.E., and Barrett, A.J. (1985) Neuroendocrinology 4 1,400-404. 17. Tavera, C., Guillemot, J.C., Capdevielle, J., Fermra, P., Leung-Tack, J., and Collt, A. (1989) Prep. Biochem. 19,279-291. Esnard, A., Esnard, F., Faucher, D., and Gauthier, F. (1988) FEBS Lett. 236,475-478. :;: Esnard, F., Esnard, A., Faucher, D., Capony, J.P., Derancourt, J., Brillard, M., and Gauthier, F. (1990) Biol. Chem. Hoppe-Seyler 371, 161-166. 40. Cole, T., Dickson, P.W., Esnard, F., Averill, S., Risbridger, G.P., Gauthier, F., and Schreiberg, G. (1989) Eur. J. B&hem. 186-35-42. 21. Tavern, C., Prkot, D., Girolami, J.P., Leung-Tack, J., and Collt, A. (1990) Biol. Chem. Hoppe-Seyler 371, 187-192. 22. Chapman, H., Reilly, J., Yee, R., and Grubb, A. (1990) Am. Rev. Respir. Dis. 141, 698705. 23. Ghiso, J., Saball, E., Leoni, J., Rostagno, A., and Frangione, B. (1990) Proc. Natl. Acad. Sci. USA 87, 1288-1291. Leung-Tack, J., Tavern, C., Martinez, J., and Collt, A. (1990) Inflammation 14,247-258. E: Leung-Tack, J., Tavern, C., Gensac, M.C., Martinez, J., and Collt, A. (1990) Exp. Cell. Res. 188, 16-22. 26. Leung-Tack, J., Tavira, C., Er-Raki, A., Gensac, M.C., and Collt, A. (1990) Biol. Chem. Hoppe Seyler 37 1,255-258. Sun, Q. (1898) Exp. Cell. Res. 180, 150-160. 2287:Solem, M., Rawson, C., Linburg, K., and Barnes, D. (1990) Biochem. Biophys. Res. Commun. 172, 945-95 1. 29. Collt, A., Tavern, C., Laurent, P., Leung-Tack, J., and Girolami, J.P. (1990) J. Immunoassay 11,199-214. 30. Bonnaud, B., Castro, B., Cousse, H., and Martinez, J. ( 1982) Boll. Chim. Farm. 121, 550-556. 31. Foidart, J.B., Duchenne, C., Dubois, C., Deheneffe, J., and Mahieu, D. (1981) Adv. Nephrol. 10, 267-292. 32. Emond, C., Bascands, J.L., Pecher, C., Cabos-Boutot, G., Pradelles, P., Regoli, D., and Girolami, J.P. (1990) Europ. J. Pharmacol. 190, 381-392. 33. Mene, P., Simonson, M.S., and Dunn, J.M. (1989) Physiol. Rev. 69, 1347- 1424. Bradford, M. (1976) Anal. Biochem. 72,248-254. E: Tonnelle, C., Collt, A., Fougereau, M., and Manuel, Y. (1979) Biochem. Biophys. Res. Commun. 86, 613-619. 36. Baricos, W.H., Cortez, S.L., Le, Q.C., Zhou, Y., Dicarlo, R.M., O’Connor, S.A., and Shah, S.V. (1990) Kidney International 38,395-401. 37. Striker, L.J., Peten, E.P., Elliot, S.J., Doi, T., Striker, G.E. (1991) Lab. Invest. 64,446456.
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CYSTATIN C SECRETION BY RAT GLOMERULAR MESANGIAL CELLS: AUTOCRINE LOOP FOR in vitro GROWTH-PROMOTING ACTIVITY C. TavCra I, J. Leung-Tack l, D. Prevot I, M.C. Gensac l, J. Martinez *, P. Fulcmnd *, and A. Colle l* 1 INSERM U 133, Faculte deMtdecine, 133route de Narbonne,31062 Toulouse,France 2 CCIPE, Faculti de Pharmacie,avenueCharlesFlahaut, 34060Montpellier, France Received
December
16,
1991
Cystatin C, the major inhibitor of the cysteineproteinasesfound in humanandmt body fluids, is particularly abundantin seminalplasmaand cerebrospinalfluid. In a precedentreport, we have evidenced noteworthy levels of cystatin C in tat kidney cortex. In the presentstudy, we show that rat mesa&al glomerular cells produce cystatin C. Immunoprecipitation of extracts of metabolically labeledcells and culture mediashowedthat the synthesizedcystatin C is a 15.5 + 0.5 kDa protein. The protein was releasedinto the culture supematant(1.6 + 0.26 ug/ 106 cells / 24h). Urinary rat cystatin C and PPPR synthetic peptide (5-8 N-terminal sequenceof rat cystatin C) increasedmesangialcell proliferation. Affinity chromatography on Ultrogel-avidinbiotin-PPPR of extracts of metabolically labeledcells indicate the existenceof a PPPR binding protein of 46 kDa. The resultsdescribedin this work suggest,for glomerular rat mesa&al cells in vitro, an autocrine regulationof proliferation by cystatin C. 0 1992 Academic Press, Inc.
