Abstract
ABSTRACT. Interactions between inflammatory infiltrates and resident tubular epithelial cells may play important roles in the development of tubulointerstitial fibrosis, by promoting epithelial cell-myofibroblast transdifferentiation (EMT). Human proximal tubular epithelial cells transdifferentiated to myofibroblasts after treatment with activated PBMC conditioned medium. mRNA and protein levels for α-smooth muscle actin, collagen I, and fibronectin EDA+ (markers for the myofibroblastic phenotype) were increased, whereas those for E-cadherin and cytokeratin 19 (markers for the epithelial phenotype) were decreased. cDNA microarray analysis was used to identify other changes in gene expression that might point to novel molecular mechanisms driving EMT. Of 1176 array genes, 61 demonstrated at least a twofold change at at least two consecutive time points, of the five time points examined (0.5, 4, 8, 16, and 48 h). Of these genes, 59% were upregulated and 41% were downregulated. The array indicated upregulation of expression of the oncostatin M (OSM)-specific receptor β subunit from 4 to 48 h after exposure of kidney epithelial cells to activated PBMC conditioned medium, which contained high levels of OSM. In additional experiments, it was demonstrated that OSM induced EMT. OSM activated the Jak/Stat signaling pathway in epithelial cells, and a specific inhibitor of Jak2 blocked both its phosphorylation after exposure to OSM and the induction of α-actin and loss of cytokeratin 19 expression. Therefore, OSM is a novel inducer of EMT and is likely to be one of several cytokines produced by inflammatory infiltrates that contribute to this and subsequent tubulointerstitial fibrosis. E-mail: zzhang@tech.mrc.ac.uk
Tubulointerstitial fibrosis is the principal hallmark of most types of progressive renal disease. Initial renal injury stimulates various types of kidney cells to produce inflammatory mediators. These mediators activate renal tubule epithelial cells and peritubular capillary endothelial cells and facilitate infiltration of mononuclear cells into the interstitium. Histologically, progressive renal disease is characterized by an interstitial infiltrate of mononuclear cells. The interactions between inflammatory infiltrates and resident tubular cells contribute to ongoing inflammation, formation and activation of myofibroblasts, and ultimately interstitial fibrosis. The pivotal role of inflammatory infiltration in the progression of tubulointerstitial fibrosis is supported by the strict correlation between tubular atrophy, interstitial fibrosis, and the extent of interstitial infiltration (1,2⇓).
The appearance of myofibroblasts is thought to play a key role in the progression of chronic renal fibrosis (3). These cells express the mesenchymal marker α-smooth muscle actin (αSMA) and are a major source of extracellular matrix (ECM) proteins in kidney fibrosis. αSMA expression is widely regarded as a molecular marker for these cells. Despite some debate regarding the origins of kidney myofibroblasts, emerging evidence suggests that, under pathologic conditions, myofibroblasts derived from tubular epithelial cells through epithelial cell-myofibroblast transdifferentiation (EMT) play an important role in interstitial fibrosis (4–6⇓⇓). The precise mechanisms that initiate EMT remain unclear, but it is unlikely that a single cytokine acting in isolation is responsible. Multiple cytokines activating a number of cellular receptors are likely to be involved in vivo. However, most previous in vitro studies of EMT investigated the effects of single cytokines on the induction of EMT (5–8⇓⇓⇓).
To model the effects of multiple stimulating cytokines on EMT in vitro, Healy et al. (9) stimulated human tubular epithelial cells with activated PBMC conditioned medium (aPBMC-CM). This medium provides a physiologically relevant mixture of cytokines, for assessment of the effects of an inflammatory infiltrate on tubular epithelial cells. Anticipating that this stimulation would trigger complex changes in gene expression, we used cDNA microarray technology for dynamic global assessment of such changes during EMT. This enabled us to identify a pathway not previously implicated in EMT. In this report, we demonstrate that oncostatin M (OSM)-specific receptor β subunit (OSMRβ) expression is upregulated by aPBMC-CM and that OSM can induce EMT of human kidney cells via the Jak/Stat1/3 signaling pathway.
Materials and Methods
Materials
Mouse monoclonal anti-human E-cadherin antibody was purchased from BD Bioscience (Oxford, UK), anti-human αSMA antibody from Chemicon International (Temecula, CA), and anti-human fibronectin EDA+ antibody and anti-human cytokeratin 19 antibody from Abcam (Cambridge, UK). Rabbit polyclonal anti-human Stat1 and Stat3 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and rabbit anti-human phospho-Stat1 and phopspho-Stat3 antibodies were obtained from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase-conjugated rabbit anti-mouse Ig and peroxidase-conjugated goat anti-rabbit Ig were obtained from Dako (Cambridgeshire, UK). mAb used to neutralize human TGF-β1, TNF-α, IFN-γ, EGF, GM-CSF, and OSM were obtained from R&D Systems (Minneapolis, MN). Jak3 inhibitor [4-(4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline], genistein, AG490, and H-7 were obtained from Calbiochem (CN Biosciences, Nottingham, UK).
