CD40L Proinflammatory and Profibrotic Effects on Proximal Tubular Epithelial Cells: Role of NF-B and Lyn
Paola Pontrelli*,
Michele Ursi,
Elena Ranieri*,
Carmen Capobianco,
Francesco P. Schena,
Loreto Gesualdo and
Giuseppe Grandaliano
* Clinical Pathology, Department of Biomedical Sciences, and Division of Nephrology, Department of Biomedicine University of Foggia, Foggia, and Division of Nephrology, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
Address correspondence to: Dr. Giuseppe Grandaliano, Division of Nephrology, Department of Emergency and Transplantation, University of Bari, Piazza Giulio Cesare 11, 70124 Bari, Italy. Phone: +39-080-5593234; Fax: +39-080-5575710; E-mail: g.grandaliano{at}nephro.uniba.it
Received for publication February 22, 2005.
Accepted for publication December 16, 2005.
Chronic allograft nephropathy (CAN) is the main cause of renalgraft loss, but its pathogenic mechanisms are still unclear.Immune system activation has been suggested as a key event inthe development of CAN. CD40 is a co-stimulatory protein whoseexpression is upregulated in proximal tubular epithelial cells(PTEC) in acute rejection. This receptor interacts with CD40L,expressed by activated T cells. CD40L induces the productionby PTEC of different proinflammatory cytokines, but very littleis known of its profibrotic effects. The aim of this study wasto investigate the effect of CD40/CD40L interaction on PTECexpression of plasminogen activator inhibitor-1 (PAI-1), a powerfulprofibrotic mediator, and monocyte chemoattractant protein-1(MCP-1), a proinflammatory cytokine, and to investigate thesignaling pathways that lead to these effects. Soluble CD40Linduced a time-dependent increase in both PAI-1 and MCP-1 geneexpression and protein production in PTEC. CD40 cross-linkingon PTEC caused TNF-R–associated factors 2 and 6 membranetranslocation. This event led to NF-B activation, through theNF-B–inducing kinase, and to a significant increase inthe phosphorylation of lyn, a src-related tyrosine kinase. Lyn,upon phosphorylation, became strictly associated with caveolin-1,a scaffolding protein enriched in caveolae. Lyn inhibition didnot have any effect on CD40L-induced NF-B activation and MCP-1expression but abolished PAI-1 induction. On the contrary, NF-Binhibition significantly reduced only MCP-1 expression. In conclusion,CD40L could play a key role in the pathogenesis of CAN throughPAI-1 induction. CD40L profibrotic and proinflammatory effectsare mediated by different signaling pathways, suggesting thatdrugs that inhibit inflammation may not be equally effectivein reducing fibrosis.
CD40 is a cell surface glycoprotein that belongs to the TNF-receptor(TNF-R) superfamily, largely expressed on the cellular surfaceof antigen-presenting cells, including B lymphocytes, macrophages,and dendritic cells. It is also present in some nonlymphoidcells, such as tubular epithelial cells, where it has been suggestedto play a role in the pathogenesis of renal inflammatory response(1). This receptor interacts with CD154 or CD40L, a cell-surfaceprotein that is highly expressed by activated T lymphocytes.CD40/CD40L interaction induces in vitro production of differentproinflammatory cytokines, including IL-8, monocyte chemoattractantprotein-1 (MCP-1), and RANTES by proximal tubular epithelialcells (PTEC) (2). CD40L (CD154) expression was described ongraft infiltrating T cells and macrophages, whereas CD40 expressionis induced on glomerular and tubular epithelial cells duringchronic renal allograft rejection (3). Moreover, in differentanimal models, anti-CD154 antibody not only prevents renal structuraland functional injury but also inhibits and interrupts the developmentof chronic rejection (4). In addition, CD40/CD40L interactionwas shown to mediate the progression of other chronic rejectionmodels in limb, heart, and liver transplantation (5–7).
After CD40 receptor multimerization by its ligand, the CD40cytoplasmic domain associates with TNF-R–associated factor(TRAF) proteins and induces the subsequent activation of differentsignaling pathways (8). CD40 is also associated with caveolin-1,a structural protein of caveolae, specialized membrane microdomains,involved in several signaling events (9). Some components ofthe CD40 signaling pathway, c-jun NH2-terminal kinase, p38,and extracellular signal–regulated kinase 1/2 mitogen-activatedprotein kinase but not TRAF-6, are present within the caveolaeand dissociate after CD40/CD40L interaction (9). In PTEC, ithas been demonstrated that CD40/CD40L interaction increasesc-jun NH2-terminal kinase, p38, and extracellular signal–regulatedkinase mitogen-activated protein kinase phosphorylation, andtheir activation is required for MCP-1 and IL-8 production (2).
