Inhibitory effect of interleukin-1 on angiotensin II-induced connective tissue growth factor and...

doi: 10.1152/ajprenal.00129.2007294:F149-F160, 2008. First published 7 November 2007;Am J Physiol Renal Physiol

Marta Ruiz-OrtegaEsteban, Gisselle Carvajal, Raquel Rodrígues-Díez, Juan José Plaza, Jesús Egido and Elsa Sánchez-López, Juan Rodriguez-Vita, Cecile Cartier, Monica Rupérez, Vanesacollagen production in cultured mesangial cellsII-induced connective tissue growth factor and type IV

on angiotensinβInhibitory effect of interleukin-1

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Inhibitory effect of interleukin-1� on angiotensin II-induced connective tissuegrowth factor and type IV collagen production in cultured mesangial cells

Elsa Sanchez-Lopez,1 Juan Rodriguez-Vita,1 Cecile Cartier,1 Monica Ruperez,1 Vanesa Esteban,1

Gisselle Carvajal,1 Raquel Rodrıgues-Dıez,1 Juan Jose Plaza,2 Jesus Egido,2 and Marta Ruiz-Ortega1

1Cellular Biology in Renal Diseases Laboratory and 2Renal Unit, Fundacion Jimenez Dıaz, Universidad Autonoma Madrid,Madrid, Spain

Submitted 19 March 2007; accepted in final form 26 October 2007

Sanchez-Lopez E, Rodriguez-Vita J, Cartier C, Ruperez M,Esteban V, Carvajal G, Rodrıgues-Dıez R, Plaza JJ, Egido J,Ruiz-Ortega M. Inhibitory effect of interleukin-1� on angiotensinII-induced connective tissue growth factor and type IV collagenproduction in cultured mesangial cells. Am J Physiol Renal Phys-iol 294: F149–F160, 2008. First published November 7, 2007;doi:10.1152/ajprenal.00129.2007.—Connective tissue growth factor(CTGF) is overexpressed in kidney diseases associated with extracel-lular matrix accumulation. Angiotensin II (ANG II) participates inrenal fibrosis by the upregulation of growth factors, including CTGF,and extracellular matrix proteins, such as type IV collagen. Duringrenal injury, ANG II and the macrophage-produced cytokine interleu-kin-1� (IL-1�) may be present simultaneously in the glomerularenvironment. However, there are no studies about the interactionbetween ANG II and IL-1� in renal fibrosis. For this reason, incultured mesangial cells (MC), we investigated whether IL-1� couldregulate ANG II-mediated collagen accumulation and the mechanismsunderlying this process. In MC, CTGF is a downstream mediator oftype IV collagen production induced by ANG II. IL-1� did notincrease the production of CTGF and type IV collagen but signifi-cantly inhibited ANG II-induced CTGF and type IV collagen over-expression. Moreover, IL-1� also inhibited type IV collagen upregu-lation caused by exogenous recombinant CTGF. Matrix metallopro-teinase-9 (MMP-9) is the main enzyme involved in type IV collagendegradation. In MC, coincubation of IL-1� and ANG II caused asynergistic increase in MMP-9 gene expression and activity, associ-ated with type IV collagen inhibition. The described IL-1� effectswere dependent on activation of ERK/MAPK but independent p38-MAPK, JNK, phosphatidylinositol 3-kinase/Akt, and Rho-associatedkinase pathways. In summary, these data indicate that IL-1� inhibitedANG II-mediated type IV collagen production, via CTGF downregu-lation, and increased type IV collagen degradation, through MMP-9upregulation. Our in vitro data show that the proinflammatory cyto-kine IL-1� abrogates ANG II-induced CTGF production, describingantagonistic activities of proinflammatory cytokines on ANG II ac-tions.

transforming growth factor-�; extracellular-regulated kinase; metal-loproteinases

MANY STUDIES HAVE DEMONSTRATED activation of the renal reninangiotensin system and increased local angiotensin II (ANG II)production in human and experimental kidney diseases. Block-ade of ANG II, by angiotensin-converting enzyme inhibitors orAT1 antagonists, is one of the best options to treat renaldiseases (35, 43). ANG II plays an important role in theregulation of kidney functions and participates in the progres-sion of renal damage through the modulation of cell growth,

fibrosis, and inflammation (23, 35, 37). These responses aremediated by the endogenous production of growth factors andcytokines (23, 33–35, 37). ANG II contributes to fibrosis bypromoting extracellular matrix (ECM) protein production,through endogenous synthesis of transforming growth factor-�(TGF-�) and connective tissue growth factor (CTGF) (23,33–35, 37).