Cell Culture and Treatment
PBMC were prepared from acid-citrate-glucose-treated buffy coats (North London Blood Transfusion Centre, London, UK) (10). The PBMC pellet was resuspended in RPMI 1640 medium (Invitrogen, Paisley, UK) supplemented with 10% (vol/vol) FCS, 290 μg/ml glutamine, 100 U/ml penicillin, and 50 μg/ml streptomycin. Cells (1 × 107) were activated by culture for 5 d in serum-free medium with the supplements described above, with or without concanavalin A (5 μg/ml; Sigma, Dorset, UK). The culture was then centrifuged (1000 × g for 10 min), and the conditioned medium was collected, sterilely filtered, divided into aliquots, and stored at −70°C until used.
Primary human proximal tubular epithelial cells (PTEC) were obtained from BioWhittaker Inc. (Walkersville, MD). Cells were cultured in renal epithelial cell growth medium (REGM) (BioWhittaker) supplemented with 10 μg/ml human recombinant EGF, 5.0 mg/ml insulin, 0.5 mg/ml hydrocortisone, 0.5% FCS, 0.5 mg/ml epinephrine, 6.5 μg/ml triiodothyronine, 10 mg/ml transferrin, 10 mg/ml gentamicin, and 50 μg/ml amphotericin-B. HK2 cells, a human kidney PTEC line, were a gift from Dr. D. Newman (SW Thames Renal Institute, London, UK) and were cultured under the same conditions as PTEC.
Epithelial cell cultures at approximately 70% confluence were treated with various concentrations (5 to 30%) of aPBMC-CM in REGM. aPBMC-CM obtained in the absence of concanavalin A stimulation and RPMI 1640 medium alone were used as control samples, but neither had any effect on EMT. Therefore, RPMI 1640 medium alone was used as a control sample in later experiments. After treatment for 0.5, 4, 8, 16, or 48 h, the medium was collected and the cells were processed for RNA isolation or SDS-PAGE and Western blotting analyses.
For investigation of the effects of recombinant human OSM (R&D Systems), epithelial cells were cultured as described above, with additional time points (5, 10, 15, 30, 60, 120, and 240 min). Cells were then harvested in either mammalian protein extraction reagent (Pierce, Rockford, IL), for cultures treated for 0.5 to 48 h, or RIPA buffer supplemented with complete protease inhibitor cocktail (Roche Diagnostic, East Sussex, UK), 1 mM activated sodium vanadate, and 1 mM sodium fluoride, for cultures treated for 5 to 240 min.
Human Cytokine Measurements
Quantikine ELISA kits (R&D Systems) were used to measure the levels of activated TGF-β1, EGF, GM-CSF, IFN-γ, TNF-α, IL-1β, IL-2, IL-6, and OSM in the aPBMC-CM, according to the manufacturer’s protocols.
RNA Isolation, cDNA Preparation, Array Hybridization, and Analysis
Paired RNA samples were prepared for each experiment, to minimize experimental variation. Total RNA was extracted from cells with Trizol reagent (Invitrogen), the RNA yield was evaluated with a GeneQuant II kit (Amersham, Buckinghamshire, UK), and the quality was assessed by electrophoresis through a 1% agarose/formaldehyde gel. Poly(A)+ RNA was purified from 30 μg of total RNA with two rounds of oligo(dT) magnetic bead chromatography, according to the instructions for the Atlas Pure total RNA isolation kit (Clontech, Hampshire, UK). One microgram of poly(A)+ RNA was used to prepare 32P-labeled cDNA, as specified in the Atlas expression array user manual (Clontech). A total of 1.2 × 106 dpm of each cDNA was hybridized with Atlas human cancer cDNA expression array 1.2 membranes (Clontech), according to the protocols. After hybridization, membranes were washed as specified in the instruction manual and then exposed to a Storm 860 PhosphorImager (Amersham). All hybridization experiments were repeated, and only gene expression changes observed in both arrays were analyzed further.
The phosphorimage of each membrane was analyzed with AtlasImage software (Clontech). The template elements were aligned over the true array spot, and the spot intensity value was quantified after subtraction of background levels. The adjusted intensities for all gene spots were obtained by normalization of the hybridization signals, with housekeeping genes as the references. A change in gene expression was considered significant only if the ratio between treated and control samples was >2.0 or <0.5 at two consecutive time points, of the total of five time points tested.