Although the proinflammatory effect of CD40L is well described,very little is known of its potential profibrotic effects. Theplasminogen activator inhibitor-1 (PAI-1) is an antifibrinolyticpeptide that plays a key role in extracellular matrix deposition,significantly reducing its turnover (10,11). PAI-1 gene expressionis strikingly induced in several chronic tubulointerstitialdiseases that are characterized by progressive interstitialfibrosis, including chronic allograft nephropathy (CAN) (12,13),but the link between CD40/CD40L interaction and PAI-1 productionfrom tubular epithelial cells is still largely unclear. Thus,the aim of our study was to investigate the role played by CD40/CD40Linteraction on PTEC expression of PAI-1, a powerful profibroticmediator, and MCP-1, a proinflammatory cytokine, and to studythe intracellular mechanisms that lead to these effects.
Reagents
DMEM/F12 medium, FBS, and trypsin were obtained from Sigma CellCulture (Milan, Italy). Penicillin/streptomycin and L-glutaminewere from Life Technologies (Milan, Italy). PP1 was from Biolmol(Plymouth Meeting, PA). CAPE was from Calbiochem (Darmstadt,Germany). sCD40L was from Alexis (Lausen, Switzerland). ThepLucNF-B vector (containing the firefly luciferase cDNA underthe control of three NF-B consensus sequences) was providedby Dr. M. Fresno (14). The dual luciferase assay kit was fromPromega (Milan, Italy). The DNA-free kit was from Ambion (Austin,TX). The protein G immunoprecipitation kit was from Sigma Aldrich(St. Louis, MO). The monoclonal anti–NF-B p65 antibody,the polyclonal anti–phospho-NIK antibody, the monoclonalanti-NIK antibody, the polyclonal anti–phospho-lyn antibody,the monoclonal anti-lyn antibody, the polyclonal anti–c-srcantibody, the monoclonal anti-TRAF2 antibody, and the monoclonalanti-TRAF6 antibody were obtained from Santa Cruz BiotechnologyInc. (Santa Cruz, CA). The polyclonal anti–phospho-Y418Srcantibody was purchased from BioSource International Inc. (Camarillo,CA). The monoclonal anti–phospho caveolin (Y14) antibodywas from BD biosciences (San Diego, CA). The polyclonal anti-caveolinantibody was from Upstate (Lake Placid, NY). The horseradish-peroxidase(HRP)-conjugated sheep anti-mouse and sheep anti-rabbit antibodieswere supplied from Amersham Biosciences (Buckinghamshire, UK).[32P]dCTP was purchased from ICN (Milan, Italy). All other chemicalswere reagent grade.
Cell Isolation and Culture
HK2, an immortalized PTEC line from normal adult human kidney(15), was obtained from American Type Culture Collection (ATCC,Rockville, MD). Cells were grown to confluence in DMEM/F12 mediumsupplemented with 10% FBS, 100 U/ml penicillin, 100 µg/mlstreptomycin, and 2 mM L-glutamine. For passage, confluent cellswere washed with PBS, removed with 0.05% trypsin/0.02% EDTAin PBS, and plated in DMEM/F12.
Mouse fibroblast L cells, stably transfected with human CD40L(provided by Dr. M. Shurin, Department of Surgery, Pittsburgh,PA), were used to confirm all of the results that were obtainedwith soluble CD40L. Nontransfected L cells were used as thenegative control. For co-culture experiments, trypsinized PTECand trypsinized human CD40 ligand transfected mouse L cells(CD40L-cells) or untransfected L cells were mixed in suspensionin a 1:1 ratio. Cells were cultured in complete 1% FBS-RPMImedium. L cells were irradiated 10,000 rads to prevent theirovergrowth.
The concentration of the soluble form of CD40L that was usedin all of the experiments was defined on a dose range that wasdemonstrated previously to be effective in the same cell type(16). The dose- and time-dependent effect of the inhibitorsused, CAPE and PP1, was reported previously in the same celltype (17). In addition, any toxic effect of the two inhibitorsat the dose used in this study was excluded by thiazolyl bluetetrazolium bromide (MTT) (data not shown).
