Role for TGF- in Cyclosporine-Induced Modulation of Renal Epithelial Barrier Function
Gemma Feldman,
Breda Kiely,
Natalia Martin,
Gavin Ryan,
Tara McMorrow and
Michael P. Ryan
Department of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland
Address correspondence to: Dr. Michael P. Ryan, Department of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland. Phone: +353-1-7166549; Fax: +353-1-71612157; E-mail: michael.p.ryan{at}ucd.ie
Received for publication May 25, 2006.
Accepted for publication March 12, 2007.
It was previously shown that cyclosporine A (CsA) increasestransepithelial resistance in MDCK cells. Activation of theextracellular signal–regulated kinase 1/2 (ERK1/2) mitogen-activatedprotein kinase (MAPK) cascade seems to be pivotal to the CsA-inducedincrease in transepithelial electrical resistance (TER). Thisstudy examined the role played by TGF- in mediating the CsA-inducedactivation of ERK1/2 and the resulting increase in TER in MDCKcells. Paracellular permeability across MDCK monolayers aftervarious treatments was assessed by measurement of TER. TGF-secretion was measured by Western blot and ELISA. Activationof the ERK1/2 pathway and tight junction protein expressionwere also assessed by Western blot analysis. CsA increased productionand secretion of TGF- and expression of the TGF- receptor II.Exogenous addition of TGF-1 activated ERK1/2 and increased TERacross MDCK monolayers, both of which were attenuated by theMEK inhibitor U0126. Neutralizing antibodies against TGF-1 andthe TGF- receptor II significantly reduced the CsA-induced increasein TER. Both CsA and TGF-1 increased expression of tight junctionproteins claudin-1 and zonula occludens 2. Inhibition of thep38 MAPK pathway also attenuated the TGF-1–induced increasein TER. The results presented here suggest that the CsA-inducedmodulation of paracellular permeability may be mediated, atleast in part, by an increase in TGF- production.
Cyclosporine A (CsA) is an immunosuppressive drug that is usedin organ transplantation and in the treatment of some primaryrenal and nonrenal immune-mediated diseases. However, its clinicaluse is limited by adverse effects, the most important of whichis nephrotoxicity (1). This condition is manifested by renalinsufficiency as a result of glomerular and vascular changes,abnormalities of tubular function, and hypertension. Renal biopsyreveals arteriolar hyalinosis, focal interstitial fibrosis,tubular atrophy, and glomerulosclerosis with focal atrophy.Mechanisms of CsA-induced nephrotoxicity were thought to behemodynamic in origin, but there is accumulating evidence fora direct tubular effect. CsA-induced tubular damage is not wellunderstood; however, alterations in tubular morphology havebeen seen in acute studies in rats (2–4).
Epithelial tissues serve as selective permeability barriers,separating fluid compartments with different chemical compositions.Renal epithelium tight junctions mediate this barrier role andseal cells together to impede the paracellular leakage of smallmolecules. In renal tubules, the paracellular pathway playsan important role in vectorial transport with some selectivityfor transported ions such as magnesium, calcium, sodium, andchloride. In mammalian kidney, the complexity of the tight junctionincreases from the proximal to the collecting tubule. In mammaliankidney, the transepithelial resistance (TER) and complexityof the tight junctions increase from the proximal tubule alongthe nephron to the distal tubule. Examination of the differentialexpression of tight junction proteins along isolated rabbitrenal tubules showed that the levels of zonula occludens 1 (ZO-1),ZO-2, and occludin increased from the proximal tubules to thedistal tubules, mirroring the increase in complexity of thejunctions (5). Tight junctions are subject to physiologic andpharmacologic regulation/modulation. We previously showed thatCsA alters the barrier function of renal epithelial cells, andthe mechanism may involve activation of the extracellular signal–regulatedkinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK)pathway (6).
