TGF- 1 -induced EMT can occur independently of its proapoptotic effects and is a - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:44 |只看该作者 |倒序浏览 |打印

作者：Neil G. Docherty, Orfhlaith E. O‘Sullivan, Declan A. Healy, Madeline Murphy, Amanda J. O‘Neill, John M. Fitzpatrick, and R. William G. Watson作者单位：School of Medicine and Medical Sciences, Conway Institute, University College Dublin, Dublin, Ireland

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      【摘要】
   Apoptosis and epithelial-mesenchymal transdifferentiation (EMT) occur in stressed tubular epithelial cells and contribute to renal fibrosis. Transforming growth factor (TGF)- 1 promotes these responses and we examined whether the processes were interdependent in vitro. Direct (caspase inhibition) and indirect [epidermal growth factor (EGF) receptor stimulation] strategies were used to block apoptosis during TGF- 1 stimulation, and the subsequent effect on EMT was assessed. HK-2 cells were exposed to TGF- 1 with or without preincubation with ZVAD-FMK (pan-caspase inhibitor) or concomitant treatment with EGF plus or minus preincubation with LY-294002 (PI3-kinase inhibitor). Cells were then assessed for apoptosis and proliferation by flow cytometry, crystal violet assay, and Western blotting. Markers of EMT were assessed by microscopy, immunofluorescence, real-time RT-PCR, Western blotting, PAI-1 reporter assay, and collagen gel contraction assay. TGF- 1 caused apoptosis and priming for staurosporine-induced apoptosis. This was blocked by ZVAD-FMK. However, ZVAD-FMK did not prevent EMT following TGF- 1 treatment. EGF inhibited apoptosis and facilitated TGF- 1 induction of EMT by increasing proliferation and accentuating E-cadherin loss. Additionally, EGF significantly enhanced TGF- 1 -induced collagen I gel contraction. EGF increased Akt phosphorylation during EMT, and the prosurvival effect of this was confirmed using LY-294002, which reduced EGF-induced Akt phosphorylation and reversed its antiapoptotic and proproliferatory effects. TGF- 1 induces EMT independently of its proapoptotic effects. TGF- 1 and EGF together lead to EMT. EGF increases proliferation and resistance to apoptosis during EMT in a PI3-K Akt-dependent manner. In vivo, EGF receptor activation may assist in the selective survival of a transdifferentiated, profibrotic cell type. , s: A' h0 _5 d
      【关键词】 proliferation fibrosis transdifferentiation tubular epithelium
   THE PROGRESSIVE ACCUMULATION of extracellular matrix (ECM) in the glomeruli (glomerulosclerosis) and/or the interstitial space (tubulointerstitial fibrosis) occurs during chronic renal disease. The severity of tubulointerstitial fibrosis correlates well with the risk for renal failure progression ( 1 ). Epithelial-mesenchymal transdifferentiation (EMT) of tubular epithelial cells may contribute to the generation of renal fibrosis (reviews in Refs. 16, 24, 54 ).

EMT describes a phenotypical change induced in epithelial cells, which in the setting of tissue injury, gives way to a cell type termed the myofibroblast, characterized by increased motility, ECM protein synthesis, proliferation, and invasiveness. EMT contributes to embryonic patterning and the transition from carcinoma in situ to metastasis ( 11, 42 ). EMT is proposed to be involved in the wound-healing response ( 16, 24, 54 ). Transdifferentiating epithelial cells lose their defined cell-cell-basement membrane contacts and their structural/functional polarity to become spindle shaped and morphologically similar to activated fibroblasts. Loss of epithelial cell marker expression occurs concomitantly with, and as a driver of, these changes.