Western Blot
PTEC were plated in six-well dishes and grown to confluencein DMEM/F12 supplemented with 10% FBS. The cells were incubatedfor 40 h in serum-free medium and then exposed to sCD40L (0.1mg/ml) and sCD40L enhancer (1 mg/ml) or the enhancer alone forthe indicated times. In separate sets of experiments, cellswere preincubated with PP1 (50 µM) for 18 h before sCD40Lwas added. At the end of the treatment, the cell monolayer wasrinsed twice rapidly with ice-cold PBS and lysed in 100 µlof RIPA buffer (1 mM PMSF, 5 mM EDTA, 1 mM sodium orthovanadate,150 mM sodium chloride, 8 µg/ml leupeptin, 1.5% NonidetP-40, and 20 mM tris-HCl [pH 7.4]). The lysates were kept onice for 30 min and centrifuged at 10,000 x g at 4°C for5 min. The supernatants were collected and stored at –80°Cuntil used. Aliquots that contained 40 µg of proteinsfrom each lysate were subjected to SDS-PAGE on a 10% gel underreducing conditions and then electrotransferred onto nitrocellulosemembrane (Hybond C; Amersham). The filter was blocked overnightat room temperature with 2% BSA in PBS that contained 0.1% tween-20(TBS) and then incubated with the polyclonal anti–phospho-NIKantibody (1:500 dilution in TBS at room temperature for 2 h)or the polyclonal anti–phospho-lyn antibody (1:700 dilutionin TBS at room temperature for 2 h) or the polyclonal anti–phospho-Y418-srcantibody (1:650 dilution in TBS at room temperature for 2 h).The membranes were washed twice in TBS and incubated for 1 hat room temperature with HRP-conjugated sheep anti-rabbit IgGat 1:1500 dilution in TBS. The membranes were washed three timesat room temperature in TBS and then once with 0.1% SDS in PBS.The ECL enhanced chemiluminescence system (Amersham) was usedfor detection. The same membranes then were stripped and immunoblottedagain with anti-human NIK or anti-lyn mAb (1:500 dilution inTBS at room temperature for 2 h) or anti–c-src-polyclonalantibody (1:1500 dilution in TBS at room temperature for 2 h).The ECL enhanced chemiluminescence system (Amersham) was usedfor detection.
Immunoprecipitation
Confluent PTEC in 60-mm2 culture dishes were placed in serum-freemedium for 40 h. sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml)or the enhancer alone then was added for the indicated times.Cells were washed twice with ice-cold PBS and lysed in situwith RIPA buffer. The cell lysate was incubated for 30 min onice and than centrifuged at 10,000 x g for 5 min at 4°C.The supernatants were collected and stored at –80°Cuntil used. A total of 200 µg of protein from the supernatantwere immunoprecipitated using the protein G immunoprecipitationkit (Sigma). The proteins first were incubated with 2.5 µgof anti–phospho-lyn polyclonal antibody, overnight ona rocking platform at 4°C, and then with protein G Sepharosefor 3 h at 4°C. The immunoprecipitated proteins were elutedin sample buffer (2--mercaptoethanol, 10% SDS, 10% glycerol,0.5 M Tris-HCl [pH 6.8], and 0.05% blue-bromophenol) and boiled.The immunoprecipitated proteins were separated by electrophoresison a 10% polyacrylamide gel and transferred onto a nitrocellulosemembrane. The membrane was blocked as described previously andincubated with anti-caveolin polyclonal antibody (1:400) for2 h at room temperature, washed, and incubated with HRP-conjugatedsheep anti-rabbit IgG (1:1500) for 1 h at room temperature TheECL system was used for detection. The same membrane then wasstripped and immunoblotted again with the anti–phospho-lynpolyclonal antibody that was used for the immunoprecipitation(1:700).
Transient Transfections and Luciferase Assay
Transient transfection was carried out by electroporation usingthe Gene Pulser II RF module (Biorad, Hercules, CA). ConfluentPTEC were trypsinized, and 5 x 106 cells were resuspended in0.5 ml of medium that contained 7.5 µg of pLuc3X NF-Band 2.5 µg of pCMVGal and kept on ice for 10 min. Electroporationwas carried out at 50 µF and 1.2 Kv. After electroporation,cells were placed on ice for 5 min and then plated in a six-wellplate at a concentration of 8 x 105/well. At 24 h, the mediumwas removed, 2 ml of fresh medium was added, and incubationcontinued for an additional 24 h. For determination of the effectof sCD40L stimulation, the cells were rinsed once with DMEMand triplicate wells were incubated with or without sCD40L (0.1mg/ml) and sCD40L enhancer (1 mg/ml) or the enhancer alone inthe presence or in the absence of specific inhibitors in 1 mlof serum-free DMEM. After incubation for the indicated times,the cells were rinsed once in PBS, then scraped and lysed in100 µl of reporter lysis buffer supplied with the LuciferaseReporter Assay System (Promega). The extracts were incubatedat room temperature for 10 min and centrifuged at 12,000 x gfor 5 min. Twenty microliters of the supernatant was assayedfor luciferase activity using a DIGENE DCR-1 luminometer (AbbotLaboratories, Abbot Park, IL). Luciferase activity was normalizedto -galactosidase.
Evaluation of PAI-1–and MCP-1–Secreted Proteins on Culture Supernatants by ELISA
PTEC were plated in 60-mm2 Petri dishes and cultured as detailedabove. After reaching confluence, cells were serum-starved for40 h and then incubated for the indicated times with sCD40L(0.1 mg/ml) and sCD40L enhancer (1 mg/ml) or the enhancer alone.Cell culture supernatants were collected at the end of eachincubation. PAI-1 and MCP-1 concentrations were assessed byELISA, using commercially available kits (American DiagnosticInc., Greenwich, CT, and Apulia Biotech, Valenzano, Italy, respectively).Concentration of PAI-1 and MCP-1 in supernatants were normalizedto cellular proteins and expressed as pg PAI-1/µg cellularproteins and pg MCP-1/µg cellular proteins.