For further analysis, the data were imported into the GeneSpring program (Silicon Genetics, Redwood City, CA) as an Excel (Microsoft, Seattle, WA) spreadsheet, formatted as a tab-delimited text file, and ratio measures of aPBMC-CM-treated versus control samples were generated. A hierarchical cluster analysis with a standard correlation of 0.95 and a distance of 0.01 (dendrogram) for the expression measure was performed with GeneSpring.
SDS-PAGE and Western Blotting
For protein analysis, cells were washed twice with ice-cold PBS and lysed in ice-cold mammalian protein extraction reagent or RIPA buffer supplemented with inhibitors. After 30 min on ice, the lysate was centrifuged at 14,000 × g at 4°C for 15 min. Total protein levels in the supernatant were measured with a bicinchoninic acid protein assay kit (Pierce).
Samples (10 to 20 μg) were subjected to 10% SDS-PAGE, transferred to a Hybond ECL membrane (Amersham), and probed with primary antibodies in wash buffer, followed (after washing) by appropriate peroxidase-conjugated secondary antibodies in wash buffer (11). After further extensive washing, the immunoblots were observed with ECL assays (Amersham); band densities were quantified with QuantityOne software (Bio-Rad, Hercules, CA), after scanning with a GS-710 calibrated imaging densitometer (Bio-Rad).
Reverse Transcription-PCR
A one-step reverse transcription (RT)-PCR was performed with the Titanium One-Step RT-PCR kit (BD Biosciences), with the primers presented in Table 1. The target transcript was reverse-transcribed at 50°C for 1 h. The PCR products for B4-2, VIM, CDH6, GBP2, ERF1, and GAPDH were amplified with 20 PCR cycles, whereas the other genes of interest (SOCS3, NGAL, PAI1, and OSMRβ) were amplified with 35 cycles (initial denaturation at 94°C for 5 min, followed by PCR amplification at 94°C for 30 s, 65°C for 30 s, and 68°C for 1 min). PCR products and molecular weight markers were separated on 2 or 4% E-gels (Invitrogen), after which bands were observed with GelDoc 1000 software (Bio-Rad) and quantified with a scanning densitometer.
Table 1. Sequences of PCR primers used in this study
Fibronectin and Collagen Type I Measurements
Culture media were analyzed for fibronectin and collagen type I with sandwich ELISA, with purified collagen type I (Chemicon) and fibronectin (Chemicon) as standards (12). After incubation with biotinylated primary antibodies for 1 h at 37°C, plates were washed and reacted with alkaline phosphatase-conjugated anti-Extravidin (Sigma), followed by p-nitrophenyl phosphate substrate tablets (Sigma), according to the instructions provided by the manufacturer. The product was detected at 405 nm with an ELISA plate reader (Anthos, Austria).
Statistical Analyses
Data are expressed as mean ± 2 SD, unless otherwise stated. Comparisons between control and aPBMC-CM-treated cultures were performed with t tests for paired data. Values of P < 0.05 were considered significant.
Results
Growth Factor and Cytokine Composition of aPBMC-CM
Table 2 lists the concentrations, determined with Quantikine ELISA kits, of TGF-β1, EGF, IL-1β, IL-2, IL-6, OSM, GM-CSF, TNF-α, and IFN-γ in aPBMC-CM. The total activated TGF-β1 level was approximately 0.5 ng/ml, but the levels of OSM, TNF-α, IFN-γ, and GM-CSF were 2.0, 5.7, 22, and 32 ng/ml, respectively. The levels of growth factors and cytokines produced by PBMC in response to concanavalin A are generally comparable to, or greater than, those produced in response to other cell activators (phytohemagglutinin, PMA, and LPS), as indicated in the data sheets published by R&D Systems (Table 2).