The mechanisms of progressive glomerulonephritis remainmostly unknown. During renal injury, ANG II and proinflamma-tory cytokines, such as interleukin-1� (IL-1�), released by infil-trating inflammatory cells and intrinsic mesangial cells (MC), maybe simultaneously present in the glomerular environment. Thesefactors can activate MC to increase ECM production through therelease of growth factors and therefore contribute to renal damageprogression. However, data about the interrelation between theproinflammatory cytokine IL-1� and ANG II on MC behavior arescarce. IL-1� is an important cytokine with a broad range ofbiological activities (7) involved in kidney injury and repair (39,42). In MC, IL-1� regulates cell growth, inflammation, and ECMproteins (6, 17, 28, 30), although there are no data about CTGFregulation.

Our aim was to investigate the effect of IL-1� in ANG IIresponses, evaluating in MC the effects on ECM regulation, aswell as some potential molecular mechanisms involved. ANG IIbinds to specific receptors (AT1 and AT2) to activate cellularresponses. AT1 receptor mediates upregulation of growth factorsand fibrosis (23, 37). The AT1 signaling mechanisms are similarto those activated by cytokines, such as IL-1�, and includeactivation of protein kinases, for example, MAPK cascade, Rho-associated kinase (ROCK), and phosphatidylinositol 3-kinase(PI3-kinase)/Akt pathway (7, 8, 13, 16, 35). These in vitro exper-iments may help to elucidate the mechanisms of renal damageperpetuation and could suggest novel therapeutic strategies formodulating the biology of renal injury.

MATERIALS AND METHODS

Cell culture studies. Rat MC were cultured from isolated glomeruliby several sieving techniques and differential centrifugations (32).Glomerular MC were characterized by phase-contrast microscopy,positive staining for desmin and vimentin, and negative staining forkeratin and factor VIII antigen, excluding epithelial and endothelialcontamination, respectively. Cells were trypsinized, counted, grownin RPMI 1640 medium (GIBCO, Grand Island, NY), supplementedwith 100 U/ml penicillin, 100 �g/ml streptomycin, and 2 mM glu-tamine in the presence of 20% heat-inactivated FBS, and cultured at

Address for reprint requests and other correspondence: M. Ruiz-Ortega,Renal Unit, Fundacion Jimenez Dıaz, Avda Reyes Catolicos 2, 28040 Madrid,Spain (e-mail: mruizo@fjd.es).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Renal Physiol 294: F149–F160, 2008.First published November 7, 2007; doi:10.1152/ajprenal.00129.2007.

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Fig. 1. Effect of IL-1� on connective tissue growth factor (CTGF) regulation in mesangial cells (MC). MC were treated with 1 ng/ml IL-1� and/or 10�7 mol/lANG II for different times. A: CTGF gene expression was evaluated by Northern blot. Data were normalized by GAPDH expression. Similar results were foundby real-time PCR. Top: representative Northern blot experiment. Bottom: results (means � SE) of 4 experiments. *P � 0.05 vs. control. #P � 0.05 vs. ANGII. B: cells were treated with 1 ng/ml IL-1� and/or 10�7 mol/l ANG II for 24 h. Total CTGF protein levels were measured by Western blot. A representativeexperiment of 6 is shown.

F150 INTERLEUKIN-1� INHIBITS ANG II-INDUCED FIBROSIS

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37°C in 5% CO2 atmosphere. At confluence, cells were made quies-cent for 48 h in RPMI 1640 with 0.5% FBS medium, and thendifferent studies were performed. The experiments were done withcultured MC at passage 0 or 1.

Reagents. All culture reagents were purchased from GIBCO BRL(Paisley, Scotland, UK). ANG II was obtained from Bachem. Recom-binant IL-1� was from Peprotech (London, UK). Human recombinantCTGF was from MBL International (Woburn, MA).

The inhibitors used were PD-98059 (ERK1/2 inhibitor), SB-203580(p38-MAPK inhibitor), and SP-600125 (JNK1, JNK2, and JNK3inhibitor), all from Stressgen Bioreagents (Victoria, BC, Canada);wortmannin (PI3-kinase/Akt inhibitor) and matrix metalloprotein-ase-9 (MMP-9) inhibitor I, both from Calbiochem; and fasudil andY-27632 (ROCK inhibitors) from Tocris Cookson (Bristol, UK).Cells were preincubated with all inhibitors 1 h before the stimulus wasadded. None of the inhibitors was toxic at the doses used, and nonehad an effect on basal CTGF levels.

The oligodeoxynucleotides (ODNs) were synthesized by Metabion(Martinsried, Germany). Cells were preincubated for 30 min withODN antisense CTGF (5�-TACTGGCCGGCGGTCAT-3�), whichblocks endogenous CTGF production, and ODN sense CTGF (5�-ATGACCGCCGCCAGTA-3�) as control.