EMT is believed to occur in response to a number of environmental stresses and associated cytokine/growth factor stimuli. Environmental cues that have been shown to induce EMT in tubular epithelial cells include mechanical stretch ( 40 ), cyclosporine treatment ( 28 ), exposure to advanced glycation end products (AGE-consequence of hyperglycemia) ( 23 ), hypoxia ( 27 ), oxidative stress ( 39 ), activated monocyte supernatant ( 35 ), interleukin-1 ( 47 ), and oncostatin M ( 35 ), as well as the culturing of cells on collagen I ( 53 ).+ ]$ Y7 o; l! h+ z0 S) a# N- N6 z
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Transforming growth factor- 1 (TGF- 1 ) is a key mediator of EMT, principally via the activation of the smad signaling pathway ( 37 ). Indeed, EMT occurring in response to stimuli such as mechanical stretch, cyclosporine, and interleukin-1 treatment has been shown to rely on the activity of TGF- 1 ( 28, 40, 47, 48 ). Hypoxia is known to induce TGF- 1 in cultured cells of various types ( 6, 7, 19 ) and is likely to play an important role in hypoxia-related EMT.
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Increases in TGF- 1 following renal injury are likely to originate from increased bioavailability of the sequestered latent form and upregulated expression of TGF- 1 in both the injured tubule and in infiltrating immune cells. Interestingly, inflammatory cells, particularly macrophages, are a major source of not only TGF- 1 but other cytokines that have been implicated in the EMT process such as oncostatin M ( 35 ). TGF- 1 has been implicated as a key mediator of EMT not only in progressive renal disease, but also in other fibrotic diseases including cirrhosis ( 5 ) and chronic pancreatitis ( 12 ).$ w- t3 I% R1 b, q7 e- {
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Some reports suggest that tubular EMT can also proceed in a TGF- 1 -independent manner. AGE have been shown to induce tubular EMT via activation of the ERK 1/2 MAP kinase pathway downstream of the RAGE receptor, and this is unaffected by the presence of a TGF- 1 -neutralizing antibody ( 23 ).( n6 n+ M* x( y0 e2 M, f2 @. C: P9 o
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TGF- 1 induces tubular apoptosis via activation of the caspases ( 4 ), contributing to both profibrotic and proapoptotic events in tissue remodeling during renal disease. These observations in vitro are supported by evidence in vivo wherein the use of anti-TGF- 1 antibodies has been shown to reduce both tubular cell death and fibrosis ( 30 ) in a rat model of renal fibrosis. Amelioration of the apoptotic and fibrotic responses to renal injury also occurs in animal models using growth factors known to antagonize TGF- 1 activity ( 9 ).( A# r# g' B0 J8 a8 C
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Proapoptotic effects of TGF- 1 have variously been described as occurring via both smad-dependent and -independent mechanisms. Dai et al. ( 4 ) demonstrated that the susceptibility of a tubular epithelial cell line (HKC) to staurosporine-induced apoptosis was significantly enhanced by TGF- 1 in a p38 MAP kinase-dependent and smad-independent manner. A role for p38 in TGF- 1 -induced apoptosis of murine hepatocytes has also been demonstrated, but was shown to be dependent on the formation of smad transcriptional complexes and the upregulation of the immediate early response gene GADD45b ( 51 ). GADD45b overexpression has been shown to induce a G 2 /M cell cycle arrest by sequestering cyclin B1 outside the nucleus ( 15 ). Interestingly, cultured podocytes have been shown to undergo a p38 MAP kinase-dependent G 2 /M cell cycle arrest and subsequent apoptosis in response to treatment with TGF- 1, suggesting a similar mechanism may be in operation ( 49 ). Other reports have suggested a critical role for smad transcriptional activity in TGF- 1 -induced apoptosis. TGF- 1 failed to induce growth arrest and apoptosis in colonocyte cultures derived from smad 3 null mice while treatment of wild-type colonocytes resulted in growth inhibition and induction of apoptosis ( 29 ). Cultured mammary epithelial cells from Smad 3 null mice also show decreased apoptosis in response to TGF- 1 treatment ( 50 ). Importantly, Smad 3 null mice show reduced tubular apoptosis and subsequent fibrosis following ureteric obstruction ( 14 ).7 m7 m& ]8 S) T+ ]) z# |
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Conflicting evidence exists regarding a role for smad 7 in modulating epithelial apoptosis in response to TGF- 1. Stable overexpression of smad 7 has been shown to decrease the activity of the prosurvival NF- B transcription factor in Madin-Darby canine kidney cells, thereby sensitizing them to the induction of apoptosis by TGF- 1 ( 21 ). However, in a study using a gastric epithelial cell line, TGF- 1 was seen to induce apoptosis via the smad-dependent induction of Bim, subsequent induction of mitochondrial instability and caspase activation, an effect blockable via transfection with smad 7 ( 36 ).8 h7 o' t- |, n% {! q
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Early administration of epidermal growth factor (EGF) in models of renal injury aids tubular cell reproliferation and causes a resistance to apoptosis ( 2 ). This complements findings in vitro where EGF treatment leads to reduced tubular cell apoptosis in response to mechanical stretch ( 20 ). However, the observation that transgenic renal expression of a dominant-negative EGF receptor reduces renal fibrosis following 5/6 renal mass reduction, prolonged renal ischemic insult, and ANG II infusion ( 22, 46 ) suggests that EGF receptor activation may actively participate in the fibrotic process. Studies in vitro have shown that TGF- 1 in combination with EGF effectively induces EMT in cultured renal tubular cells ( 37 ).- k4 y0 w: X  @% k