Table 2. Concentrations of active cytokines present in concanavalin A (5 μg/ml)-activated PBMC conditioned medium
Induction of Tubular Epithelial Cells to EMT by aPBMC-CM
Renal tubular epithelial cells undergo transdifferentiation to myofibroblasts in response to aPBMC-CM (9). To establish a firm basis for the investigation of novel changes in gene expression during EMT, we first determined the optimal concentration and time required for our aPBMC-CM to induce this change. Expression of the phenotypic markers E-cadherin and cytokeratin 19 for epithelial cells and αSMA and the EDA+ splice form of fibronectin (fibronectin EDA+) for myofibroblasts was assessed in PTEC treated with various concentrations (5 to 30%) of aPBMC-CM for 48 h (Figure 1, A and B) or with 30% aPBMC-CM for different times (0.5 to 48 h) (Figure 1C). Changes in cell morphologic features were also assessed (Figure 2, A and B). Although 15% aPBMC-CM induced the expression of both αSMA and fibronectin EDA+, a significant reduction in E-cadherin and cytokeratin 19 expression occurred only with higher concentrations (25 to 30%) (Figure 1, A and B). aPBMC-CM (30%) significantly induced both αSMA and fibronectin EDA+ expression after 8 h of incubation, but an almost total loss of E-cadherin expression required 48 h of treatment (Figure 1C). PTEC cultured in REGM alone maintained classic cobblestone epithelial morphologic features and growth patterns (Figure 2A), but cells stimulated with 30% aPBMC-CM for 48 h adopted fibroblastic morphologic features (Figure 2B). Treatment of PTEC with 30% aPBMC-CM for 48 h significantly increased the levels of collagen type I (3.8-fold) (Figure 2C) and fibronectin (9-fold) (Figure 2D) in the culture medium, compared with control values. aPBMC-CM also caused HK2 cells to undergo EMT in a manner identical to that of PTEC (data not shown), as previously reported by Healy et al. (9). Therefore, in some parts of this study, HK2 cells were used in place of PTEC. Collectively, these data indicate that our 30% aPBMC-CM induces EMT in a period of 48 h, in agreement with data reported by Healy et al. (9).
Figure 1. Activated PBMC conditioned medium (aPBMC-CM) induction of concentration- and time-dependent changes in cellular markers expressed by proximal tubular epithelial cells (PTEC). (A) Cells were treated for 48 h with different doses of aPBMC-CM, and both epithelial cell (E-cadherin and cytokeratin 19) and myofibroblast [α-smooth muscle actin (αSMA) and fibronectin (Fn) EDA+] markers were assessed with SDS-PAGE and Western blotting. Twenty micrograms of total protein were loaded in each lane. Lane 1, control; lane 2, 5% aPBMC-CM; lane 3, 10% aPBMC-CM; lane 4, 15% aPBMC-CM; lane 5, 20% aPBMC-CM; lane 6, 25% aPBMC-CM; lane 7, 30% aPBMC-CM. (B) Densitometric analyses demonstrated the changes in band intensities for each marker. Data are presented as the mean ± 2 SD of three experiments. (C) Cells were treated with 30% aPBMC-CM for different times (0.5 to 48 h), and both epithelial cell (E-cadherin and cytokeratin 19) and myofibroblast (αSMA and fibronectin EDA+) markers were determined as A. The band intensities were quantified in densitometric analyses. Data are presented as the mean ± 2 SD of three experiments.
Figure 2. aPBMC-CM induction of changes in morphologic features and synthesis of matrix proteins by cultured PTEC. (A and B) Histologic analyses. Cells cultured in renal epithelial cell growth medium (REGM) with 30% RPMI 1640 medium maintained classic epithelial morphologic features (A), whereas cells cultured for 48 h with 30% aPBMC-CM exhibited significant alterations in cellular morphologic features (B). Magnification, ×100. (C and D) Quantification of protein levels. Cells were stimulated with 30% aPBMC-CM, in the presence of 5 μg/ml ascorbic acid, for different time periods (0.5 to 48 h). Collagen type I (C1) (C) and fibronectin (Fn) (D) protein levels in aPBMC-CM were measured with sandwich ELISA. ⧫, protein levels in control samples; ▪, protein levels in aPBMC-CM-treated samples. Data represent the mean ± 3 SEM of three experiments, each performed in duplicate.
Specific antibodies were used to neutralize TGF-β1, TNF-α, IFN-γ, EGF, GM-CSF, and OSM in aPBMC-CM, before exposure of HK2 cells to the medium for 4 d. The effects of antibody neutralization of individual cytokines and growth factors on E-cadherin expression and ECM protein production were analyzed. None of the antibodies provided significant protection against the changes initiated by aPBMC-CM, as indicated by changes in E-cadherin expression and fibronectin production (Figure 3; data for IFN-γ and GM-CSF not shown). These results suggest that aPBMC-CM-induced changes in epithelial cells are initiated by two or more cytokines.
Figure 3. Failure of neutralizing antibodies to individual cytokines to restore the aPBMC-CM-induced decrease in E-cadherin expression (A) and increase in fibronectin (Fn) production (B) to control levels. aPBMC-CM (30%) was incubated with each neutralizing antibody [5 and 25 μg/ml anti-TGF-β1 mAb, 1 μg/ml anti-TNF-α, 1 μg/ml anti-EGF, and 1 μg/ml anti-oncostatin M (OSM)] for 30 min at 37°C and then added to HK2 cells for an additional 4 d. Media were collected for fibronectin measurements with ELISA. Cell lysate E-cadherin levels were assessed with SDS-PAGE and Western blotting. Results are plotted as percentage changes, compared with control values. Con, control; PBMC, aPBMC-CM-treated samples; 5TGF and 25TGF, media supplemented with 5 and 25 μg/ml anti-TGF-β1 mAb, respectively; TNF, EGF, and OSM, media supplemented with anti-TNF-α, anti-EGF, and anti-OSM antibody, respectively.