Gene and protein studies. RNA was isolated by Trizol (Invitrogen).CTGF gene expression was evaluated by Northern blot (36). Real-timePCR reactions were performed on ABI Prism 7500 sequence-detectionPCR system (Applied Biosystems, Foster City, CA), and cDNA wassynthesized with 2 �g of total RNA, according to manufacturer’s protocol.Assay identifications are as follows: Rn00573960_g1 for CTGF,Rn01401018_m1 for type IV procollagen, Rn00579162_m1 for MMP-9,Rn00587558_m1 for tissue inhibitor of metalloproteinase-1 (TIMP-1),Rn01513693_m1 for thromsbospondin-1, and Rn00572010_m1 for TGF-�.Data were normalized with GAPDH and 18S ribosomal RNA expression(assay identifications: Rn99999916_s1 and Hs99999901_m1, respectively).

Cells were homogenized in lysis buffer (170 mmol/l Tris �HCl,22% glycerol, 2.2% SDS, 0.1 mmol/l PMSF, orthovanadate, andprotease inhibitor cocktail) and then separated by SDS-PAGE. CTGFprotein and the phosphorylation levels of ERK1/2, p38, JNK, and Aktwere determined in total protein extracts by Western blot (37). TypeIV collagen and MMP-2 were analyzed in cell-conditioned medium,and 50 �g of proteins (quantified by bicinchoninic acid method) wereloaded in each lane. The loading controls used were tubulin (for totalproteins), total protein levels (in phosphorylation studies), or redPonceau staining (for soluble proteins). Antibodies employed wereCTGF from Torrey Pines Biolabs (Houston, TX); phospho-JNK1/2from Stressgen Bioreagents; MMP-2, phospho-ERK1/2, ERK1/2,phospho-p38, p38, phospho-Akt, Akt, JNK1/2, and type IV collagenfrom Santa Cruz Biotechnology (Santa Cruz, CA); and peroxidase-conjugated secondary antibodies from Amersham. Type IV collagenproduction was also determined by ELISA (Exocell).

MMPs activity was measured by zymography (44). Supernatantswere loaded in 7.5% SDS-PAGE containing 1 mg/ml gelatin undernonreducing conditions. Next, gels were first incubated in developingbuffer, then with Coomassie blue, and finally with 20% methanol-10% acetic acid. Total protein synthesis was evaluated by [3H]leucineincorporation (31).

Immunofluorescence. To determine type IV collagen production,cells were trypsinized and seeded in 24-well plates over crystalcoverslips. After a 24-h serum-starvation step, cells were pretreatedwith several inhibitors for 1 h and then cells were stimulated forincreasing times (24–72 h) with ANG II and/or IL-1�. Cells were thenfixed in Merckofix (Merck), treated with 0.1% Triton X-100, andincubated with primary antibody against type IV collagen. After cellswere washed, they were incubated with FITC-conjugated secondaryantibody, and nuclei were stained with 1 �g/ml propidium iodide. Theabsence of primary antibody was the negative control. Samples weremounted in Mowiol 40-88 (Sigma) and examined by a laser scanningconfocal microscope (Leika).

Statistical analysis. The autoradiographs were scanned using theGS-800 calibrated densitometer (Quantity One, Bio-Rad). Results, ex-pressed as n-fold over control, are means � SE of experiments made.One-way ANOVA was used to compare gene and/or protein expressionlevels between groups. When statistical significance was found, Bonfer-roni post hoc comparison test was used to identify group differences.Differences were considered significant at P � 0.05. Statistical analyseswere conducted using SPSS statistical software version 11.0.

RESULTS

IL-1� inhibits ANG II-induced CTGF gene expression andprotein production in MC. We have previously demonstratedthat in MC ANG II elicited a rapid and sustained CTGF mRNAupregulation and protein production (37); however, there are nostudies about CTGF regulation by IL-1�. In MC, IL-1� caused aslight but not significant increase in CTGF mRNA expressionafter 3 h, which diminished to control levels at 18 h but did notincrease CTGF protein synthesis (Fig. 1). Surprisingly, IL-1� wasfound to suppress ANG II-induced CTGF mRNA expression atall times studied, between 3 and 24 h. This effect was dosedependent, with a maximal response at 10 ng/ml IL-1�. CTGFproduction caused by ANG II was also significantly decreased byIL-1� (Fig. 1B). This effect was not due to a general inhibition ofprotein expression because tubulin or total protein levels were notmodified (determined by red Ponceau staining and [3H]leucineincorporation, respectively, not shown).