CsA stimulates renal and systemic overproduction of TGF-, whichis thought to be an important factor in the development of CsAchronic nephrotoxicity (7–9). More than 70% of the renalbiopsies from CsA-treated transplant patients with chronic allograftfibrosis expressed high levels of TGF- (10). Using Affymetrixgene microarrays, we demonstrated upregulation of TGF- in humanrenal tubular cells that were treated with CsA (11). TGF- hasbeen implicated in the modulation of paracellular permeability.TGF-1 enhanced the barrier function of intestinal epithelialmonolayers and promoted intestinal epithelial restitution (12–16).A recent study demonstrated that TGF- lowered endothelial permeabilityin mouse brain capillary endothelial cells, suggesting thatcellular constituents that produce TGF- in the brain may keepthe blood–brain barrier functioning (17). TGF-1 not onlyupregulates colonic epithelial barrier function but also curtailsthe effects of barrier-reducing cytokines such as IFN-, IL-4,and IL-10 (12). In contrast, TGF-1 prevented glucocorticoid-stimulatedtight junction formation and reduced TER in murine mammary epithelialcells (18). Therefore, it is probable that TGF-1 may produceconverse effects on epithelial barrier function in various tissues.
There is evidence linking activation of the MAPK pathways tothe TGF-–induced modulation of tight junctions and paracellularpermeability. Co-stimulation of thyroid epithelial cells withEGF and TGF-1 drastically reduced TER and increased paracellularflux of inulin (19). Reduced levels of claudin-1 and occludinaccompanied the loss of barrier function. The MEK inhibitorU0126 prevented residual ERK phosphorylation and abrogated thesynergistic responses to TGF-1 and EGF. TGF- enhanced epithelialbarrier function of T84 and HT-29 monolayers up to three-fold(16). The barrier function could be restored by treatment withSB203580, an inhibitor of the p38 MAPK pathway, but not by inhibitorsof c-Jun N-terminal kinase, ERK1/2 MAPK, or phosphatidylinositol3-kinase (16). In contrast, TGF-3 reduced Sertoli cell tightjunction function (20). This TGF-3–mediated effect ontight junction barrier and the TGF-3–induced phospho-p38MAPK production could be blocked by a specific p38 MAPK inhibitorbut not by a specific MEK1/2 kinase inhibitor (21). These resultsdemonstrate that TGF-3 uses the p38 MAPK pathway to regulateSertoli cell tight junction dynamics. In this study, we examinedwhether the CsA-induced effects on paracellular permeabilityare mediated by TGF-.
Cell Culture and Treatment
MDCK II cells (22) that were obtained from the American TypeCulture Collection (Middlesex, UK) were cultured as before (6).CsA was prepared as a stock solution (4.2 mM) in 100% EtOH.U0126 and SB203580 were prepared as stock solutions of 10 mMin DMSO. TGF-1 was reconstituted with 4 mM HCl that contained1 mg/ml BSA to a stock of 5 µg/ml. TGF-1–neutralizingantibody (nAb) was prepared as a stock solution of 500 µg/mlin PBS. TGF- receptor II (TGF--RII) nAb was prepared as a stocksolution of 0.25 mg/ml in PBS. Cells were preincubated for 1h with inhibitors before treatment with CsA.
TER
The intactness of paracellular pathways of the MDCK monolayersto small ions was monitored by measurement of TER using theREMS automated system (World Precision Instruments, Stevenage,UK). Cells were grown on Costar HTS-Transwell (Corning, NY)cell culture inserts (pore size 0.4 µm). MDCK cells thatwere seeded at a density of 1 x 105 cells/ml were seen to reacha stable TER, representing a confluent monolayer, 5 d afterseeding. Cells were exposed, on the apical side only, to thevarious treatments. The point of drug addition was taken astime 0, and TER was monitored over a range of times. TER wasnormalized to the area of the filter after removal of backgroundresistance of a blank filter that contained only medium. TERwas thus measured as ohms x cm2 (·cm2) and results wereexpressed as the change in TER with respect to time matchedcontrols [TER (·cm2)].
FITC-Dextran Flux Measurements
The intactness of the paracellular pathway across monolayerswas assessed by measurement of FITC-dextran flux. MDCK cellswere seeded at a density of 1 x 105 cells/ml onto Costar Transwellfilters. After treatment, medium was replaced with serum-freemedium that contained FITC-dextran on the apical surface. Afteran appropriate incubation period, the FITC fluorescence in eachwell of the base plate was determined by using a Wallac VictorV plate reader (Perkin Elmer Life Sciences, Waltham, MA). Theresults were expressed as mean FITC flux (ng/cm2 per h).