Despite decreases in EGF ligand expression during renal injury ( 43 ), the EGF receptor can be stimulated via transactivation by other ligands such as ANG II, through the release of ligands sequestered in the ECM and oxidative stress ( 31, 41, 54 ). Signaling through the EGF receptor is known to influence the apoptotic resistance and invasive potential of certain cancers, and this may be associated with the adoption of a transdifferentiated phenotype ( 45 ). EGF provides resistance to the proapoptotic effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in breast cancer cells via a mechanism involving mitochondrial stabilization ( 13 ). The activity of Fas ligand has also been shown to be abolished by EGF treatment in vitro in breast cancer cell lines ( 10 ).
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Aside from its role in proliferation and resistance to apoptosis, one key piece of evidence that may link EGF receptor stimulation to the promotion of EMT is its negative effect on E-cadherin expression.
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The loss of E-cadherin is associated with increases in the invasive potential of cancer cells and has also been identified as a key step in EMT. EGF can lead to a reduction in E-cadherin expression by both transcriptional and nontranscriptional pathways. EGF has been shown to downregulate caveolin-1 function, leading to endocytosis of E-cadherin ( 25 ). Additionally, the tyrosine kinase activity of the ligand-bound EGF receptor has been shown to target -catenin for release from E-cadherin-containing junctional complexes, leading to their degradation ( 8 ).1 `! ~* l- ]+ r* b9 U6 G* P. n

Long-term stimulation of the EGF receptor, as well as TGF- 1 treatment, leads to the induction of Snail transcription factor expression, which can lead to the binding of regulatory E-boxes on the E-cadherin promoter and cause transcriptional repression ( 8, 52 ). TGF- 1 may also induce a loss of E-cadherin expression via the snail-dependent induction of matrix metalloprotease (MMP) expression and its subsequent cleavage of E-cadherin ( 52 ).- ?# E4 U  n! e+ Y/ n

It is unknown whether the apoptotic remodeling effect of TGF- 1 is integral to the development of EMT in a cellular population. We hypothesized that during in vitro EMT, TGF- 1 could induce EMT independent of its proapoptotic effects. We first examined whether TGF- 1 could induce EMT following a general inhibition of its proapoptotic activity via the use of a pan caspase inhibitor. Second, we examined whether EGF receptor activation would block the proapoptotic effects of TGF- 1 while enhancing proliferation and EMT.