IL-1� does not modify TGF-� expression caused by ANG IIin MC. ANG II and TGF-� share some responses involved inmatrix regulation (23). ANG II increases TGF-� mRNA ex-pression, protein production, and activation of latent TGF-�,through a thrombospondin-1-mediated process (25). In MC, wehave observed that IL-1� and ANG II induced TGF-� geneexpression that was not modified when both stimuli were addedtogether, at the times studied until 24 h (Fig. 2). We have foundthat coincubation of IL-1� and ANG II also upregulatedthrombospondin-1 expression, showing no differences to eachstimuli alone (Fig. 2). These data suggest a different regulationof CTGF and TGF-� by IL-1�.

IL-1� attenuates type IV collagen gene expression andprotein release induced by ANG II in MC. ANG II contributesto ECM accumulation (23, 35, 37). Stimulation of resting MC

Fig. 2. Coincubation of IL-1� and ANG II does not modify transforminggrowth factor-� (TGF-�) and thrombospondin-1 gene expression. Cells werecostimulated with 1 ng/ml IL-1� and 10�7 mol/l ANG II for 24 h. Values aremeans � SE of 4 experiments of real-time PCR. *P � 0.05 vs. control.

F151INTERLEUKIN-1� INHIBITS ANG II-INDUCED FIBROSIS

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with ANG II increased type IV procollagen gene and proteinlevels, as expected (Fig. 3). IL-1� has also upregulated severalECM proteins (28, 39), but we found that it had no effect ontype IV collagen gene and protein production. Moreover,IL-1� markedly inhibited ANG II-induced type IV collagengene expression, as well as cell-associated and soluble protein

levels (at all times studied) (Fig. 3), showing a regulationsimilar to that observed with CTGF.

The next set of experiments was designed to demonstrate thelink between CTGF and type IV collagen synthesis. CTGFincreases ECM proteins, including type IV collagen (14). InMC, treatment with human recombinant CTGF (10 ng/ml)

Fig. 3. A: effect of simultaneous presence of IL-1� andANG II on type IV collagen gene expression. Cells werecostimulated with 1 ng/ml IL-1� and 10�7 mol/l ANG IIfor 18 and 24 h. Values are means � SE of 6 experimentsof real-time PCR. IL-1� diminishes ANG II-inducedcell-associated and soluble type IV collagen. B: cellswere costimulated with 1 ng/ml IL-1� and 10�7 mol/lANG II for 72 h. Values are means � SE of 3 experi-ments of type IV collagen production. *P � 0.05 vs.control. #P � 0.05 vs. ANG II. C: representative West-ern blot of soluble and cell-associated type IV collagenof 3 Western blot experiments. Tubulin was used asloading control.

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increased type IV collagen deposition (Fig. 4A). The blockadeof endogenous CTGF by ODN antisense CTGF, but not withcontrol sense ODN, markedly diminished ANG II-inducedtype IV collagen mRNA overexpression (44% inhibition, P �

0.05, n � 3, real time PCR, not shown) and protein production(Fig. 4A). These data show that inhibition of CTGF has a keyrole in the regulation of type IV collagen by ANG II in MC. Inthese cells, IL-1� also inhibited type IV collagen accumulation

Fig. 4. A: role of CTGF on inhibitory effect of IL-1� on ANG II-induced type IV collagen regulation. Cells were stimulated with 10�7 mol/l ANG II, 10 ng/mlrecombinant CTGF, and/or 1 ng/ml IL-1� for 72 h. In some points, cells were pretreated with 20 �g/ml oligodeoxynucleotide (ODN) antisense CTGF and ODNsense CTGF or matrix metalloproteinase-9 (MMP-9) inhibitor I for 30 min and 1 h before stimulation, respectively. Confocal microcopy images are arepresentative experiment of 4 inmunofluorescence stainings of type IV collagen. B: ANG II upregulation of CTGF and type IV collagen is mediated byp38-MAPK activation. MC were pretreated with 10�6 mol/l SB-203580 (p38 inhibitor), 10�5 mol/l PD-98059 (ERK p42/44 inhibitor), or 10�5 mol/l SP-600125(JNK inhibitor) 1 h before stimulation with 10�7 mol/l ANG II for 24 h. Left: values are means � SE of 4 experiments of Western blot analysis of CTGF.Right: values are means � SE of 4 experiments of real-time PCR analysis of type IV procollagen gene expression. *P � 0.05 vs. control. #P � 0.05 vs. ANG II.

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caused by recombinant CTGF, indicating that IL-1� regulatesCTGF at two levels, by inhibiting its synthesis and its cellularresponses.