cDNA Microarray Analysis of Changes in Gene Expression during EMT
To elucidate dynamic changes in gene expression during EMT, we performed a series of cDNA microarray analyses at different times (0.5, 4, 8, 16, and 48 h) after stimulation of PTEC with 30% aPBMC-CM. A total of 61 of the 1176 genes included in the Atlas human cancer cDNA expression array (see http://atlasinfo.clontech.com/atlasinfo for complete list) were ranked as upregulated or downregulated, as defined by an at least twofold change in expression levels at at least two consecutive time points, of the five tested. Changes in the expression levels of a number of these genes were validated with RT-PCR (Figure 4). The RT-PCR data were in good agreement with the cDNA microarray data.
Figure 4. Time course of mRNA expression of 10 selected genes during epithelial cell-myofibroblast transdifferentiation (EMT). mRNA was isolated at the indicated times after 30% aPBMC-CM treatment, and the expression of mRNA for B4 protein (B4-2), TIS11B protein (ERF1), IFN-induced guanylate-binding protein 2 (GBP2), neutrophil gelatinase-associated lipocalin precursor (NGAL), plasminogen activator inhibitor-1 (PAI1), Stat-induced Stat inhibitor 3 (SOCS3), OSM-specific receptor β subunit (OSMRβ), vimentin (VIM), kidney cadherin (CDH6), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were analyzed with reverse transcription-PCR. The number of PCR cycles was optimized for each gene, so that no products were saturated (see Materials and Methods). Ethidium bromide-stained PCR products were separated on 2 or 4% E-gels.
The 61 EMT-related genes encode predominately transcription factors, signaling molecules, metabolic enzymes, receptors, and ECM proteins. They were profiled on the basis of the similarity of their temporal expression patterns, with hierarchical cluster analysis (GeneSpring). Figure 5 presents the clustering algorithm, in which each color block represents the expression value of a particular gene at one of the five time points during EMT. Genes with similar patterns of expression in response to aPBMC-CM stimulation are grouped together by means of common branches in the dendrogram, with shorter branches representing closer relationships between genes. Similarities in temporal expression patterns for particular EMT-related genes are thus revealed.
Figure 5. Cluster analysis of gene expression profiles for human epithelial cells in the presence or absence of 30% aPBMC-CM, at five different time points. A total of 61 of 1176 genes were regarded as significantly upregulated or downregulated (i.e., at least a twofold change in the ratio of expression at a given time after aPBMC-CM treatment to control expression) at at least two consecutive time points of the five tested, as described in the text. For each gene, the ratio of expression is represented by a color, according to the color scale at the bottom. The 61 genes plotted vertically, with the five time points plotted horizontally, indicate the statistical relatedness of genes in the cluster, with shorter branches representing closer relationships between genes. Definitions of gene abbreviations can be found at http://www.atlasinfo.clontech.com/atlasinfo.
OSM Induction of EMT
Array analysis indicated changes in the expression of a number of genes that may play important roles in EMT. Among these, OSMRβ expression was induced 4 h after stimulation with aPBMC-CM and remained elevated for the next 44 h (Figure 5). Because the concentration of OSM in aPBMC-CM is high (Table 2) and OSM has been reported to stimulate fibroblast production of collagen (13), we investigated its possible role in EMT. HK2 cells were treated with different doses of OSM (0.2, 2, 10, or 25 ng/ml) for 48 h (Figure 6, C and D) or with 25 ng/ml OSM for different times (0.5 to 48 h) (Figure 6E), after which the cellular levels of αSMA and E-cadherin were assessed. Exogenous OSM stimulated αSMA expression by HK2 cells in a dose-dependent manner and simultaneously induced loss of E-cadherin expression (Figure 6, C and D). With 25 ng/ml OSM, αSMA expression was induced as early as 30 min but a decrease in E-cadherin expression was observed only after 4 h, with total loss by 48 h (Figure 6E). Consistent with these changes, the OSM-treated cells lost their epithelial morphologic features and displayed elongated, spindle-shaped, fibroblast-like, morphologic features (Figure 6, A and B). In addition, HK2 cells treated with 25 ng/ml OSM for 48 h increased fibronectin synthesis fourfold (data not shown), consistent with the hypothesis that the cytokine induces EMT.