Cellular Viability
Confluent monolayers were treated for appropriate periods oftime. Monolayers were incubated in 10% AlamarBlue reagent (Biosource,Nivelles, Belgium) at 37°C for 2 h. Fluorescence was readat 545 nm on a Wallac Victor V multiwell plate reader. The amountof fluorescence detected is proportional to the percentage ofviable cells.
Cellular Proliferation
Confluent monolayers were treated for appropriate periods oftime. Cell numbers were determined using the CyQUANT cell proliferationassay (Molecular Probes, Eugene, OR) according to the suppliedprotocol.
Preparation of Whole Extracts for SDS-PAGE
Whole-cell extracts were prepared as before (6). Briefly, afterappropriate treatment, cells were scraped into radioimmunoprecipitationassay buffer (20 mM Tris [pH 7.4], 50 mM NaCl, 5 mM EDTA, 50mM NaF, 20 mM sodium pyrophosphate, 1 mM orthovanadate, 1% [vol/vol]Triton X-100, 0.1% [wt/vol] SDS, 1 mM PMSF, 1 µg/ml pepstatin,0.5 µg/ml leupeptin, and 0.5 µg/ml aprotinin). Proteinconcentration was determined using a BCA protein assay kit (Pierce,Rockford, IL).
Preparation of Samples for TGF- Protein
Supernatants were collected, and 9 µl of Strataclean resinwas added to 1 ml of each supernatant sample. After centrifugation,supernatants were discarded and the pellets were resuspendedin 10 µl of 3x sample buffer. Proteins were denaturedby boiling at 100°C for 3 min.
SDS-PAGE and Western Blotting
Equal amounts of cell extracts were electrophoresed on SDS-polyacrylamidegels, and proteins were transferred to nitrocellulose membrane.For ensuring equal loading, membranes were stained with Ponceau-S.Membranes were blocked with 5% (wt/vol) milk proteins/Tris-bufferedsaline and incubated overnight at 4°C with the primary antibody:rabbit anti-TGF- (Pan specific), mouse anti-TGF--RII, rabbitanti-phospho-ERK1/2, rabbit anti-occludin, rabbit anti–junctionaladhesion molecule-A (JAM-A), rabbit anti–atypical PKCisotype-specific interacting protein/partitioning defective3 (APIS/PAR3), rabbit anti–claudin-1, rabbit anti–claudin-2,rabbit anti–claudin-3, rabbit anti–claudin-4, rabbitanti–claudin-16, rabbit anti–ZO-1, or rabbit anti–ZO-2.Bound antibody was detected with appropriate secondary antibodiesand enhanced chemiluminescence.
TGF-1 ELISA
After treatment of confluent monolayers, 100 µl of supernatantwas transferred to a 96-well microtiter plate. This plate hadbeen prepared by coating it with a monoclonal mouse TGF-1 antibodyand blocking with 4% (wt/vol) BSA/PBS overnight at 4°C.The supernatant or TGF-1 standards (recombinant human TGF-1)were incubated for 2 h. After washing, each well was incubatedwith the biotinylated secondary antibody, the streptavidin-conjugatedhorseradish peroxidase detection reagent, and the substratesolution. The reaction was halted by the addition of 1.8 M H2SO4.Absorbance was measured at 450 nm using a Wallac Victor V platereader. The mean absorbance of each standard was plotted againstTGF-1 concentration to allow quantification of TGF-1 (pg/ml).
Statistical Analyses
Statistical analyses were performed using the statistical programGraphPad Prism 2.1 (GraphPad, San Diego, CA). Data were analyzedby one-way ANOVA, and multiple comparisons between control andtreatment groups were made using the Dunnett posttest. Comparisonsbetween treatments were made using the Bonferroni posttest.Alternatively, the unpaired t test was used to test for statisticalsignificance. Results were expressed as means ± SEM.P 0.05 was deemed statistically significant.