Previous studies have demonstrated that ANG II upregulatesCTGF via AT1 receptors and activation of MAPK, PKC, andROCK pathways (15, 18, 38), but there are no studies in MC.The involvement of MAPK cascade was evaluated with the useof specific inhibitors of the p38-MAPK (SB-203580), ERK1/2(PD-98059), and JNK (SP-600125) pathways (9, 19). Wefound that only the p38-MAPK inhibition, but not ERK andJNK inhibition, diminished ANG II-induced CTGF productionin MC (Fig. 4B). In fibroblasts, the inhibitors of ERK1/2-MAPK and JNK-MAPK, but not p38-MAPK, decreased ANGII-stimulated CTGF expression (22), showing a different reg-ulation depending on the cell type. In addition, only p38inhibition diminished type IV collagen gene expression in-duced by ANG II (Fig. 4B). All of these results suggest that

CTGF is a downstream mediator of ANG II-induced type IVcollagen production and that this regulation requires p38-MAPK activation in MC.

IL-1� inhibits ANG II-induced CTGF and type IV collagenthrough ERK-dependent pathway. Next, we investigated themolecular mechanisms involved in CTGF and type IV collageninhibition caused by IL-1�. IL-1� activates several intracellu-lar pathways shared with ANG II, including MAPK, ROCK,and PI3-kinase/Akt pathways (7, 8, 13, 16, 35). First, weevaluated the potential role of several intracellular signals inthe inhibitory effect of IL-1� on CTGF regulation, using apharmacological approach. Only the ERK1/2 inhibitor PD-98059, but not the p38 or JNK inhibitors, significantlyblocked IL-1� effect, at both the gene (Fig. 5A) and proteinlevels (Fig. 5, B and C). The two ROCK inhibitors (fasudil andY-27632) did not attenuate the IL-1� effect. PI3-kinase inhi-bition by wortmannin and LY-294002 blocks ANG II-stimu-

Fig. 5. Inhibitory effect of IL-1� on ANG II-inducedCTGF regulation is mediated by ERK/MAPK pathway.MC were pretreated with 10�6 mol/l SB-203580 (p38inhibitor), 10�5 mol/l PD-98059 (ERK p42/44 inhibitor),10�5 mol/l SP-600125 (JNK inhibitor), 10�6 mol/l fa-sudil or Y-27632 (Rho-associated kinase inhibitors), or10�9 mol/l wortmannin [phosphatidylinositol 3-kinase(PI3-kinase) inhibitor] for 1 h before treatment with 1ng/ml IL-1� and/or 10�7 mol/l ANG II for 24 h.A: values are means � SE of 7 experiments of real-timePCR. B: values are means � SE of 5 experiments ofWestern blot. C: representative gel. *P � 0.05 vs. control.#P � 0.05 vs. ANG II. ¶P � 0.05 vs. IL-1� � ANG II.

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lated hyperplasia in cultured rat cells (40). Wortmannin did notaffect CTGF inhibition caused by IL-1�. These data suggestthat the IL-1� inhibitory effect seems to be mediated byactivation of ERK cascade.

We also evaluated whether the same intracellular signalswere involved in type IV collagen downregulation by IL-1�.As shown in Fig. 6, only pretreatment with the ERK1/2inhibitor PD-98059, but not with the other MAPK inhibitors orwith the ROCK and PI3-kinase inhibitors, significantly re-stored type IV procollagen gene levels, showing a similarresponse to CTGF regulation.

These results suggest that in MC ANG II regulates CTGFand type IV collagen via activation of p38 MAPK, whereasERK1/2 participates in the IL-1� inhibitory effect.

IL-1� and ANG II coincubation synergistically increasesERK1/2, but not p38-MAPK, JNK, ROCK, or Akt activation. InMC, IL-1� and ANG II triggered phosphorylation of all threeMAPKs, including ERK, p38, and c-JNK (10, 13, 16). Wecompared the effect of IL-1� and ANG II on ERK activation (Fig.7, A and B). Significant activation of ERK was detected within 2min of ANG II stimulation, showed by enhanced phosphorylationof ERK1/2, remaining elevated at 30 min, but diminished tocontrol levels after 50 min. IL-1� presented a delayed response,increasing ERK phosphorylation after 30 min and remainingelevated after 50 min. Coincubation of IL-1� and ANG II mark-edly enhanced ERK activation after 30 min. We also evaluated theeffect of long-term incubation on ERK phosphorylation, showingno activation after 24 h with any stimulus.

Furthermore, we studied the activation of p38, JNK, andPI3-kinase/Akt pathways caused by ANG II and IL-1� alone orin combination. In MC, both ANG II and IL-1� activate p38,JNK, and PI3-kinase/Akt pathways (Fig. 7C). However, onlythe ERK pathway shows a synergistic activation when bothstimuli are added together. Moreover, p38, JNK, and PI3-kinase/Akt pathways do not participate in the IL-1� inhibitoryeffect on ANG II-induced CTGF and type IV collagen expres-sion, as shown in Figs. 5 and 6. These data suggest that ANGII and IL-1� caused a synergistic activation of ERK1/2 path-way that turns into blockade of CTGF and type IV collagenexpression and protein production.