Proteins of the cysteine proteinasefamily participate in numerous cellular processes including protein catabolism (l), proteolytic processingof prohormones(2), penetration of normal tissuesby malignant cells (3). Cystatin C is a major cysteine proteinaseinhibitor (4,5) belonging to family 2 of the cystatin superfamily (6). Human cystatin C, previously known as gamma-traceor post-gammaglobulin, is an alkaline low molecular weight protein (13 kDa) described for the first time in 1961, in cerebrospinal fluid (7,8), and isolated from urine of patients with tubular disorders (9, IO). It was shown to be a 120 amino-acid polypeptide containing 2 disullide bridges. Under normal physiological conditions, cystatin C is presentin all testedhuman biological fluids ( 11,12) and is particularly abundantin seminalplasma(13) and cerebrospinal fluid (14). Immunohistochemical studies of human cystatin C have also shownthat this protein is particularly expressedin neuroendocrinecells( 15,16). Rat cystatin C is an alkaline low molecularmassprotein, it hasbeenisolatedfrom urine after induction of a tubular dysfunction with sodium chromate, and from seminal vesicles (17,18). The primary structure of rat cystatin C was determinedby nucleotide and amino-acid sequencing (19,20). In rat, all tissuesand biological fluids already examined contained measurablelevels of cystatin C. Seminalvesiclesanddifferent compoundsof the central nervous * To whom correspondenceshouldbe addressed. 0006-291X/92 Copyright All rights
$1.50
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
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system contained the highest amount of cystatin C. Except these compartments, the kidney cortex area demonstrated noteworthy levels of cystatin C (21). Secretion of cystatin C by monocytes or macrophages (22) and the specific interaction between cystatin C and the fourth component of complement may be of particular interest at tissue sites of inflammation (23). Cystatins C and the 5-8 N-terminal synthetic tetrapeptides (KPPR in human and PPPR in rat), modulate leukocyte chemotaxis and phagocytosis-associated respiratory burst and were thus suggested to play a regulatory role in inflammatory processes (24, 25, 26). Treatment of mouse 3T3 tibroblasts with chicken cystatin was able to induce a stimulation of cell proliferation in culture (27). Lastly, an increase in cystatin C mRNA levels was observed in serum-free mouse embryo cells, after treatment with picomolar concentrations of TGFB (28). In this work, we have investigated the synthesis and secretion of cystatin C by rat glomerular mesa&al cells. The effects of rat cystatin C and synthetic peptide PPPR have been studied on the growth of rat mesangial cells. Finally we have investigated the possible presence of specific binding site(s) for PPPR. MATERIALS
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Cystatin C measurement.Purification and identification of rat cystatin C have been described in details (17). Briefly, urine from sodium chromate intoxicated rats was chromatographied on an affinity column of Sepharose coupled to carboxymethyl-papain (Cm-papain). The second purification step consisted of an anion-exchange chromatography column equilibrated in 0.02M Tris-HCl, pH 9.2, as a starting buffer. Gradient elution was performed using the same buffer containing 0 to 0.3M NaCl. Antisera against purified rat cystatin C were raised in rabbits. Rat cystatin C levels were determined using the radioimmunoassay previously described (29). Fetal calf serum (FCS) was shown not to react in this rat cystatin C quantification method. Peptide synthesis. Peptides PPPR and biotinylated PPPR were synthesized according to a technique already described for tuftsin and postin (KPPR). Their purification was undertaken by a preparative HPLC system (PREP LC 500, Waters) on a 500 Cl8 column, and controlled by analytical HPLC (Waters 440) in a Cl8 column eluted with 0.1% TFA, 20% acetonitril and 80% H,O, at a flow rate of 1mVmin (25,30). Cell culture. -Rat mesa&al cells were obtained from