MATERIALS AND METHODS

Materials. Human proximal tubular cells (HK-2) were from the American Type Culture Collection (ATCC). ZVAD-FMK, TGF- 1, TNF-, and EGF were obtained from R&D Systems. Protein concentrations were determined using the DC protein assay kit (Bio-Rad). Anti-pAkt and total Akt primary antibodies and goat anti-mouse IgG secondary antibody were from Cell Signaling. Mouse anti-human E-cadherin and fibronectin monoclonal antibodies were from Becton Dickenson (Transduction Laboratories). The Ki-67 antibody was from DAKO. The AlexiFluor 488 FITC-conjugated anti-mouse IgG and Texas red-labeled phalloidin and Super Fas ligand were from Alexis biochemicals. Collagen I was from Becton-Dickinson. The Immobilon-P PVDF membranes were from Millipore. Total RNA extracts were made using a TRIzol-based method. The chemiluminescent detection kit (ECL) was from Amersham. Synthesis of cDNA was performed using a kit (Invitrogen). Citifluor anti-quench mounting medium was from AGAR Scientific. The dual-luciferase reporter assay was from Promega. Fugene 6 came from Roche Pharmaceuticals.
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All remaining chemicals and reagents were purchased from Sigma.9 ?  E6 z; f5 Z. F0 T0 t) |2 M
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Cell culture. HK-2 cells (1.6 x 10 6 ) were cultured in 75-cm 2 flasks in DMEM-F12 containing 10% fetal bovine serum, 2 mM glutamine, and 1% penicillin-streptomycin. At 80% confluence, cells were trypsinized and 5 x 10 4 cells/well seeded onto 6-well plates, 1 x 10 4 cells/well seeded onto 24-well plates, and 2 x 10 4 cells/well seeded onto 8-well chamber slides for experimental procedures. After 24 h, cells were serum starved in defined medium containing: 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite, 36 ng/ml hydrocortisone for a further 24 h. At this point, cells were treated before analysis.) ]1 q* h4 ~% T; b1 A/ t" }
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Quantification of apoptosis (SubG 0 ) and proliferation (G 2 /M) using propidium iodide flow cytometry. Briefly, cells from each well of a six-well plate were trypsinized and centrifuged at 1,300 rpm for 5 min and then resuspended in 300 µl of a hypotonic fluorochrome solution (50 µg/ml PI, 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA, and 0.1% Triton X-100 in PBS) and then lysed in the dark for 15 min. Samples were analyzed using an XL flow cytometer (Coulter). A minimum of 5,000 events was analyzed per sample. Apoptotic nuclei were distinguished from normal nuclei by their hypodiploid DNA and assessment of percentage apoptosis was established. Cellular debris was excluded from analysis by raising the forward threshold. The G2/M phase peak was also analyzed as an indicator of percentage proliferation, and when combined with cell number and Ki-67 expression data, allowed for reliable comparison of proliferation between treatments. All measurements were performed under the same instrument settings.

Morphological assessment of HK-2 cell transdifferentiation. Following growth factor treatment, cells were photographed in culture using a JVC KY-FSSBE digital camera coupled to a Nikon TMS microscope. Representative images were then captured and saved using the Matrox Intellicam program.
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Real-time RT-PCR. Total RNA was isolated using TRIzol and cDNA was generated by reverse transcription using random hexamers. Transcript levels were determined by real-time Taq man PCR using a PerkinElmer 7700 analyzer. Probe and primer sequences for fibronectin were: sense primer: 5'-CCGCCGAATGTAGGACAAGA-3'; antisense primer: 5'-TGCCAACAGGATGACATGAAA-3; probe: 5'-CAACCATCTCATGGGCCCCATTCC-3. E-Cadherin and 18S rRNA primers were as per predeveloped assays (PerkinElmer Life Sciences).

Cycling conditions were as follows: 1 ) 2 min at 50°C; 2 ) 10 min at 95°C; 3 ) 15 s at 95°C; 4 ) 1 min at 60°C; 5 ) repeat from 3 for an additional 39 times. Probes were labeled with 5'-FAM and with 3'-TAMRA as quencher, with the exception of the ribosomal probe, which was labeled with 5'-VIC to facilitate multiplexing. All results were normalized to 18S rRNA.