Effect of CsA on TGF- Secretion and TGF--RII Expression
Treatment of MDCK cells with 4.2 µM CsA resulted in asignificant increase in TGF- secretion at 24, 48, and 72 h (Figure 1A),as demonstrated by Western blotting using a PAN-specific antibody.There was also a significant increase in TGF-1 secretion, asdetermined by a specific ELISA (Figure 1B). Because productionof the TGF-1 isoform was increased by CsA treatment, we investigatedthe effect of CsA on TGF--RII expression: Treatment with 4.2µM CsA significantly increased TGF--RII expression after24 and 72 h (Figure 1C).
Figure 1. TGF- expression and TGF- receptor II (TGF--RII) expression in MDCK cells. (A) Western blot analysis of TGF- secretion after cyclosporin A (CsA) treatment. Confluent monolayers were treated with vehicle or 4.2 µM CsA for 24, 48, and 72 h. Supernatants were collected, and proteins were separated electrophoretically on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Expression of TGF- was identified using a polyclonal rabbit anti–TGF- (Pan-specific) antibody and appropriate secondary reagents. (B) TGF-1 secretion as measured by ELISA. Confluent monolayers were treated with vehicle () or 4.2 µM CsA () for 72 h. Supernatants were collected at various time points and assayed for TGF-1. *P < 0.05, **P < 0.01, and ***P < 0.001 versus time-matched control. (C) Confluent monolayers were treated with vehicle or 4.2 µM CsA for 24 and 72 h. Whole-cell lysates were then made using radioimmunoprecipitation assay (RIPA) lysis buffer. Cellular proteins were separated electrophoretically on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Expression of TGF--RII was identified using a polyclonal mouse anti–TGF--RII antibody and appropriate secondary reagents.
Effect of TGF-1 on TER
Exposure of MDCK cells to TGF-1 (5 or 10 ng/ml) significantlyincreased TER at 24 h after treatment, and this effect was maintainedup to 72 h (Figure 2A). Treatment with a TGF-1–neutralizingantibody (10, 20, or 30 µg/ml) resulted in no significantchanges of basal TER over 72 h (data not shown). However, co-treatmentof the monolayers with 4.2 µM CsA plus 30 µg/mlTGF-1 nAb significantly reduced the CsA-induced increase inTER at 24 h, and this effect was maintained up to 72 h (Figure 2B).Treatment with a TGF--RII nAb (10, 50, or 100 µg/ml) didnot significantly alter the basal TER (data not shown). However,co-treatment with CsA (4.2 µM) and TGF--RII nAb (100 µg/ml)resulted in significantly lower TER readings at 24, 48, and72 h compared with treatment with CsA alone (Figure 2C). Treatmentwith the TGF--RII nAb did not abolish the CsA-induced increasein TER but only partially ameliorated the CsA-induced effect.
Figure 2. Effect of TGF-1 on transepithelial electrical resistance (TER). (A) Confluent monolayers were treated, on the apical side alone, with increasing concentrations of TGF-1. (B) Confluent monolayers were pretreated for 1 h with 30 µg/ml TGF-1–neutralizing antibody (nAb) before treatment with 4.2 µM CsA, from the apical side. (C) Confluent monolayers were pretreated for 1 h with 100 µg/ml TGF--RII nAb before apical treatment with 4.2 µM CsA. TER was monitored over 72 h. Results are expressed as the change in TER (TER) compared with time-matched control filters and are given as means ± SEM of five independent experiments, each performed in duplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 versus time-matched control or between different treatments, when indicated.
Effect of CsA on FITC-Dextran Flux, Cell Proliferation, and Cell Viability
When confluent monolayers were exposed to CsA for periods upto 72 h from the apical side only, there was no significantchange in the rate of flux of 4000, 10,000, or 20,000 FITC-dextranacross the monolayers (Figure 3A).