RNA Isolation and Northern Blot Analysis
PTEC were plated in 60-mm2 Petri dishes and cultured as detailedabove. After reaching confluence, cells were serum-starved for40 h and then incubated for the indicated times with sCD40L(0.1 mg/ml) and sCD40L enhancer (1 mg/ml) or the enhancer alone.In separate sets of experiments, cells were preincubated withPP1 (50 µM) and CAPE (10 µg/ml) for 18 h beforesCD40L was added. At the end of incubation, cells were lysedwith 1 ml of TRIzol Reagent (Life Technologies, Milan, Italy).
PAI-1 gene expression was studied by Northern blotting. Electrophoresisof 20 µg of total RNA from each experimental conditionwas carried out in 1.1% agarose gel with 2.2 M formaldehyde.The RNA then was transferred overnight onto a nylon membrane(Schleicher & Schuell, Dassel, Germany). The membrane wasstained with ethidium bromide to evaluate the 28S and 18S ribosomalbands and prehybridized at 42°C for 4 h in 50% Formamide,0.5% SDS, 5x SSC, and 0.1 mg/ml salmon sperm DNA. A 1253-bpfragment of the human PAI-1 cDNA was used as probe. The DNAfragment was labeled by random priming using a commerciallyavailable kit (Amersham) and [32P]dCTP (specific activity 3000Ci/mmol). The probe (106 cpm/ml) was added to 10 ml of prehybridizationsolution, and the blots were hybridized for 16 h at 42°C.The membranes then were washed once in 2x SSC at room temperaturefor 5 min, once in 2x SSC and 0.1% SDS at room temperature for20 min, once in the same buffer at 55°C for 30 min, andin SSC and 0.1% SDS at 55°C for an additional 30 min. Afterdrying, membranes were exposed to a Kodak X-OMAT film (Rochester,NY) with intensifying screens at –70°C.
Real-Time PCR
MCP-1 RNA expression was investigated by real-time PCR. To thispurpose, 10 µg of total RNA was treated with DNA-freekit (Ambion) to remove DNA contamination. One microgram of treatedRNA was used in a reverse transcription reaction, as describedpreviously (17,18). Real-time PCR analysis for MCP-1 gene expressionwas performed using a specific TaqMan Probe and analyzed withthe i-Cycle thermal cycle (Biorad, Hercules, CA) by the ApuliaBiotech (Valenzano, Bari, Italy).
Confocal Microscopy
TRAF2, TRAF6, p65 NF-B subunit, and phospho-caveolin proteinexpression and cell distribution were evaluated in PTEC by indirectimmunofluorescence and confocal microscopy analysis using specificantibodies. For each experiment, 1.5 x 104 cells were platedon a coverslip and then stimulated with sCD40L (0.1 mg/ml) andsCD40L enhancer (1 mg/ml) for the indicated times. In separatesets of experiments, cells were preincubated with CAPE (10 µg/ml)for 45 min before sCD40L was added. PTEC then were fixed withparaformaldehyde 4% for 15 min, treated with Triton-X 100 0.2%in PBS for 5 min, incubated for 1 h in blocking buffer (1% BSAin PBS), and then incubated with monoclonal (1:500 dilution)specific antibodies for 1 h. The immune complexes then wereidentified by incubating for 1 h the PTEC with the secondaryantibodies Alexa Fluor 488 goat anti-mouse IgG-FITC-conjugated(1:300 dilution; Molecular Probes). The cells were washed threetimes with PBS between each step. The slides then were mountedin Gel/Mount (Biomeda, Foster City, CA) and sealed. Negativecontrol was obtained by incubating PTEC with the blocking solutionand then omitting the primary antibody.
The cell-specific fluorescence was analyzed by confocal laserscanning microscopy using the Leica TCS SP2 (Leica, Wetzlar,Germany), equipped with an argon-krypton (488 nm) laser. Thelaser allows the acquisition of FITC (green). Confocal imageswere taken at 500-nm intervals on the z axis of the cells. Imagesfrom individual optical planes and multiple serial optical sectionswere analyzed and sequentially scanned. The images were recordedusing the Leica imaging software. Image analysis was performedon all acquired fields.
Statistical Analyses
Data are presented as mean ± SD and compared by ANOVA.P < 0.05 was considered statistically significant.