Figure 6. OSM induction of HK2 cells to undergo EMT. (A) Cells cultured in REGM alone maintained classic epithelial morphologic features. Magnification, ×100. (B) OSM induced morphologic changes in HK2 cells cultured for 48 h with 25 ng/ml OSM. Magnification, ×100. (C to E) OSM caused concentration- and time-dependent changes in cellular markers expressed by HK2 cells. (C) HK2 cells were treated for 48 h with different doses of OSM (0.2 to 25 ng/ml), and E-cadherin and αSMA levels were determined with SDS-PAGE and Western blotting. Lane 1, control (0 ng/ml OSM); lane 2, 30% aPBMC-CM; lane 3, 25 ng/ml OSM; lane 4, 10 ng/ml OSM; lane 5, 2 ng/ml OSM; lane 6, 0.2 ng/ml OSM. (D) The band intensities were determined densitometrically. □, E-cadherin; ▪, αSMA. Data are represented as mean ± 1 SD of two experiments. (E) Cells were treated with 25 ng/ml OSM for different times (0.5 to 48 h), and E-cadherin and αSMA levels were determined as in C. The band intensities were determined densitometrically. Data are represented as mean ± 1 SD of two experiments.
OSM signaling involves the Jak/Stat pathway (14). After addition of OSM (25 ng/ml) to HK2 cells, both Stat1 and Stat3705 were phosphorylated as early as 10 min, with a peak at 15 min (Figure 7A). Stat1 phosphorylation declined after 30 min of OSM treatment, whereas Stat3705 phosphorylation decreased after 1 h. The total Stat1 and Stat3 protein levels remained constant throughout the experiment (data not shown). Stat1 and Stat3705 phosphorylation levels were dependent on the OSM concentration (Figure 7B).
Figure 7. OSM induction of time- and concentration-dependent Stat phosphorylation in HK2 cells. (A) Cells were treated with OSM (25 ng/ml) for various times, and phosphorylated Stat1 and Stat3 levels were determined with SDS-PAGE and Western blotting with specific antibodies. (B) HK2 cells were treated with a range of concentrations of OSM (0.2 to 25 ng/ml) for 15 min, and phosphorylated Stat1 (▪) and Stat3705 (□) levels were determined with SDS-PAGE and Western blotting. The band intensities were determined densitometrically. Data are represented as mean ± 1 SD of two experiments.
Genistein, a general inhibitor of tyrosine phosphorylation, completely abolished OSM-induced Stat1 and Stat3705 phosphorylation (Figure 8A). A specific inhibitor of Jak2, AG490, had the same inhibitory effect on OSM-induced phosphorylation of Stat1 and Stat3705 (Figure 8A). However, only a small inhibitory effect on OSM-induced Stat1 phosphorylation was observed with the addition of a Jak3 inhibitor to HK2 cells (Figure 8A). The serine/threonine phosphorylation inhibitor H-7 had no effect on Stat phosphorylation. Similar inhibitory profiles were observed when 30% aPBMC-CM, instead of OSM, was used to stimulate HK2 cells (Figure 8B). Moreover, when HK2 cells were treated with 30% aPBMC-CM for 48 h in the presence of these inhibitors, genistein (Figure 8C, left, lane 3) and AG490 (Figure 8C, left, lane 5) prevented aPBMC-CM-induced changes in cytokeratin 19 and αSMA levels (Figure 8C, left, lane 2). Similar inhibitory data were obtained when OSM (25 ng/ml), instead of aPBMC-CM, was used to induce EMT (Figure 8C, right). Collectively, these data suggest that the EMT that occurs in response to aPBMC-CM is dependent on signaling through the Jak/Stat pathway and that one factor in the aPBMC-CM that initiates EMT is OSM.
Figure 8. Inhibition of Stat phosphorylation and EMT. (A and B) HK2 cells were treated with 100 μM genistein, 100 μM H-7, 100 μM AG490, or 100 μM Jak3 inhibitor for 4 h before stimulation with either 25 ng/ml OSM (A) or 30% aPBMC-CM (B) for 15 min. Total Stat1 and Stat3 and phosphorylated Stat1 and Stat3 levels were determined with SDS-PAGE and Western blotting with specific anti-Stat1, anti-Stat3, anti-phospho-Stat1, and anti-phospho-Stat3 antibodies, respectively. Band intensities were determined densitometrically (A and B, lower). □, phosphorylated Stat1; ▪, phosphorylated Stat3. Lane 1, untreated HK2 cells; lane 2, OSM (A)- or aPBMC-CM (B)-treated HK2 cells; lane 3, 100 μM genistein with OSM or aPBMC-CM; lane 4, 100 μM H-7 with OSM or aPBMC-CM; lane 5, 100 μM AG490 with OSM or aPBMC-CM; lane 6, 100 μM Jak3 inhibitor with OSM or aPBMC-CM. Data are represented as mean ± SEM of three experiments. (C) For detection of changes in cellular markers, HK2 cells were stimulated for 48 h with aPBMC-CM (left) or OSM (right) and the same inhibitors as described above, after which they were analyzed for α-SMA and cytokeratin 19 with SDS-PAGE and Western blotting. Left: lane 1, untreated HK2 cells; lane 2, 30% aPBMC-CM-treated HK2 cells; lane 3, 100 μM genistein with aPBMC-CM; lane 4, 100 μM H-7 with aPBMC-CM; lane 5, 100 μM AG490 with aPBMC-CM; lane 6, 100 μM Jak3 inhibitor with aPBMC-CM; right: lane 1, untreated HK2 cells; lane 2, 25 ng/ml OSM-treated HK2 cells; lane 3, 100 μM AG490 with OSM. Equal amounts of total protein were loaded in each lane.