Figure 3. Effect of CsA on MDCK cell monolayers. (A) Flux of FITC-dextran across intact monolayers was assessed after CsA treatment. After treatment, medium on the apical side was replaced with 20 µg/ml 4000, 10,000, or 20,000 FITC-dextran, and the fluorescence levels of the basolateral medium were read after 90 min. Results are expressed as FITC flux (ng/cm2 per h) compared with time-matched control filters and are given as means ± SEM of three independent experiments performed in triplicate. (B) Cellular proliferation was assessed using the CyQUANT cell proliferation assay. (C) Cellular viability was assessed using the AlamarBlue assay. Results are expressed as percentage of control (100%) and are given as means ± SEM of three independent experiments, each performed in triplicate. 95% confidence intervals were constructed, and a t test was performed to assess statistical significance.
MDCK cells were counted using the CyQUANT cell proliferationassay after treatment with 4.2 µM CsA (Figure 3B). Therewas a small but significant decrease in cell proliferation inCsA-treated cells at all time points: 24, 48, and 72 h. We alsonoted that TGF-1 (5 and 10 ng/ml) significantly reduced MDCKcell proliferation at 48 and 72 h (data not shown).
The viability of MDCK cells was analyzed after treatment for24, 48, and 72 h with 4.2 µM CsA (Figure 3C). CsA hadno significant effect on the cellular viability at any timepoints. Cell viability was significantly reduced at 72 h aftertreatment with TGF-1 (5 and 10 ng/ml; data not shown).
Effect of CsA on ERK1/2 Activation
Treatment of MDCK cells with CsA (4.2 µM), for periodsup to 72 h, resulted in significant activation of ERK1/2 signalingas demonstrated by Western blotting using activated ERK-specificantibody (Figure 4). Significant activation of ERK1/2 was observedat 3, 24, and 72 h of exposure (Figure 4).
Figure 4. Western blot analysis of phosphorylated extracellular signal–regulated kinase 1/2 (ERK 1/2) expression in MDCK II cells in the presence or absence of CsA. MDCK II cells were grown to confluence on Falcon six-well plates and treated as described for up to 72 h. Cells were treated with 4.2 µM CsA. Whole-cell lysates were then made, using RIPA lysis buffer, at 30 min (A), 3 h (B), 24 h (C), and 72 h (D) after treatment. Cellular proteins were separated electrophoretically on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Expression of phospho-ERK1/2 was identified using a polyclonal rabbit anti–phospho-ERK1/2 antibody and an anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody. Representative blots from one of three separate experiments are shown. Lane 1, control; lane 2, 4.2 µM CsA.
TGF-1 Activates ERK1/2
Both CsA (4.2 µM) and TGF-1 (5 ng/ml) activated ERK1/2(Figure 5). The MEK inhibitor U0126 reduced basal activationof ERK1/2 and also prevented the CsA-induced and the TGF-1–inducedactivation of ERK1/2 (Figure 5). No effects of ERK1/2 activationwere seen after treatment with a control IgG antibody for aperiod up to 72 h (data not shown).
Figure 5. Western blot analysis of phospho-ERK1/2 expression after treatment with CsA or TGF-1. Monolayers were pretreated for 1 h with U0126 (10 µM) or TGF-1 nAb (30 µg/ml) before treatment with 4.2 µM CsA or 5 ng/ml TGF-1 for periods up to 24 h. Whole-cell lysates were made using RIPA lysis buffer, and proteins were separated electrophoretically on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Expression of phospho-ERK1/2 was identified using a polyclonal rabbit anti–phospho-ERK1/2 antibody and appropriate secondary reagents. Lane 1, control; lane 2, 4.2 µM CsA; lane 3, 5 ng/ml TGF-1; lane 4, 10 µM U0126; lane 5, 10 µM U0126 plus 4.2 µM CsA; lane 6, 10 µM U0126 plus 5 ng/ml TGF-1.
Effect of the MEK Inhibitor U0126 and the p38 Inhibitor SB203580 on TER
U0126 (10 µM) induced a significant decrease in TER. Co-treatmentof the MDCK cells with 5 ng/ml TGF-1 plus 10 µM U0126significantly reduced the TGF-1–induced increase in TERthat was observed when cells were treated with TGF-1 alone (Figure 6A).