We first investigated the effects of CD40L on the gene expressionof PAI-1, a powerful profibrotic mediator, and MCP-1, a proinflammatorycytokine, in PTEC. CD40/CD40L interaction was able to induceboth PAI-1 and MCP-1 mRNA expression in a time-dependent manner,with a peak at 6 h for PAI-1 (Figure 1A) and at 24 h for MCP-1(Figure 1B), as demonstrated by Northern blot and real-timePCR, respectively. MCP-1 gene expression came down after CD40cross-linking on PTEC for 48 h (data not shown). To confirmthat the CD40L-induced MCP-1 and PAI-1 gene expression resultedin an increase of protein production, we evaluated, by ELISA,the protein concentration of both PAI-1 and MCP-1 in the cellsupernatant at 24 and 48 h. As shown in Figure 1, C and D, therelease of PAI-1 protein was significantly increased at 48 h,whereas MCP-1 protein production was induced at 24 as well as48 h by CD40 cross-linking.
Figure 1. The effect of CD40L on plasminogen activator inhibitor-1 (PAI-1; A and C) and monocyte chemoattractant protein-1 (MCP-1; B and D) gene and protein expression in proximal tubular epithelial cells (PTEC). (A and B) Confluent, quiescent PTEC were stimulated with sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) or enhancer alone (basal) for the indicated times and then harvested. PAI-1 gene expression was evaluated by Northern blotting (A, top), whereas MCP-1 gene expression was evaluated by real-time PCR (B), as described in Materials and Methods. 28S and 18S ribosomal RNA bands on ethidium bromide–stained gel were used to control the RNA loading (A, bottom). The figure is representative of three experiments. *P < 0.001 versus basal. (C and D) Confluent, quiescent PTEC were stimulated with sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) or enhancer alone (basal) for 24 and 48 h. Supernatants were collected and used to evaluate PAI-1 (C) and MCP-1 (D) concentration by ELISA. The concentrations were normalized with cellular proteins. Data represent mean ± SD of triplicate wells. *P < 0.01 versus time-related basal.
To clarify the intracellular mechanisms that lead to these proinflammatoryand profibrotic effects of CD40L, we investigated the signalingpathways that potentially are activated by CD40 cross-linking.We first studied TRAF membrane translocation and subsequentactivation by confocal microscopy. As shown in Figure 2, TRAF2and 6, both present as a perinuclear fluorescence in basal conditions,quickly translocated to the cell surface with a discrete distributionafter CD40 ligation in a time-dependent manner. No other TRAFproteins presented any significant membrane translocation (datanot shown). TRAF6 has been shown in other cell types to be associatedwith NF-B activation. Thus, we investigated whether CD40L couldactivate this transcription factor in PTEC. CD40 cross-linkingcaused a significant increase in luciferase activity after transienttransfection of PTEC with an expression vector that containedthe luciferase cDNA under the control of three NF-B consensussequences (Figure 3A). NF-B activation and nuclear translocationwere confirmed by confocal microscopy using a mAb that specificallyrecognizes the NF-B subunit p65. The incubation of PTEC withsoluble CD40L induced a significant and time-dependent nucleartranslocation of p65 when compared with basal conditions witha peak at 30 min (Figure 3B). CD40L-induced NF-B nuclear translocationwas blocked by incubation with CAPE, a specific NF-B inhibitor(Figure 3C). Because Nomura et al. (19) suggested that NIK mayrepresent the link between TRAF and NF-B activation, we investigatedthe phosphorylation levels and subsequent activation of thisserine kinase. CD40 cross-linking induced a marked increasein NIK serine phosphorylation already after 5 min and with apeak at 15 min, as demonstrated by Western blotting (Figure 3D).NIK phosphorylation was unaffected by CAPE preincubation(data not shown).
Figure 2. CD40L effect on TNF-R–associated factor 2 (TRAF2) and TRAF6 distribution in PTEC. TRAF2 and TRAF6 distribution was analyzed by confocal microscopy using respectively a mouse monoclonal anti-TRAF2 antibody and a mouse monoclonal anti-TRAF6 antibody (green). Nuclei were stained with To-pro (bleu). PTEC were incubated without (B) or with sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) for 5, 15, and 30 min and than processed as described in Materials and Methods. The figure is representative of three experiments.
Figure 3. CD40L-induced NF-B (A through C) and NIK (D) activation in PTEC. (A) Confluent, quiescent PTEC were trypsinized, and 5 x 106 cells were resuspended in 0.5 ml of medium that contained 7.5 µg of pLucNF-B vector and 2.5 µg of pCMVGal. Electroporation was carried out as described in Materials and Methods. Transfected cells were incubated with or without sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) or enhancer alone (basal) in serum-free medium for 24 h. The figure is representative of three experiments. *P < 0.01 versus basal. (B) NF-B activation was analyzed by confocal microscopy using an antibody that recognizes the p65 subunit of the nuclear factor. PTEC were stimulated with sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) for 30 min and then processed as described in Materials and Methods. The figure is representative of three experiments. (C) Quiescent PTEC that were preincubated with CAPE (10 µg/ml) for 45 min were stimulated with sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) for 30 min. NF-B nuclear translocation was performed as in B, and NF-B specific fluorescence within the nuclear area (identified by TO-PRO counterstaining) was acquired and quantified using the LEICA image software. *P < 0.01 versus basal; **P < 0.01 versus sCD40L alone. (D) Confluent, quiescent PTEC were incubated with sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) for 5, 15, 30, and 60 min and then lysed in RIPA buffer. Equal amounts of protein from each cell lysate (40 µg) were separated by SDS-PAGE, transferred onto nitrocellulose filter, and then blotted with rabbit polyclonal anti–phospho-NIK antibody as described in Materials and Methods (top). The same membrane then was stripped and immunoblotted again with mouse monoclonal anti-NIK antibody (bottom). The figure is representative of three experiments.