cultures of glomeruli isolated by graded sieving of kidney cortex suspension obtained after collagenase digestion (0.35 U/l) (31). For primary cultures, about 400 isolated glomeruli were plated into 25 cm2 flask (NUNC) in a humidified atmosphere of 95% air-5% CO, and overlayed with complete medium consisting of RPM1 1640 supplemented with 10% fetal calf serum (FCS), 50 U/ml penicillin, 5Opg/ml streptomycin and 2mM glutamine. After 3 weeks of culture, mesangial cells were identified by morphological and functional criteria and were shown to constitute 95% of the total cells (32,33). -Protein synthesis : After washing, subconfluent cultures ( lo6 cells per 25 cm2 flask) were incubated for 24h at 37°C in leucine-free medium added with 100 pCi of X-leucine (140 Ci/ mmol, Amersham). Conditionned media were harvested and cells were solubilized by sonication (2 x 15 s). These protein solutions were used for protein characterization. -For cystatin C characterization, proteins were chromatographied (Cm-papain and anion exchange columns) or selectively immunoprecipitated as follows: lml of Ultrogel-Protein A (IBF, France) was coupled to 5 ml of anti-rat cystatin C rabbit antiserum according to the manufacturer’s recommendations; 50 ~1 of Ultrogel-Protein A-IgG were incubated overnight at 4°C with cystatin C containing samples ( 3H-leucine mdiolabeled protein solutions); 100 1.11 of Sephacryl S-300 were added and the samples were centrifuged at 2,000g for 10 min. The pellets were washed until supematant radioactivity approached zero. The radiolabeled proteins, specifically bound in the pellet, were dissolved into 100 ~1 of electrophoresis buffer containing 0.06M Tris-HCl, pH 6.7, 2% SDS, 8% glycerol, 2% B-mercaptoethanol and 0.005% bromophenol blue. Samples were then boiled for 3 min at 90°C. Proteins were analyzed by electrophoresis on 4.5-12% (wb) discontinuous SDS - polyacrylamide slab gels. After protein fucation, the gels were dried and exposed to Hyperfilm-MP (Amersham). 1083
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-For binding site partial characterization, the solution of solubilized cells was chromatographied on an affinity column consisting of Ultrogel-avidin (IBF, France) coupled to biotin-PPPR according to the manufacturer’s recommendations. Proteins presenting afftnity for PPPR were specifically retained on the column and eluted with 0.2M Glycine buffer, pH 2.8 containing 0.5M NaCl. The eluted proteins were analyzed by SDS-polyacrylamide gel electrophoresis (4.5 10%) and autoradiographied. Specificity of binding sites for PPPR was demonstrated by eluting radiolabeled proteins bound on the afftnity column with lo-6M PPPR in PBS. Mesangial cell growth assays. 105mesangialcells were seededon 60mmdiameterculture dishesin 2 ml of RPM1 medium supplementedwith 10% FCS. After 48h of incubation, the cultureswere serum-depletedduring the following 48h. The mediawere removed and replaced by 2 ml of serum-freemedium, supplementedor not with the various moleculesto be tested.To estimate cell proliferation, cells were either trypsinized and counted in a Coulter Counter (Coultronics France) or submitted to 3H-thymidine uptake as follows : 5 uCi methyl- 3Hthymidine (42 Wmmol, Amersham)were addedto eachwell; after 24h, cells were washedand 5 ml of 5% TCA were addedfor 30 min.; the preparationswere rlnced with ethanol, lysed with 1 ml of 1% SDS, O.lM NaOH, 2% Na,C03 and their l3radioactivity, incorporatedinto DNA, wascounted(19OOADLiquid Scintillation Analyzer; Packard). Protein concentrationswere determinedaccordingto the Bradford method(34). Data were expressedasmeans+ SEM. RESULTS In isolatedtat glometuli cultures,the highestlevel of secretedrat cystatin C wasobtained after a 20 day period of culture, when mesangialcellscomposed95% of the cultured cells (data not shown). In conventional mesangialcell cultures i.e. in presenceof 10% FCS, a cystatin C secretionwas detectedin the conditionedmedium,and wasshownto be time-dependentvarying from 0.21 f 0.09 ug / 106 cells at 6h to 2.2 -+0.3 pg/ 106 cells at 48h (fig.lA).