Figure 6. Role of ERK1/2 and p38 mitogen-activated protein kinase (MAPK) pathways in TGF-1–induced modulation of TER. (A) Confluent monolayers were pretreated for 1 h with 10 µM U0126 (MEK1/2 inhibitor) before apical treatment with 5 ng/ml TGF-1. (B) Confluent monolayers were pretreated for 1 h with 10 µM SB203580 (p38 MAPK inhibitor) before apical treatment with 5 ng/ml TGF-1. TER was monitored over 72 h, and results are expressed as TER compared with time-matched control filters, given as means ± SEM of five independent experiments, each performed in duplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 versus time-matched control or between different treatments, when indicated.
The p38 inhibitor SB203580 (10 µM) did not significantlyalter basal TER. However, co-treatment with 5 ng/ml TGF-1 and10 µM SB203580 significantly reduced the TGF-1–inducedincrease in TER at 48 and 72 h (Figure 6B).
Effect of CsA on the Expression of Tight Junction–Associated Proteins
The effect of CsA (4.2 µM) on expression of various tightjunction–associated proteins is shown in Figures 7 and8. Western blot analysis showed increased occludin expressionafter 24 and 72 h of treatment (Figure 8). Expression of JAM-Asignificantly increased after 72 h of treatment, whereas therewas no significant change in the level of ASIP/PAR3 after treatmentfor 72 h (Figure 7). Expression levels of claudin-1 (Figure 8)and claudin-3 significantly increased after treatment with CsAfor 24 h (Figure 7). However, there was no detectable changein expression of claudin-2, claudin-4, or claudin-16 after 24h of CsA treatment. ZO-1 expression levels did not change after24 h of CsA treatment; however, levels of ZO-2 were significantlyincreased after 72 h (Figure 7).
Figure 7. Expression of tight junction proteins after CsA treatment. Confluent monolayers were treated with CsA (4.2 µM) for periods of 24 or 72 h. Whole-cell lysates were made using RIPA lysis buffer. Cellular proteins were separated electrophoretically on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Expression of tight junction proteins was identified using specific antibodies. A representative blot from one of three separate experiments is shown.
Figure 8. (A) Effect of CsA on occludin, claudin-1, and claudin-2 expression in whole-cell MDCK II cellular lysates at 24 h. Cells were grown on Falcon petri dishes. When confluent, they were exposed to vehicle (EtOH) or 4.2 µM CsA for 24 h. Whole-cell lysates were then made using RIPA lysis buffer. Cellular proteins were separated electrophoretically on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Expression of the proteins was identified using a polyclonal rabbit anti–claudin-1 or -2 or occludin antibody and an anti-rabbit HRP-conjugated secondary antibody. (i) Representative blot
Effect of TGF-1 on Tight Junction Protein Expression
Having demonstrated that CsA altered expression of some butnot all tight junction–associated proteins, we decidedto examine the effect of TGF-1 on ZO-2 and claudin-1 expression(Figure 9). Treatment of MDCK cells for 72 h with 5 ng/ml TGF-1significantly increased the expression of the integral tightjunction–associated protein claudin-1 and the peripheraltight junction–associated protein ZO-2 (Figure 9).
Figure 9. Tight junction protein expression after treatment with TGF-1. Confluent monolayers were treated with 4.2 µM CsA or 5 ng/ml TGF-1 for 72 h. Whole-cell lysates were then made using RIPA lysis buffer. Cellular proteins were separated electrophoretically on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Expression of claudin-1 was identified using a polyclonal rabbit anti–claudin-1 antibody, whereas expression of zonula occludens 2 (ZO-2) was identified using a polyclonal rabbit anti–ZO-2 antibody. Band intensity was quantified using densitometric analysis (n = 3). **P < 0.01 and ***P < 0.001 versus time-matched control or between different treatments, when indicated.
Regulation of cellular barrier permeability is a vital and complexprocess that involves intracellular signaling and rearrangementof tight junction proteins. We investigated the signaling mechanismthat is triggered by CsA and results in enhanced tight junctionbarrier function. Our data suggest that the CsA-induced increasein TER may be mediated, at least in part, by an increase inTGF- production.