Western blotting. Total cellular protein was extracted using NP-40 protein lysis buffer (0.5% NP-40, 10 mM Tris, pH 8.0, 60 mM KCl, 1 mM EDTA, pH 8.0, 1 mM DTT, 10 mM PMSF, 10 µl/ml phosphatase inhibitor cocktail, 1 mM leupeptin, 1 mM sodium orthovanadate, 1 mM aprotinin, and 2 mM pepstatin). The cell lysate was centrifuged at 13,000 rpm for 10 min at 4°C, and the supernatant was removed, snap-frozen, and stored at -80°C.- ?6 f$ x. V$ f9 p1 w; z* r, {/ c- x
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To obtain pure nuclear extracts for Ki-67 immunoblotting, a modified extraction procedure was used. Briefly, cells were harvested by scraping into cold PBS, then pelleted by centrifugation at 1,200 rpm for 5 min at 4°C. Thereafter, pellets were resuspended in cell lysis buffer (10 mM HEPES, 1.5 mM MgCl 2, 10 mM KCl, 10 mM PMSF, 10 µl/ml phosphatase inhibitor cocktail, 1 mM leupeptin, 1 mM sodium orthovanadate, 1 mM aprotinin, and 2 mM pepstatin) and allowed to swell on ice for 15 min before centrifugation at 14,000 rpm for 10 min at 4°C. Thereafter, the supernatant was stored as a cytosolic fraction and the remaining pellet was resuspended in nuclear extraction buffer (20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl 2, 1 mM EDTA, 10 mM PMSF, 10 µl/ml phosphatase inhibitor cocktail, 1 mM leupeptin, 1 mM sodium orthovanadate, 1 mM aprotinin, and 2 mM pepstatin). Nuclei were lysed on ice for 30 min, samples were centrifuged at 14,000 rpm for 30 min at 4°C, then resulting supernatants were snap-frozen and stored at -80°C./ l, k7 C3 r3 w3 F
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Total protein in extracts was quantified by Bradford assay. Protein samples (50 µg) were resolved in SDS polyacrylamide gels for 80-180 min at 140 V, then electrophoretically transferred to a PVDF membrane at 100 V for 80 min.; ]! ?7 G0 U1 M
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Membranes were then incubated in blocking buffer (5% BSA in Tris-buffered saline, pH 7.4, 0.1% Tween 20) for 1 h at room temperature, then incubated in primary antibody (1:1,000) for a further 1 h in antibody incubation buffer (1% BSA in Tris-buffered saline, pH 7.4, 0.1% Tween 20). After being washed for 5 x 5 min in TTBS buffer (Tris-buffered saline, pH 7.4, 0.1% Tween 20), membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-goat IgG at a dilution of 1:5,000. Blots were washed for a further 5 x 5 min in TTBS and developed using an enhanced chemiluminescence substrate system for detection of horseradish peroxidase (ECL). Where possible, membranes were reprobed with a monoclonal anti- -actin antibody to confirm equal loading. When it was not possible to reprobe with anti- -actin, membranes were stained with Commasie blue to check equal loading and transfer.