In the same
conditions,the intracellular cystatin C levels remainedstablearound 0.10 f 0.04 ug/ 106cells. The radiolabeledcystatin C secretedby rat mesar@alcellsduring a 24h incubationperiod with 3H-leucinepresentedidentical characteristicsto thoseof the rat urine cystatin C we already characterized (17). Both the secretedand the cellular radiolabeled cystatin C lysate had an apparent molecular mass of 15.5 f 0.5 kDa, as determined by SDS-PAGE after immunoprecipitation (fig. 1B). This molecule was retained on a Cm-papain affinity column, whereasits major part was not retained on an anion exchangechromatography at pH 9.2. As shown by RIA, cystatin C was eluted in the peaks 1 and 2 (fig.lC), the peaks 3, 4 and 5 representedother cysteineproteinaseinhibitors. The effects of native urinary rat cystatin C (PI: 10.2) and of synthetic peptide PPPR were testedon the proliferation of rat mesangialcells cultured in the absenceof FCS. The two compounds(l&M)
stimulatedthe cell proliferation, this effect wassimilar to that obtainedwith
IOM~MEGF (fig.;lA). The increasein cell proliferation, estimatedby two different methods(cell counting and 3H-thymidine incorporation) was dose-dependent.The stimulation of DNA synthesisis shownin figure 2 B. Affity chromatography,wasperformed on Ultrogel-avidine coupled to biotin-PPPR It retained radiolabeledproteins from lysates of mesangialcells cultured with 3H-leucine, thus indicating the existenceof binding proteins. Thesemoleculescould be eluted by low pH buffer (0.2M Glycine, pH 2.8, 0.5M NaCl) or more specifically by 10m6MPPPR (Bg.3A). No radiolabeledproteins unspecifically bound the affinity column. One major protein was eluted 1084
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-33-w
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Fkure 1. Synthesis andsecretion of cystatinC by ratmesangial cellsin culture. A. Rat cystatinC levelwasmeasured by RIA methodin theculturemediumof rat mesangial cellsafter different incubationtimes.For eachpoint, the numberof cellswasdetermined. CystatinC secretionwastimedependent. B. Radiolabeledproteinsfrom culture medium(lane 1) or cellular lysate(lane 2) were analyzed by SDS-polyacrylamide gel electrophoresisand autoradiography after immunoprecipitation by anti-ratcystatinC antisera.CystatinC synthesized by rat mesangial cellsshoweda molecularmassof 15.5* 0.5 kDa. C. Anion exchangechromatography: characterization of cystatinC from themediumof rat mesangial cellsculturedwith 3H-leucine. The culturemediawerechromatographied on Cmpapainaffinity column.Cysteineproteinase inhibitors,retainedon the affinity columnand thenelutedby O.OlMNaOHsolution,wereloadedonanionexchangechromatography. The two first peaksweredetectableby bothradioactivitycountandRIA method(shaded areas) indicatingthat theycontaincystatinC synthesized by mesangial cells.Peaks3,4 and5 were constituted by cysteineproteinase inhibitorsdifferentfromcystatinC.
from the affinity column by low pH buffer and was detected as a 46 kDa protein following analysisof the radiolabeledmaterial, by 10%SDS-PAGE and autotadiography (fig.3B).
DISCUSSION Our resultsdemonstratedthat conventional culturesof tat glomerular meaangialcells(in presenceof 10% FCS) were able to secreteconsiderableamountsof cystatin C (1.6 f 0.3 pg/ 106 cells / 24h). The maximum of secretion arose between 6h and 24h of culture. By comparison,much lower amountsof cystatin C (26.5 + 6.8 ng/ 106 cells/ 24h) were shown to be secretedby alveolar macrophages(22). 1085
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0%) 6 0 500 2 P 8 400 e8; *z 3 300
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Time
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Figure 2. Effect of tat cystatin C and of synthetic peptide PPPR on the induction of rat mesangial cell proliferation. A. Rat mesangial cells, cultured without FCS, were counted after various periods of treatment with purified rat cystatin C (IO-‘M) or synthetic peptide PPPR (lo-‘M). 10e9M EGF (Sigma) was used as a positive control. The two molecules stimulated mesangial cell proliferation after 48h of treatment. B. Dose dependent effect of rat cystatin C and PPPR on mesangial cell proliferation determined by 3H-thymidine incorporation. Molecules are added to culture medium at various concentrations ( 10mgMto lo-‘M ). Results are expressed in percent of control. 10 -‘M of rat cystatin C and PPPR induced a maximum incorporation of 426% and 349% respectively and 10e9M EGF stimulated the mesangial cell proliferation by 480%.