Synergy between IL-1� and ANG II on MMP-9 activity.Deposition of mesangial ECM is regulated by the balance be-tween synthesis and degradation, the latter being regulated byMMPs. By zymography, we compared the effect of ANG II andIL-1� in two different MMPs: MMP-9 (with specificity toward

type IV and V collagen) and MMP-2 (involved in fibronectin andlaminin degradation) (20). IL-1�, but not ANG II, markedlyincreased MMP-9 gene expression. Coincubation of ANG II andIL-1� caused a synergistic upregulation on MMP-9 mRNA levels(Fig. 8A). Only IL-1� treatment significantly increased MMP-9activity, shown by one lytic band corresponding to the inactiveproform, which was not detectable under control conditions or inANG II-treated cells. Coincubation of ANG II and IL-1� actedsynergistically to increase MMP-9 activity (Fig. 8B). In contrast,MMP-2 activity and protein levels were not modified by bothstimuli alone or in combination (Fig. 8C). Finally, we determinedTIMP-1 expression levels, the main inhibitor of MMP-9 (2). Asfound with MMP-9, only IL-1� upregulated TIMP-1 gene butwas not significantly modified by the coincubation with ANG II(Fig. 8D). The ratio of MMP-9 to TIMP-1 indirectly definesMMP-9 activity (3). When ANG II and IL-1� are coincubated,the MMP-9-to-TIMP-1 ratio is more than 30-fold (not shown),showing a clear matrix-degrading environment. Moreover, ourdata showed a time correlation between MMP-9 upregulation andcollagen type IV inhibition, suggesting that this protein isbeing degraded. To further determine the direct contributionof MMP-9 on type IV collagen inhibition caused by IL-1�,we used MMP-9 inhibitor I (21). MC were pretreated withMMP-9 inhibitor I before stimulation with ANG II andIL-1� for 72 h. Inhibition of MMP-9 restored type IVcollagen levels, abrogating the IL-1� inhibitory effect onANG II response (Fig. 8E and 4A), suggesting that upregu-lation of MMP-9 by IL-1� plays a key role in type IVcollagen degradation.

The synergistic effect on MMP-9 is mediated by ERK-dependent pathway. In MC, IL-1� elicited high levels ofMMP-9 regulated by MAPK (10). We further evaluated the roleof these intracellular signaling systems in the synergistic activa-tion of MMPs caused by coincubation of IL-1� and ANG II.Pretreatment with the ERK inhibitor PD-98059 diminished thesynergistic gene upregulation and activation of MMP-9 (Fig. 8, Aand B). In contrast, the inhibitors of p38-MAPK, ROCK, andPI3-kinase/Akt had no effect on this process.

DISCUSSION

Renal damage is associated with overexpression of a largevariety of growth factors and cytokines, which influence theprogression of kidney diseases. ANG II actively participates inrenal fibrosis through the overexpression of growth factors,

Fig. 6. Inhibitory effect of IL-1� on ANG II-inducedtype IV collagen regulation is mediated by ERK/MAPK pathway. MC were pretreated with 10�6 mol/lSB-203580 (p38 inhibitor), 10�5 mol/l PD-98059(ERK p42/44 inhibitor), 10�5 mol/l SP-600125 (JNKinhibitor), 10�6 mol/l fasudil or Y-27632 (Rho-asso-ciated kinase inhibitors), or 10�9 mol/l wortmannin(PI3-kinase inhibitor) for 1 h before treatment with 1ng/ml IL-1� and/or 10�7 mol/l ANG II for 24 h.Values are means � SE of 5 experiments of real-timePCR analysis of type IV procollagen gene expression.*P � 0.05 vs. control. #P � 0.05 vs. ANG II. ¶P �0.05 vs. IL-1�.

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Fig. 7. Coincubation of IL-1� and ANG II markedly increases ERK activation in MC. Cells were coincubated with 1 ng/ml IL-1� and 10�7 mol/l ANG II fordifferent times. A: representative Western blot of phospho-ERK (p-ERK1/2) and ERK1/2 (used as control). B: values are means � SE of 4 experiments. C: effectof coincubation of IL-1� and ANG II in activation of several kinases (p38-MAPK, JNK, PI3-kinase/Akt). Left: values are means � SE of 3 experiments.Right: representative Western blot. *P � 0.05 vs. control. #P � 0.05 vs. ANG II. †P � 0.05 vs. IL-1�.