Immunohistochemical detection of F-actin rearrangement and Ki-67 localization. Chamber slides were washed 3 x 3 min each time with 500 µl of PBS, then fixed for 25 min in 3.7% paraformaldeyde. Fixed monolayers were again washed with PBS and then permeabilized by a 25-min incubation with 0.2% Triton X-100. Slides were again washed three times in PBS and then blocked for 1 h with 5% goat serum in PBS. Primary antibody was added at a dilution of 1:100 in 5% goat serum and incubated for 1 h. A 1:200 dilution of FITC-labeled anti-mouse IgG or a 3:100 dilution of Texas red was added for 1 h in the dark. Slides were then washed, and in the case of F-actin staining, nuclei stained for 5 s with DAPI were then washed again. Slides were mounted with Citifluor anti-quench. DAPI, FITC, and rhodamine fluorescence were detected using a Zeiss Axioplan 2 microscope and analyzed using AxioVision 3.0.6 software (Carl Zeiss, Thornwood, NY).3 z0 g' R/ N, X. H
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PAI-1 promoter luciferase assay. HK-2 cells (1 x 10 4 /well) diluted in 10% FBS were adhered to the wells of a 24-well plate for 3 h. Vector encoding the promoter-reporter 3TP-lux was kindly donated by Dr. J. Massague (Sloan Kettering Institute, New York). This construct contains three consecutive AP-1 sites fused to a fragment of the PAI-1 promoter containing two smad response elements and an additional AP-1 site. Transfections were performed with the Fugene 6 reagent. The pRL-CMV, renilla luciferase reporter plasmid was cotransfected and its activity was used to correct for variation in transfection effeciency. Cells were subsequently lysed and luciferase assays were performed as per the supplier's instructions.
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Collagen I gel contraction assay. Following 24 h of 1% FBS treatment to induce quiescence, HK-2 cells were trypsinized and seeded into 24-well plates at a density of 1 x 10 5 cells/ml in 1% FBS medium containing 1.25 mg/ml of rat tail collagen 1. Vehicle, TGF- 1 (3 ng/ml), EGF (10 ng/ml), or TGF- 1 /EGF (3 and 10 ng/ml) were added to gels. Gels were set for 1 h at 37°C, then supplemented with 800 µl of relevant control or growth factor-containing medium. Forty- eight hours after seeding, gels were photgraphed, and contraction rates were calculated as percentages of the total original gel area, this being equal to the area of a single well of a 24-well plate at time 0 (2 cm 2 ). Quantification was carried out using the ImageJ program.

Crystal violet assay assessment of cell number. HK-2 cells were grown in 24-well plates to 70% confluence, serum starved for 24 h, and then incubated with TGF- 1 (3 ng/ml), EGF (10 ng/ml), and TGF- 1 /EGF (3/10 ng/ml) for 48 h. Cells were then fixed in 1% gluteraldehyde solution, washed in PBS, incubated with 1% crystal violet, washed, solubilized with 1% Triton X-100, and analyzed at an absorbance of 590 nm.
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Statistical methods. Results are expressed as means ± SE and were analyzed for significance by one-way ANOVA with Bonferroni and Scheffé's post hoc testing. Statistical significance was set at P - ]! k7 r# I. n" U  d

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Effect of pan-caspase inhibition and EGF treatment inhibition on TGF- 1 -induced apoptosis and EMT. HK-2 cells treated with TGF- 1 for 48 h showed a 60% increase in rates of apoptosis vs. control ( P = 0.023) and increased the apoptotic response of the cells to the G2/M arresting agent, staurosporine ( P
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Fig. 1. TGF- 1 treatment promotes apoptosis in a caspase-dependent manner. A : HK-2 cells were treated with TGF- 1 (3 ng/ml) for 48 h and then exposed to 2 µM staurosporine (STP) or DMSO vehicle (VEH) for a further 24 h. Propidium iodide staining of the nucleic acid component of solubilized cells was carried out and 5,000 events per sample were analyzed by flow cyotometry. Cellular debris was excluded by raising the forward threshold and apoptosis was calculated as the percentage of all events localizing to the sub G 0 region of histograms; n = 3 per treatment. Values are expressed as means ± SE. * P

As EGF receptor activation may occur in stressed tubular cells in vivo, we examined how stimulation of cells with EGF might affect apoptosis, proliferation, and EMT induced by TGF- 1 treatment.
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Spontaneous rate of apoptosis in control and EGF-treated cells was similar over a 48-h period. A 78% increase in apoptosis was observed in TGF- 1 -treated cells ( P