The use of 3H-leucineallowed us to show that cystatin C was de nova synthesizedby mesangialcells in vitro. Moreover, the synthesizedcystatin C exhibited the samecharacteristics to those of rat urinary cystatin C, i.e. chromatographic behaviour on Cm-papain and anion
B PPPR
0 0
I 50 Elution
(IO -‘+A)
I 100 volume
1 150 (ml)
3. Binding site for PPPR on mesangial cellular lysates. A. Affinity chromatography on Ultrogel-avidine-biotine-PPPR retained radiolabeled proteins from mesangial cellular lysates. These proteins were eluted by 10 dM PPPR indicating specificity of the binding proteins for PPPR. B. The material eluted from the affinity column by low pH buffer (0.2M Glycine, pH 2.8, 0.5M NaCl) then analyzed by 10% SDS-PAGE and autoradiography consists in one major protein of 46 kDa. Lane 1: sample in loading buffer. Lane 2: sample reduced with 2mM dithiothreitol in 50mM sodium phosphate pH 7, 2% (w/v) SDS at 100°C for Sm, and alkylated by iodoacetamide (IOOmM fmal).
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exchange chromatography (peak 1: native form with pIB9.2; peak 2: truncated form with pIC9.2) (17), apparentmolecularmass(15.5 f 0.5 kDa) andimmunological characteristics. Cell growth assaysshowedthat 10%l and lo-7M of native cystatin C (17) induced an increaseof rat mesangialcell proliferation. The maximum of stimulation wasobtainedwith 10-7 M rat cystatin C. Besides,eggwhite cystatin, anothermemberof the secondfamily of cystatins, hasalready been shownto have a proliferative effect on mouse3T3 fibroblasts (27), but much higher concentrations( 10m5M)of cystatin were required. Interestingly, differencesbetweenegg white cystatin or human and rat cystatin C especially concerned the N-terminal parts of the molecules: the egg white cystatin doesnot contain the particular KPPR sequencepresent in human cystatin C (35) or the PPPR present in rat cystatin C ( 17,18). Otherwise, we have already suggestedthat thesetwo tetrapeptidescould exert a possibleimmunomodulatingactivity, similar to that of the native cystatins C (24,25,26). In the present study, we evidenced proliferative effect of PPPR ( 10-7M), i.e.the synthetic peptide corresponding to the 5-8 Nterminal sequenceof rat cystatin C. This effect wasquite identical to that of the native molecule. Lastly, affinity chromatography on Ultrogel-avidin-biotin-PPPR allowed us to detect specific PPPR binding site of 46 kDa in mesangialcell lysates. The resultsdescribedhere, namely: a significant secretionof cystatin C, a proliferative effect of both cystatin C and PPPR and lastly the existenceon mesangialcell lysatesof PPPR binding proteins, suggesta possible autocrine regulation by cystatin C of rat glomerular mesangialcell proliferation. Moreover, the 5-8 N-terminal sequenceof rat cystatin C (PPPR) could play a major role in this cell proliferation. Recent studiesdocumentedthe presenceof the cysteine proteinases,cathepsinesB, H and L in glomeruli and demonstratedthe ability of endogenousglomerular cysteine proteinases to degradeintact glomerular basementmembranes(36). Thus, the local secretionof cystatin C may be an important factor in glomerular diseasescharacterized by matrix expansion and mesangialcell proliferation. Analogous effects have already beendescribedfor other substances andparticularly for growth factors (37).
Acknowledgments We wish to thank Dr. Jean-Marie DARBON for reading the paper and for valuable suggestions.We are grateful to Dr. Nelly BLAES for the critical review of the manuscript. We thank Jean-ClaudeLEPERT andChristianMORA for assistancewith photography. REFERENCES 1.
Barrett, A.J., and Kirschke, H. (1981) in Methods in Enzymology (L. Lorand, ed.) Vol 80, pp. 535-561, Academic Press,New York. 2. Marks, N., Berg, M.J., and Benuck, B. (1986) Arch. Biochem. Biophys. 249,489-499. 3. Maciewicz, R.A., Wardale R.J., Etherington, D.J., and Paraskeva,C. (1989) Int. J. Cant. 43,478-484. 4. Bnin, J., Popovic, T., Turck, V., Borchart, U., and Machleidt, W. (1984) Biochem. Biophys. Res. Commun. 118, 103-109. 5. Barrett, A.J., Davies, M.E., and Grubb, A. (1984) B&hem. Biophys. Res. Commun. 120, 631-636. 6. Barrett, A.J., Fritz, H., Grubb, A., Isemura, S., Jarvinen., M., Katunuma, N., Machleidt, W., Muller Esterl, W., Sasaki,M., and Turk, V. (1986) B&hem. J. 236, 311-312. 7. Clausen,J. (1961) Proc. Sot. Exp. Biol. (N.Y.) 107, 170-172. 1087
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