Discussion
Transdifferentiation is a switch from one type of differentiated cell to another type of differentiated cell (15). Analyses of kidney biopsies from patients with a variety of renal diseases indicate that tubular epithelial cells transdifferentiate to myofibroblastic cells in vivo and that the number of such myofibroblastic cells is associated with the degree of interstitial damage (16). Myofibroblasts are probably the main source of the ECM proteins that are laid down in interstitial fibrosis (3). Cytokines produced by infiltrating inflammatory cells in renal diseases may play important roles in the development of tubulointerstitial fibrosis, by inducing EMT (9). For example, IL-1β, IL-4, IL-10, and IFN-γ expression were observed to be significantly elevated in the PBMC of patients with IgA nephropathy or non-IgA mesangial proliferative glomerulonephritis (17). In this in vitro study, activated PBMC produced a variety of growth factors and cytokines, including TGF-β1, EGF, IL-1β, IL-2, IL-6, OSM, GM-CSF, TNF-α, and IFN-γ (Table 2). Both TGF-β (7) and IL-1β (6,8⇓), acting through a TGF-β-dependent mechanism (8), induce EMT in vitro. However, the final concentration of TGF-β1 in our experiments investigating EMT induced by aPBMC-CM was only 0.15 ng/ml. Yang and Liu (5) reported that 0.1 ng/ml TGF-β stimulated αSMA expression in a human epithelial cell line (HKC-8) but 4 ng/ml was required to provoke a change in cell morphologic features. Our PTEC clearly underwent a morphologic change in response to treatment with aPBMC-CM, with a much lower final concentration of TGF-β (Figure 2). Moreover, immunoneutralization of TGF-β in the aPBMC-CM reduced the loss of E-cadherin expression in treated cells by only approximately 20%. Therefore, it seemed likely that aPBMC-CM contained EMT-inducing factors in addition to TGF-β1.
Because the response of tubular cells to the multiple cytokines and growth factors in aPBMC-CM and the process of EMT itself are likely to involve changes in the expression of many different genes, we used cDNA microarray analysis to assess the changes. The Clontech human cancer array contains 1167 genes, representing approximately 4% of the human genome (18,19⇓); however, the list includes a wide range of genes involved in cancer cell transformation, a process that might have some features in common with EMT. Therefore, the array is an appropriate tool for the investigation of changes in gene expression during EMT. With examination of a series of time points during the treatment of PTEC with aPBMC-CM, a total of 61 genes were observed to demonstrate altered expression during EMT. Of these, 59% were upregulated and 41% were downregulated. The role, if any, of most of these genes in EMT is currently unknown and requires further investigation. However, among the 61 genes detected, the expression of IL-6, IL-15 receptor, IFN-γ-induced protein, and OSMRβ were elevated at almost all time points tested. Furthermore, expression of the IFN-γ receptor and its accessory factors was increased after 4 h of aPBMC-CM treatment and expression of the EGF receptor was enhanced after 8 h. The elevated expression of cytokine receptors in response to aPBMC-CM suggests that these cytokines might be involved in the process of EMT.