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such as CTGF and TGF-�, and ECM proteins (23). Upregula-tion of IL-1� has been described in human and experimentalrenal injury (39, 42). These data suggest that, during renalinjury, ANG II and IL-1� may be present simultaneously in theglomerular environment. In this paper, we investigated the

interaction between ANG II and IL-1� in renal fibrosis. Al-though the direct in vivo effect of IL-1� in the kidney has notbeen evaluated, several studies have shown that IL-1� activelyparticipates in the early inflammatory response and that theblockade of IL-1� receptor exerts renal protective effects (39,

Fig. 8. A: coincubation of IL-1� and ANG II increases MMP-9 gene expression in MC. MC were treated with 1 ng/ml IL-1� and/or 10�7 mol/l ANG II for 18 h. Somecells were pretreated with 10�5 mol/l PD-98059, 10�5 mol/l SP-600125, 10�6 mol/l SB-203580, 10�6 mol/l fasudil, or 10�9 mol/l wortmannin for 1 h. Values aremeans � SE of 6 real-time PCR experiments. B: modulation of gelatinolytic activities of MMP-9 and MMP-2 after stimulation with ANG II and IL-1� for 72 h.Migration properties were determined with the use of standard molecular weight markers. Top band corresponds to MMP-9 (90 kDa), and bottom band correspondsto MMP-2 (70 kDa). Top: representative SDS-PAGE zymography. Bottom: means � SE of 3 experiments. C: IL-1� and ANG II, alone and in combination, do notincrease MMP-2 production. Cells were treated for 72 h, and conditioned media were tested for MMP-2 production. Top: protein levels are shown as means � SE of3 independent experiments. Bottom: representative Western blot. D: tissue inhibitor of metalloproteinase-1 (TIMP-1) gene levels. MC were treated with 1 ng/ml IL-1�and 10�7 mol/l ANG II for 18 h. Values are means � SE of 4 real-time PCR experiments. E: MMP-9 inhibition recovers type IV collagen released levels caused byIL-1�. Cells were pretreated with 10�6 mol/l MMP-9 inhibitor I 1 h before stimulation with 10�7 mol/l ANG II and 1 ng/ml IL-1� for 72 h. Values are means � SDof 2 experiments of ELISA analysis. *P � 0.05 vs. control. #P � 0.05 vs. ANG II. †P � 0.05 vs. IL-1�. ¶P � 0.05 vs. IL-1� � ANG II.

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42). However, its role in chronic renal injury and its contribu-tion to fibrosis is unclear. In anti-Thy-1 nephritis, IL-1� recep-tor antagonism reduced MC proliferation and glomerular mac-rophage accumulation, but it had relatively little effect onglomerular ECM deposition (39). In rats with 5/6 nephrectomy,only overexpression of CTGF and TGF-�, but not IL-1� orTNF-, was associated with interstitial fibrosis (12). In vitrostudies in renal cells have shown different responses dependingon the cell type. In tubuloepithelial cells, IL-1� participates inmesenchymal transition, through TGF-� and ECM regulation(11, 41). We found that, in cultured MC, IL-1� increasedTGF-� but did not induce CTGF or type IV collagen produc-tion, a major component of expanded ECM in glomerulardiseases. Moreover, we confirmed that IL-1� markedly up-regulates MMP-9 activity (24). These in vitro observationssuggest that IL-1� actively participates in ECM degradation,and it is reasonable to speculate that IL-1� contributes to theremodeling processes associated with the glomerular inflam-matory process.

CTGF is upregulated in many fibrotic disorders, includinghuman kidney diseases, associated with ECM accumulation(29). In cultured renal cells, the blockade of CTGF diminishedECM accumulation caused by factors involved in renal damageprogression, such as ANG II, TGF-�, and high glucose, sug-gesting that CTGF inhibition could represent a potential ther-apeutic approach to renal fibrosis (37). We observed that incultured MC an ODN antisense CTGF, which blocks endoge-nous CTGF synthesis, diminished ANG II-induced type IVcollagen accumulation, indicating that CTGF is a key factorinvolved in its regulation. We demonstrated that in MC IL-1�can suppress ANG II-induced CTGF gene expression andproduction. In fibroblasts, TNF- also suppresses TGF-�- anddexamethasone-induced CTGF mRNA (1, 5), showing thatproinflammatory cytokines abrogate CTGF overexpression indifferent cell types. In MC, we also found that IL-1� dimin-ished type IV collagen upregulation caused by ANG II andrecombinant CTGF, indicating that IL-1� regulates CTGF attwo levels: by inhibiting its synthesis and its cellular responses.

The accumulation of mesangial ECM is the result of anexcessive ECM production and an inhibition of its degradation.In MC, we observed that the simultaneous presence of IL-1�and ANG II caused a synergistic effect on MMP-9 geneexpression and activity, as well as an increase in the ratio ofMMP-9 to TIMP-1, indicating a matrix-degrading environ-ment. MMP-9 upregulation by coincubation with IL-1� andANG II is responsible for type IV collagen degradation, asshown by the time-course correlation of regulation of bothproteins and by the effect of MMP-9 inhibitor on type IVcollagen levels.