Fig. 2. Effect of EGF receptor stimulation on apoptosis during TGF- 1 induction of EMT. Propidium iodide staining of trypsinized and Triton X-100-solubilized cells was carried out and 5,000 events per sample were analyzed by flow cytometry after treatment with TGF- 1 (3 ng/ml), EGF (10 ng/ml), or a combination of TGF- 1 and EGF (3 and 10 ng/ml) for 48 h. In all cases, cellular debris was excluded by raising the forward threshold and apoptosis was calculated as the percentage of all events localizing to the sub G o region of histograms as represented in the figure; n = 9. Values are expressed as means ± SE. * P ! N  F8 r: w$ Y9 U* }

We also examined the effect of EGF receptor stimulation on the sensitivity of transdifferentiating cells to TNF- and Fas ligand. However, no statistically significant induction of apoptosis occurred following incubation with either of these agents under any of the growth factor treatment conditions (data not shown).  O( R2 S$ }7 F! N2 d
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As we had hypothesized that EGF would increase proliferation during TGF- 1 treatment, we compared the impact of EGF on cell number and proliferation in our in vitro model of EMT. Crystal violet staining demonstrated that EGF and TGF- 1 /EGF induced average increases of 40 and 42%, respectively, vs. control absorbance values, reflecting increases in cell number ( P $ \8 x9 B1 h3 C" Y8 E3 L* ]% I
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Fig. 3. Effect of EGF receptor stimulation on cell number/proliferation during TGF- 1 induction of EMT. A : HK-2 cells were treated with TGF- 1 (3 ng/ml), EGF (10 ng/ml), or a combination of TGF- 1 and EGF (3 and 10 ng/ml) for 48 h. A : adherent cells were then fixed, stained with 1% crystal violet, solubilized with Triton X-100, and analyzed at an absorbance of 590 nm; n = 6. Data are expressed as means ± SE. * P
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Flow cytometric analysis of cells in the G 2 /M phase of the cell cycle was used as an index of percentage proliferation following 48 h of treatment. An increase in cells in this phase of the cell cycle was noted in all treatment groups vs. control, being significantly higher in the TGF- 1 /EGF group ( P % v; K& g% g$ R

As a molecular marker of proliferation, we examined the expression of a nuclear antigen, Ki-67, the expression of which is tightly delineated to the S and G 2 /M phases of the cell cycle. We confirmed that expression of Ki-67 is specific to the nucleus by immunofluorescence.
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The presence of EGF was seen to be associated with increases in the level of nuclear Ki-67. Levels were highest in the TGF- 1 /EGF group, reflecting the maximal increases in cell number and percentage of cells in the G 2 /M phase found in this group. Nuclear Ki-67 levels were lowest in the TGF- 1 only treated group, despite this group having a higher percentage of cells in the G 2 /M phase than control cells. However, as the TGF- 1 only-treated group did not show a net increase in cell number vs. control over the 48-h treatment period, these data indicate that cells in this group are either 1 ) in the early phases of proliferation or 2 ) are G 2 /M arrested. Levels of Ki-67 in the EGF only-treated group were higher than in control or TGF- 1 alone-treated cells, but lower than those seen in the TGF- 1 /EGF group, reflecting the increase in cell number in this group at 48 h and correlating with a reduced proliferation rate vs. that of the TGF- 1 /EGF-treated cells ( Fig. 3 C ). Given that EGF-treated cultures continued to grow in a monolayer and approached confluence by the end of the treatment period, it appears that proliferation is likely to begin to wane as a result of contact inhibition. Correlating the data on cell number, G 2 /M phase, and nuclear Ki-67 expression, a sustained proliferatory advantage was observed in the TGF- 1 /EGF-treated group, which was able to persist, in part, due to the lack of contact inhibition resultant from their growth in multilayer as opposed to monolayer.( c1 s! t  x) Z* D! L4 `