TRAF2 was shown previously in dendritic cells to be associatedwith the activation of lyn, a cytoplasmic tyrosine kinase thatbelongs to the src family. Indeed, also in PTEC, soluble CD40Linduced a significant and time-dependent increase in lyn tyrosinephosphorylation but not in the phosphorylation of other enzymesthat belong to the same family (src, fyn, and yes; Figure 4, A and B).CD40L-induced lyn phosphorylation was blocked by incubationwith PP1, a specific src inhibitor (Figure 4C). Vidalain etal. (20) demonstrated that in dendritic cells, lyn once activatedco-localizes with membrane rafts. We then investigated whetherCD40L incubation may induce the phosphorylation of caveolin-1,a membrane-associated glycoprotein featuring specialized microdomainsof the cell surface, the caveolae. As demonstrated by confocalmicroscopy (Figure 5A), CD40 cross-linking caused a significantand time-dependent increase in membrane-associated phospho-caveolin-1.For evaluation of whether lyn upon phosphorylation interactswith phospho-caveolin also in PTEC as in dendritic cells, totalcell lysates that were obtained before and after CD40 cross-linkingwere immunoprecipitated with a specific anti–phospho-lynantibody and blotted with a mAb that recognizes caveolin-1.As shown in Figure 5B, lyn, once phosphorylated, specificallyinteracts with caveolin-1.
Figure 4. The effect of CD40L on lyn (A) and src (B) activation in PTEC. (A and B) Confluent, quiescent PTEC were stimulated with sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) for 5, 15, 30, and 60 min and then lysed in RIPA buffer. Equal amounts of protein from each cell lysate (40 µg) were separated by SDS-PAGE, transferred onto nitrocellulose filter, and then probed with rabbit polyclonal anti–phospho-lyn antibody (A, top) and anti–phospho-Y418-src antibody (B, top) as described in Materials and Methods. The same membranes then were stripped and immunoblotted again with mouse monoclonal anti-lyn antibody (A, bottom) and anti-human src polyclonal antibody (B, bottom), respectively, as described in Materials and Methods. The figure is representative of three experiments. (C) Effect of PP1 on CD40L-induced lyn phosphorylation in PTEC. Confluent, quiescent PTEC that were preincubated for 18 h with PP1 (50 µM) were stimulated with sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) for 15 min and then lysed in RIPA buffer. Immunoblotting was performed as described in A.
Figure 5. (A) CD40L effect on caveolin activation in PTEC. Phospho-caveolin distribution was analyzed by confocal microscopy using a mouse monoclonal anti–phospho-caveolin antibody (green). PTEC were stimulated with sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) for 5, 15, and 30 min and then processed as described in Materials and Methods. The figure is representative of three experiments. (B) CD40L effect on phospho-lyn and caveolin interaction in PTEC. Phospho-lyn and caveolin interaction was analyzed by immunoprecipitation of a total protein lysate with a specific polyclonal antibody anti–phospho-lyn and subsequent Western blotting with a specific monoclonal antibody anti-caveolin (top). The same membrane then was stripped and blotted again with anti–phospho-lyn antibody (bottom). PTEC were stimulated with sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) for 15, 30, and 60 min and then processed as described in Materials and Methods. The figure is representative of three experiments.
Finally, we investigated the role of lyn and NF-B in the proinflammatoryand profibrotic effects of CD40/CD40L interaction. It is interestingthat preincubation of PTEC with PP1, the specific src inhibitor,significantly reduced CD40L-induced PAI-1 gene expression, whereasCAPE, a specific inhibitor of the transcription factor NF-B,not only did not cause any reduction in PAI-1 mRNA abundancebut also slightly induced PAI-1 gene expression (Figure 6).On the contrary, CAPE completely suppressed MCP-1 gene expression,whereas PP1 caused only a partial reduction of CD40L-inducedMCP-1 (Figure 7).
Figure 6. Effect of lyn and NF-B inhibition on CD40L-induced PAI-1 gene expression in PTEC. Confluent, quiescent PTEC were pretreated with a specific src inhibitor (PP1, 50 µM) or a specific NF-B inhibitor (CAPE, 10 µg/ml) for 18 h, stimulated with sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) for 6 h, and then harvested. PAI-1 gene expression was evaluated by Northern blotting (A, top) as described in Materials and Methods. 28S and 18S ribosomal RNA bands on ethidium bromide–stained gel were used to control the RNA loading (A, bottom). Intensity of specific mRNA bands was quantified by computer-assisted densitometry (Optilab 2.6.1), normalized to the intensity of the 28S bands on the ethidium bromide–stained blots, and expressed as PAI-1/28S ratio (B; mean ± SD of three separate experiments). *P < 0.001 versus basal; °P < 0.001 versus sCD40L.
