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BASIC SCIENCE |
*Teijin Biomedical Laboratory, Medical Research Council Technology, London, UK; Division of Mycobacterial Research, National Institute for Medical Research, London, UK; Institute for Bio-Medical Research, Teijin Ltd., Tokyo, Japan; and Division of Biomedical Sciences, Faculty of Medicine, Imperial College London, London, UK.
Correspondence to Dr. Zhi Zhang, Teijin Biomedical Laboratory, Medical Research Council Technology, 1-3 Burtonhole Lane, London NW7 1AD, UK. Phone: 44-20-8906-7177; Fax: 44-20-8906-7200;
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Abstract |
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-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 
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 -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 |
Introduction |
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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 -smooth muscle actin (SMA) and are a major source of extracellular matrix (ECM) proteins in kidney fibrosis. SMA 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 subunit (OSMR) expression is upregulated by aPBMC-CM and that OSM can induce EMT of human kidney cells via the Jak/Stat1/3 signaling pathway.
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Materials and Methods |
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SMA 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-1, TNF-, IFN-
, 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 x 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 x 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-1, EGF, GM-CSF, IFN-, TNF-, IL-1, 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 x 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 x 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 OSMR) 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.
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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.
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Results |
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1, EGF, IL-1, IL-2, IL-6, OSM, GM-CSF, TNF-, and IFN- in aPBMC-CM. The total activated TGF-1 level was approximately 0.5 ng/ml, but the levels of OSM, TNF-, IFN-, 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).
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SMA 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 SMA 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 SMA 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).
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1, TNF-, IFN-, 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- and GM-CSF not shown). These results suggest that aPBMC-CM-induced changes in epithelial cells are initiated by two or more cytokines.
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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 SMA and E-cadherin were assessed. Exogenous OSM stimulated SMA 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, SMA 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.
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SMA 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.
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Discussion |
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, IL-4, IL-10, and IFN- 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-1, EGF, IL-1, IL-2, IL-6, OSM, GM-CSF, TNF-, and IFN- (Table 2). Both TGF- (7) and IL-1 (6,8), acting through a TGF--dependent mechanism (8), induce EMT in vitro. However, the final concentration of TGF-1 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- stimulated SMA 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- (Figure 2). Moreover, immunoneutralization of TGF- 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-1.
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--induced protein, and OSMR were elevated at almost all time points tested. Furthermore, expression of the IFN- 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 -catenin, and loss of -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- or OSMR, 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 OSMR 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-, 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- induction of fibronectin, p21Cip1, and TGF-1 gene transcription (29–32). Therefore, it is likely that binding of TGF- 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.
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References |
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induces human proximal tubule cell injury, -smooth muscle actin expression and fibronectin production. Kidney Int 62: 31–40, 2002[CrossRef][Medline]
regulates tubular epithelial-myofibroblast transdifferentiation in vitro. Kidney Int 56: 1455–1467, 1999[CrossRef][Medline]
1-dependent mechanism in vitro. Am J Kidney Dis 37: 820–831, 2001[Medline]
-catenin and restoration of E-cadherin function. Cancer Res 55: 4813–4817, 1995, and oncostatin M receptor by distinct mechanisms. J Biol Chem 276: 47038–47045, 2001 induces fibronectin synthesis through a c-Jun N-terminal kinase-dependent, Smad4-independent pathway. EMBO J 18: 1345–1356, 1999[CrossRef][Medline]
. J Biol Chem 274: 35381–35387, 1999 for transforming growth factor 1 production in a Smad-dependent pathway. J Biol Chem 275: 30765–30773, 2000. Proc Natl Acad Sci USA 98: 6686–6691, 2001This article has been cited by other articles:
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  A. Mozaffarian, A. W. Brewer, E. S. Trueblood, I. G. Luzina, N. W. Todd, S. P. Atamas, and H. A. Arnett Mechanisms of Oncostatin M-Induced Pulmonary Inflammation and Fibrosis J. Immunol., November 15, 2008; 181(10): 7243 - 7253. [Abstract] [Full Text] [PDF]
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N. G. Docherty, O. E. O'Sullivan, D. A. Healy, M. Murphy, A. J. O'Neill, J. M. Fitzpatrick, and R. W. G. Watson TGF-beta1-induced EMT can occur independently of its proapoptotic effects and is aided by EGF receptor activation Am J Physiol Renal Physiol, May 1, 2006; 290(5): F1202 - F1212. [Abstract] [Full Text] [PDF]
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, 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.
, E-cadherin; 