In normal wound healing, there is an elevated ECM synthe-sis attributable to activation of resident renal cells by cytokinesand growth factors until healing is completed. When persistentactivation remains uncontrolled, ECM deposition occurs, lead-ing to pathological fibrosis. However, how the wound healingprocess is normally regulated or leads to fibrosis has not beennot completely elucidated. Our in vitro findings in MC showthat IL-1� is a negative regulator of ANG II-mediated type IVcollagen accumulation through CTGF suppression and byincreasing collagen degradation via MMP-9 upregulation. Al-though the biological meaning of this in vitro finding in renalpathology is not resolved in the present paper, our data show a

novel mechanism involved in mesangial ECM regulation thatcould be involved in maintenance of tissue homeostasis or inthe regulation of ECM deposition. In this sense, IL-1� has beenshown to participate in the end of the wound healing process bykeratinocytes through the inhibition of CTGF (26).

ANG II and TGF-� share some responses involved in matrixregulation (23). Many experimental evidences have shown thatTGF-� is a mediator of ANG II actions. However, the datapresented here show a different regulatory effect by IL-1�. InMC, IL-1� did not modify ANG II-induced gene overexpres-sion of TGF-� or thrombospondin-1, the main activator oflatent TGF-�, although it inhibited CTGF and type IV collagenproduction. In contrast, IL-1� markedly increased fibronectin,TIMP-1, and TGF-�1 production caused by TGF-�1 (28).These data show a clearly different response between ANG IIand TGF-�.

Among the multiple signaling pathways involved in renaldamage, we found that ERK-MAPK seems to be a majorcomponent mediating the inhibitory effect of IL-1� on ANGII-described responses. We demonstrated that specific inhibi-tors of the classical ERK cascade, but not p38-MAPK, JNK,PI3-kinase/Akt, or ROCK pathways, reverse the IL-1�-medi-ated effects on CTGF, type IV collagen, and MMP-9 regula-tion. In contrast, p38 activation by ANG II is required for theproduction of CTGF and type IV collagen, as previouslydescribed for TGF-� (25). These data show the complexity ofthe intracellular pathways involved in the regulation of profi-brotic mediators and ECM components. Several experimentalmodels have shown that the pharmacological blockade of ERKor p38 prevents ANG II-induced renal damage (4). Futurein vivo studies are needed to know whether the modulation ofERK or p38-MAPK pathways could be important targets forthe treatment of renal fibrosis.

During renal damage, there is an upregulation of cytokinesand growth factors, with elevated local concentrations depend-ing on the renal structures and progression of the disease (7, 23,39, 42). Our in vitro data show that, if in the glomerularmesangial milieu there is a simultaneous presence of IL-1� andANG II, IL-1� antagonizes ANG II-induced profibrotic re-sponse. Our findings are based on cell culture results and donot necessarily reflect the conditions in the fibrotic tissuein vivo, where other factors could also participate in the

Fig. 9. Scheme of the proposed mechanisms of IL-1� action on ANG II-induced CTGF and type IV collagen overexpression in cultured MCs.

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regulation of ECM. In pathological conditions, the balancebetween profibrotic and antifibrotic mediators would finallydecide whether these cells take part in excessive deposition ofECM or in an active remodeling process. Only future studies inhuman renal patients that would evaluate renal levels of a largearray of cytokines in correlation with disease progression willhelp us to explain why some glomerulonephritis cases progressto end-stage kidney disease.

In summary, in cultured MC, we observed that the proin-flammatory cytokine IL-1� abrogates ANG II-induced CTGFproduction and exerts an inhibitory effect on ANG II-mediatedECM accumulation. This IL-1� inhibitory effect was mainlymediated by ERK/MAPK pathway (Fig. 9).

These observations could help us to gain knowledge regard-ing the mechanisms involved in the regulation of tissue ho-meostasis and ECM accumulation.

ACKNOWLEDGMENTS

We thank Dr. Juan Antonio Moreno-Gutierrez for help with statisticalanalysis and Alberto Puime, Mar Gonzalez Garcia-Parreno, and Sandra LopezLeon for technical help.

GRANTS

This work has been supported by grants from Fondo de InvestigacionSanitaria (PI0205513, PI020822, ISCIII-RETIC-RD06/0004), Ministerio deEducacion y Ciencia (SAF 2005-03378), and Sociedad Espanola de Nefrolo-gia. E. Sanchez-Lopez and J. Rodriguez-Vita are fellows of Fondo de Inves-tigacion Sanitaria. G. Carvajal is a fellow of Fundacion Carolina and Funda-cion Inigo Alvarez de Toledo.

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Inhibitory effect of interleukin-1 on angiotensin II-induced connective tissue growth factor and...

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