Figure 7. Effect of src and NF-B inhibition on CD40L-induced MCP-1 gene expression in PTEC. (A) Confluent, quiescent PTEC were pretreated with a specific src inhibitor (PP1, 50 µM) or a specific NF-B inhibitor (CAPE, 10 µg/ml) for 18 h, co-incubated with CD40L cells for 6 h and then harvested. MCP-1 gene expression was evaluated by real-time PCR as described in Materials and Methods. *P < 0.001 versus basal; #P < 0.001 versus CD40L cell. (B) Confluent, quiescent PTEC were pretreated with a specific src inhibitor (PP1, 50 µM) or a specific NF-B inhibitor (CAPE, 10 µg/ml) for 18 h, stimulated with sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) or enhancer alone (basal) for 24 h, and then harvested. MCP-1 gene expression was evaluated by real-time PCR as described in Materials and Methods. *P < 0.001 versus basal; #P < 0.001 versus CD40L.
To rule out the possibility that lyn may modulate NIK and/orNF-B activation, we preincubated PTEC that were transfectedwith the pLucNF-B vector with PP1 before CD40 cross-linking.PP1 did not influence CD40L-induced NF-B activation (Figure 8).
Figure 8. Effect of src inhibition on CD40L-induced NF-B activation in PTEC. Confluent, quiescent PTEC were trypsinized, and 5 x 106 cells were resuspended in 0.5 ml of medium that contained 7.5 µg of pLucNF-B vector and 2.5 µg of pCMVGal. Electroporation was carried out as described in Materials and Methods. Transfected cells were incubated with or without sCD40L (0.1 mg/ml) and sCD40L enhancer (1 mg/ml) or the enhancer alone (basal) in serum-free medium for 24 h in the absence and in the presence of PP1 (50 µM). The figure is representative of three experiments. *P < 0.001 versus basal.
CAN remains the major cause of renal graft failure, but themolecular mechanisms that lead to the development and progressionof this pathologic condition are still largely unclear. Severalobservations suggest the involvement of both alloantigen-dependentand -independent mechanisms. It has been demonstrated that theCD40-CD40L (CD154) co-stimulatory pathway plays a critical rolein the pathogenesis of both acute and chronic kidney allograftrejection. Lederer et al. (21) recently demonstrated a CD40Lupregulation in peripheral blood of patients with chronic renalallograft dysfunction. CD40L is expressed at a very high levelon graft-infiltrating T cells and macrophages, whereas CD40expression is augmented on proximal tubules in biopsies of patientsduring chronic rejection (3). CD40 ligation not only is ableto induce co-stimulatory molecules and major histocompatibilitycomplexes expression but also may modulate the production ofinflammatory cytokines and chemokines by mononuclear cells andPTEC (1,2). Our data confirmed MCP-1 production by PTEC afterCD40 cross-linking. This chemokine is a powerful chemotacticand activating factor for monocytes and has been suggested asa key mediator of acute graft dysfunction. Indeed, the expressionof both this chemokine and its specific receptor is strikinglyincreased in acute rejection biopsies (22,23). In addition,null mice for MCP-1 and its specific receptor are characterizedby a reduced rate of acute graft rejection (24). Moreover, MCP-1expression is upregulated in CAN and in this scenario may stillaccount for the inflammatory component that features this pathologiccondition.
However, the main feature of CAN is interstitial fibrosis, andvery little is known of the potential profibrotic effects ofCD40–CD40L interaction. In this study, we demonstratefor the first time that CD40 cross-linking on PTEC may inducethe expression of PAI-1, a powerful profibrotic mediator. PAI-1has been shown to play a key role in extracellular matrix accumulationand subsequent interstitial fibrosis (11). Indeed, this antifibrinolyticmolecule may exert its antagonist effect not only on plasminogenactivators but also on several proteolytic enzymes that areinvolved in extracellular matrix turnover (11). Thus, PAI-1may cause a significant deposition of extracellular matrix componentsby reducing their catabolisms. PAI-1 gene expression is markedlyincreased at the glomerular and tubular interstitial levelsin a variety of progressive renal disease. In particular, weand others reported a striking upregulation of PAI-1 gene expressionin CAN, in which expression of this antifibrinolytic moleculecorrelates directly and significantly with the degree and theextent of interstitial fibrosis (25).