Cytokines, such as TGF-, are known to perturb tight junctionsin retinal endothelial cells, 31EG4 cells, and Sertoli cellsin vitro (23). Chronic kidney fibrosis has been associated withthe ability of CsA to increase production of TGF-1 (9). We havedemonstrated that CsA increased TGF- production and TGF-1 secretionby MDCK cells. Although control cells seemed to secrete relativelyhigh levels of TGF-1, we observed that treatment with CsA significantlyupregulated TGF--RII expression, and this may increase the overallavailable number of receptors for TGF-1 to bind to in CsA-treatedcells. We subsequently showed that the CsA-induced increasein TER could be partially prevented by simultaneously blockingthe bioactivity of TGF-1 or blocking the bioactivity of theTGF--RII with neutralizing antibodies. This finding indicatesthat TGF-1 plays a part in the CsA-induced increase in tightjunction barrier function.
Two parameters of tight junction permeability, TER and paracellularflux of uncharged molecules, are considered to measure similarcharacteristics of tight junctions. Although we observed anincrease in TER (decrease in paracellular permeability to smallions) upon treatment with CsA, there was no significant changein the rate of FITC-dextran flux across the monolayers. However,paracellular flux and TER are not always inversely correlated,especially in leaky epithelia (24). As cell density increases,there is an increase in intracellular space per unit area ofmonolayer. There have been several conflicting theories regardingthe relationship between tight junction function and cell proliferation.Some investigators reported that a rise in cell number couldgenerate an increase in TER (25–28). However, contrastingreports suggested that overgrowth of cells could result in enhancedtransepithelial permeability. For example, the tumor-promotingphorbol ester increased the passage of a range of solutes acrossthe epithelium with a concurrent increase in cell proliferation(26). Because CsA seemed to induce a small reduction in cellnumber with a concomitant decrease in transepithelial permeability,it is conceivable that there were fewer tight junctions, whichsubsequently restricted transepithelial electrical conductance.However, the viability assay showed no significant effects ofCsA on MDCK cells. Therefore, other explanations for the increasein TER were investigated.
Because TGF-1 has been reported to activate ERK1/2 in epithelialcells (29) and we previously demonstrated upregulation of TGF-in response to CsA in renal tubular cells (11), we investigatedthe possibility that CsA activates ERK1/2 via the TGF-1 isoformin our model system. TGF-1 activated ERK1/2 within 30 min oftreatment. Because TGF-1 secretion was activated within 3 hof exposure to CsA, this supports our hypothesis that CsA maybe altering tight junction barrier function, at least partially,via the TGF-1-ERK1/2 route. TGF-1 also significantly increasedthe TER across MDCK monolayers. The magnitude of this increasewas similar to that observed when MDCK cells were treated withCsA. The TGF-1–induced increase confirms that TGF-1 isa promoter of tight junction integrity in MDCK cells and thatCsA is potentially acting via this TGF- isoform. Like the CsA-inducedincrease in TER, the TGF-1–induced increase in TER wasattenuated when cells were co-treated with U0126. This suggeststhat both CsA and TGF-1 are mediating their effects via theERK1/2 MAPK signaling pathway.
Recent in vivo studies revealed that TGF-3 is a crucial regulatorof blood–testis barrier (BTB) dynamics in the rat (21,30).For example, when CdCl2 was administered to adult rats, thedisruption of Sertoli cell tight junction at the BTB and thesubsequent germ cell loss from the epithelium were associatedwith a transient surge in TGF-3 (21). This subsequently activatedthe p38 MAPK downstream as well as disruption and loss of occludinand actin filaments at the BTB (31). Therefore, we investigatedwhether TGF-1 was partly mediating its effects on TER via thep38 MAPK pathway. Inhibition of this pathway significantly attenuatedthe TGF-1–induced increase in TER after 48 and 72 h oftreatment. However, we observed no significant change in theCsA-induced effect when we inhibited the p38 MAPK pathway (6),which signifies that TGF-1 may also be mediating its effecton barrier function via the p38 MAPK pathway, but this particularsignaling route is not triggered by exposure to CsA.