Cellular phenotype is determined by the specific proteins expressed by the cell and the nature of its environment. Many experimental models have demonstrated that membrane-associated adherens junctions and desmosomes are dissociated during EMT; at the same time or shortly thereafter, cytoskeletal rearrangement takes place (20,21⇓). In our study, several genes known to participate in cell-cell and cell-matrix adhesion were observed to be differentially expressed during EMT. The expression of kidney cadherin was suppressed at 16 h (Figure 5). Cadherins are major cell-cell adhesion proteins, the cytoplasmic domains of which bind to catenin proteins. Strong intercellular adhesion depends on linkage of the cadherin/catenin complex to the actin cytoskeleton via α-catenin, and loss of α-catenin affects cell-cell adhesion and promotes tumorigenicity (22). mRNA for intermediate filament genes was concurrently upregulated, which would facilitate the cell adopting a mesenchymal phenotype. It was evident that expression of vimentin was elevated 48 h after aPBMC-CM treatment (Figure 5). The mRNA levels of ECM components such as fibronectin were also elevated at 48 h. These findings support the occurrence of EMT, with myofibroblasts being the main source of the increased ECM deposition observed in renal fibrosis. The findings of increased mRNA levels for ECM proteins were supported by data demonstrating increased ECM protein secretion, as determined by ELISA (Figure 2, C and D). These data indicated that the excessive production of ECM proteins by myofibroblasts is a relatively late-stage event. Clearly, the dynamic changes in gene expression observed in this in vitro study support the concept that the transition of tubular epithelial cells to myofibroblasts is a multistep process, involving the weakening of epithelial connections and the acquisition of myofibroblastic characteristics, which probably closely models the in vivo situation (5).
OSM is a pleiotropic cytokine that belongs to the IL-6 family of cytokines (23), which also includes leukemia inhibitory factor. OSM binds to gp130 and either leukemia inhibitory factor receptor-α or OSMRβ, to form OSM signaling receptor complex I or II, respectively (24,25⇓). Receptor binding activates the Jak/Stat signaling pathway, which leads to gene transcription and negative-feedback regulation of the receptor (26). Downregulation occurs through receptor degradation, followed by upregulation through increased synthesis. The increased expression of OSMRβ in PTEC after exposure to aPBMC-CM presumably reflects this second phase of receptor regulation, after the initial interactions of the cells with the high levels of OSM present in aPBMC-CM.
The possible role of OSM in wound healing and fibrosis has been the subject of previous speculation, because OSM upregulates tissue inhibitor of metalloproteinase-1 and −3, plasminogen activator inhibitor-1, ECM components, and p21, as well as enhancing the growth of fibroblasts (27). Our data demonstrate for the first time that OSM stimulates EMT in a dose- and time-dependent manner (Figure 6). Interstitial infiltration by monocytes and macrophages is routinely present in kidneys with progressive renal diseases, and monocytes and macrophages are known to be sources of OSM production. Therefore, we speculate that OSM may contribute to renal fibrosis in vivo by promoting EMT. However, it is likely that OSM acts together with other mediators, such as TGF-β, both in vivo and when epithelial cells are exposed to aPBMC-CM in vitro.
Having established a role for OSM in EMT in vitro, we tested whether this role is dependent on intracellular signaling via the Jak/Stat pathway. Oligomerization of OSM-receptor complexes occurs with ligand binding and activates the tyrosine kinases Jak1, Jak2, and Tyk2, which in turn phosphorylate tyrosine residues in the cytoplasmic domain of the receptor. These phosphorylations create docking sites for Stat proteins, as well as linker proteins (26). The Stat proteins then undergo phosphorylation, dimerization, and translocation to the nucleus, where they regulate target genes. However, the linker proteins can propagate signals to other pathways, such as those involving ERK1/2, JNK, and phosphatidylinositol-3-kinase (26). We observed that both OSM and aPBMC-CM were able to induce Stat1 and Stat3705 phosphorylation, which could be inhibited by genistein and the Jak2 inhibitor AG490 and to a lesser extent by a Jak3 inhibitor. Moreover, these inhibitors, but not a serine/threonine kinase inhibitor, were able to prevent changes in gene expression indicative of EMT in HK2 cells treated with aPBMC-CM. In view of the specificity of AG490 and the Jak3 inhibitor, these findings strongly suggest that OSM initiates EMT via the Jak/Stat pathway. However, the involvement of other pathways downstream of the receptors remains possible.
Stable expression of MEK-1, which is upstream of mitogen-activated protein (MAP) kinase/ERK, induces EMT in renal epithelial cells (28). Moreover, MAP kinase pathways have been suggested to mediate the TGF-β induction of fibronectin, p21Cip1, and TGF-β1 gene transcription (29–32⇓⇓⇓). Therefore, it is likely that binding of TGF-β to its receptor leads to activation of MAP kinase pathways, as well as to activation of the Smad signaling pathway, and the EMT events it induces are likely to involve MAP kinase pathways. Whether MAP kinase pathways are involved in EMT, downstream of Stat activation induced by OSM, is unknown. Further investigation of the role of OSM and other cytokines in EMT and renal fibrosis is clearly justified.
Acknowledgments
This work was supported by Teijin Ltd. (Japan) and UK Medical Research Council Technology. Drs. Nightingale and Patel contributed equally to this work. We are grateful to Professor AM El Nahas for helpful discussions. We also thank Parvin Ahmed for preparing aPBMC-CM.
- © 2004 American Society of Nephrology
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