Activation of the CD40 signaling pathways is mediated primarilyby recruitment of several TRAF protein family members that areassociated with the CD40 cytoplasmic domain (8). TRAF recruitmentby CD40 is cell specific and influences the activation of downstreamsignaling pathways. We demonstrated that upon CD40 cross-linking,TRAF2 and 6 are translocated rapidly to the cell membrane. Severalpieces of evidence demonstrated that CD40 can activate NF-Bby at least two pathways, involving both TRAF2 and TRAF6, asdemonstrated in knockout mice (26,27), whereas only TRAF2 hasbeen shown in dendritic cells to underlie the activation oflyn, an src-related cytoplasmic tyrosine kinase. Indeed, alsoin our system, TRAF2 and TRAF6 membrane translocation was associatedwith NF-B and lyn activation. It is interesting that we observedthat the activation of NF-B that was induced by CD40L cross-linkingwas unchanged after preincubation of PTEC with PP1, a powerfuland selective src inhibitor, clearly suggesting that the twosignaling pathways are independent in PTEC. Whereas NF-B activationupon CD40–CD40L interaction was shown already in PTEC,the involvement of lyn in the CD40 signaling pathways was observedonly in dendritic cells. In their report, Vidalain et al. (20)demonstrated that in dendritic cells, CD40/CD40L cross-linkinginduces lyn activation through its association to sphingolipid-and cholesterol-rich plasma membrane microdomains. In our system,CD40 cross-linking caused the phosphorylation of caveolin-1,the main protein component of membrane caveolae and its associationwith lyn. Thus, PTEC and dendritic cells not only express CD40on their surface but also behave alike upon CD40 cross-linking.
Finally, we demonstrated that the two signaling pathways aredifferently involved in MCP-1 and PAI-1 gene expression. CD40L-inducedMCP-1 but not PAI-1 gene expression was completely abolishedby CAPE, a specific NF-B inhibitor. Conversely, inhibition oflyn significantly reduced PAI-1 but not MCP-1 gene expression.Because inhibition of src and NF-B did not influence TRAF translocationand NIK activation (data not shown), it is conceivable thatprofibrotic/proinflammatory signaling diverges somewhere betweenthese two steps, although the level of divergence remains tobe identified specifically. This observation suggests that theintracellular pathway that leads to the release of PAI-1 byPTEC is dependent on lyn, whereas NF-B plays a key role in theCD40 proinflammatory signal but does not influence the profibroticeffect. Motojima et al. (28) reported that uremic toxins inducethe free radical production and activate NF-B in HK2 cells,which, in turn, upregulates PAI-1 gene expression. In our hands,NF-B activation seems to be neither sufficient nor necessaryfor PAI-1 gene expression that is induced also by differentagonists (angiotensin II, angiotensin IV, activated factor XII,and thrombin; data not shown), suggesting that different stimulican selectively activate different pathways also in the samecell type. In addition, NF-B inhibition, in association withCD40 cross-linking but not by itself (data not shown), seemsto induce a slight upregulation of PAI-1 gene expression, suggestinga potential repressive effect of NF-B on this profibrotic mediator.These findings may be of clinical relevance. Indeed, corticosteroids,routinely used in the treatment of acute graft rejection, arewidely known to exert their anti-inflammatory effects to modulateNF-B activity. Our observation suggest that although blockingthe inflammatory component of the rejection, steroids mightnot influence at all the development of fibrosis. Indeed, Emeiset al. (29) demonstrated in a model of endotoxemia that theuse of dexamethasone significantly reduced proinflammatory cytokineproduction without affecting PAI-1 circulating levels.
Our data demonstrate that CD40L could play a key role in theprogression of interstitial fibrosis in CAN through PAI-1 induction.Moreover, CD40L-mediated profibrotic and proinflammatory effectsdepend on two distinct intracellular pathways. Thus, blockingthe inflammatory signal may not ensure an efficient antifibroticeffect.
Acknowledgments
This study was supported by the "Comitato Eccellenza Genomicain Campo Biomedico ed Agrario" (CEGBA), MIUR (COFIN 2002 toF.P.S. and L.G. and COFIN 2003 to G.G.) and the 5th EuropeanFramework "Quality of Life and Management of Living Resources(contract QLG1-2002-01215 to G.G.).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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B and Lyn
Top
Abstract
Introduction
B activation, through the NF-
-mercaptoethanol, 10% SDS, 10% glycerol, 0.5 M Tris-HCl [pH 6.8], and 0.05% blue-bromophenol) and boiled. The immunoprecipitated proteins were separated by electrophoresis on a 10% polyacrylamide gel and transferred onto a nitrocellulose membrane. The membrane was blocked as described previously and incubated with anti-caveolin polyclonal antibody (1:400) for 2 h at room temperature, washed, and incubated with HRP-conjugated sheep anti-rabbit IgG (1:1500) for 1 h at room temperature The ECL system was used for detection. The same membrane then was stripped and immunoblotted again with the anti–phospho-lyn polyclonal antibody that was used for the immunoprecipitation (1:700). 