Treatment with CsA induced significant changes in the expressionof several tight junction components. Although CsA increasedoccludin expression, it remains unclear whether occludin isactually a key player in regulating tight junctions. There aremany conflicting reports regarding the significance of occludinin the maintenance of a functioning tight junction complex,and evidence suggests that it is the claudin proteins that playa pivotal role in the regulation of tight junctions. After exposureto CsA, increased expression of claudin-1 and claudin-3 wasobserved, both of which are implicated in the maintenance oftight junction integrity (32–34) No alterations in claudin-2expression were detected after CsA treatment. Claudin-2 is associatedwith increased "leakiness" across epithelial cells. Significantly,claudin-2 has been detected in the low-resistance MDCK II cellsbut is absent from the high-resistance MDCK I cell strain, andintroduction of claudin-2 into the MDCK I cells results in adrop in resistance (35). It has been reported that activationof ERK1/2 results in a reduction in claudin-2 expression inMDCK cells, and this is accompanied by an increase in resistance(33,36). In our system claudin-2 expression was unaltered aftertreatment with CsA despite activation of ERK1/2, suggestingthat alterations in claudin-2 expression may be stimulus dependent.Exposure of MDCK cells to TGF-1 significantly increased claudin-1and ZO-2 expression, correlating with the effect of CsA on thesetight junction components, which further strengthens our hypothesisthat CsA regulates tight junction function, at least partially,via this cytokine. Another study (37) demonstrated that TGF-upregulated claudin-1 expression two- to three-fold in T84 monolayers,but expression of claudin-2 and claudin-4 was unaltered. Inaddition, we found that treatment of MDCK cells with the MEK1 inhibitor U0126 decreased claudin-1 expression compared withcontrol level (data not shown). Therefore, it is possible thatin MDCK II cells, CsA targets claudin-1 via the TGF-1–ERK1/2route.
Both CsA and TGF-1 upregulated expression of ZO-2. The ZO familyconsists of three peripheral membrane proteins that are membersof the MAGUK family. ZO-1, ZO-2, and ZO-3 are located in theunderlying tight junction plaque and link to the actin cytoskeleton.ZO-1 and ZO-2 seem to interact directly with each other, occludin,the claudins, and the actin-based cytoskeleton through theirPDZ domains. It is reported that subcellular localization ofZO-2 is sensitive to the state of cell–cell contact thatis exhibited by the epithelial monolayer. In sparse epithelialcultures, ZO-2 accumulates in clusters at the nucleus. Shuttlingof ZO-2 between the tight junction region and nucleus mightbe accomplished by the occurrence of putative nuclear localizationand exportation signals on its sequence (38). The functionalsignificance of the nuclear distribution of ZO-2 remains tobe determined.
What is the significance of establishing the CsA-induced signalingmechanism? Enhancement of tight junction barrier function asa result of treatment with CsA may be a cytoprotective responseby the cells to protect against injury. Determining how this"tightening" is triggered could be beneficial in the developmentof therapeutics for tight junction–related diseases oras a mechanism for drug delivery. Researchers are attemptingto identify active compounds or excipients that can reversiblyopen tight junctions, thereby permitting drugs to pass through.
This project was supported by the Conway Institute of Biomolecularand Biomedical Research and the Dublin Molecular Medicine Centre,under the Programme for Research in Third Level Institutionsadministered by the Higher Education Authority.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
G.F. and B.K. contributed equally to this work.
B.K.'s current affiliation is Cork Cancer Research Centre, BioSciencesInstitute and Mercy University Hospital, University CollegeCork, Cork, Ireland.
See the related editorial, "Opening Pandora's Box in the TightJunction," on pages 1624–1625.
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Abstract
Introduction
in mediating the CsA-induced activation of ERK1/2 and the resulting increase in TER in MDCK cells. Paracellular permeability across MDCK monolayers after various treatments was assessed by measurement of TER. TGF-
, IL-4, and IL-10 (12). In contrast, TGF-
·cm2) and results were expressed as the change in TER with respect to time matched controls [
TER (
0.05 was deemed statistically significant. 
) or 4.2 µM CsA (