Effect of EGF during TGF- 1 -induced EMT. In keeping with our hypothesis that EGF-mediated inhibition of apoptosis would not inhibit TGF- 1 -induced EMT, we compared EMT parameters across the different treatment groups. Control and EGF alone-treated HK-2 cells maintained cobblestone morphology and a cortical actin network ( Fig. 4 A ). TGF- 1 and TGF- 1 /EGF combined treatment led to morphological changes consistent with EMT. Monolayers showed a loss of cobblestone morphology and the induction of multilayer growth in which cells adopted a more elongated and spindle-shaped morphology ( Fig. 4 A ). This morphological change was also observable by F-actin rearrangement into stress fibers ( Fig. 4 A ). Morphological changes were consistently more marked in the TGF- 1 /EGF-treated group ( Fig. 4 A ).4 E9 p5 G4 y/ c& @- H* ^
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Fig. 4. Effect of EGF on TGF- 1 -induced EMT in HK-2 cells. HK-2 cells were treated with TGF- 1 (3 ng/ml), EGF (10 ng/ml), or a combination of TGF- 1 and EGF (3 and 10 ng/ml) for 48 h and then morphological, molecular, and functional parameters of EMT were assessed as follows. A : light microscopy ( x 10) and F-actin rearrangement ( x 40). Real-time RT-PCR and Western blot analyses of E-cadherin ( B ) and fibronectin ( C ) were also carried out; n = 3 per treatment for real-time RT-PCR. * P - g5 {3 P9 o: `( i! @& l% `, k

E-cadherin and fibronectin expression were examined by real-time RT-PCR and Western blotting as changes in their expression are highly indicative of tubular EMT ( 10 ). EMT in the TGF- 1 - and TGF- 1 /EGF-treated groups was characterized by the loss of E-cadherin expression at both the mRNA and protein level. Loss of E-cadherin mRNA expression during TGF- 1 treatment was significantly enhanced by the addition of EGF ( P = 0.03 vs. TGF- 1 alone). Indeed, a significant loss of E-cadherin mRNA vs. control was observed when EGF was administered singly ( P
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As the myofibroblast is proposed to contribute to fibrosis, we examined the expression of fibronectin as a marker of profibrotic gene expression. Unstimulated and EGF single-treated HK-2 cells showed a basal expression of fibronectin at the protein level with evidence of a modest upregulation of fibronectin transcription following EGF treatment ( P = 0.04). However, in both the TGF- 1 /EGF- and TGF- 1 -treated groups, a significant upregulation over control values was observed at the transcriptional level ( P
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Control HK-2 cells transfected with the PAI-1 promoter-luciferase construct showed a low baseline activity. TGF- 1 caused a more marked increase in promoter activity ( P - y* t% G9 r. `/ F6 s6 V" G. U
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Fig. 5. Effect of EGF on TGF- 1 -mediated activation of a PAI-1 promoter construct. HK-2 cells were grown on 24-well plates and transiently transfected with a PAI-1 promoter luciferase reporter construct. Cells were treated with TGF- 1 (3 ng/ml), EGF (10 ng/ml), or a combination of TGF- 1 and EGF (3 and 10 ng/ml) for 48 h and then promoter activity was measured as a function of luciferase fluorescence; n = 9 per treatment. * P
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We next examined the relative abilities of TGF- 1, EGF, and a TGF- 1 /EGF combined treatment to affect a contractile response in HK-2 cells seeded in collagen I gels. HK-2 cells were able to efficiently contract collagen gels under basal conditions with an average percentage contraction rate of 48% in control cultures. TGF- 1 and EGF, when administered individually, led to significantly increased contraction rates of 67 and 69%, respectively ( P 3 ]8 \0 m4 W5 h

Fig. 6. Collagen I gel contraction by HK-2 cells. HK-2 cells were seeded into 24-well plates at a seeding density of 1 x 10 5 cells/ml of 1% FBS medium containing 1.25 mg/ml of rat tail collagen 1 ± TGF- 1 (3 ng/ml), EGF (10 ng/ml), or TGF- 1 /EGF (3 and 10 ng/ml). A : control. B : TGF- 1. C : EGF. D : TGF- 1 /EGF. Gels were set for 1 h at 37°C and then supplemented with 800 µl of relevant control or growth factor-containing medium. Forty eight hours after seeding, contraction rates were expressed as percentages of the total original gel area, this being equal to the area of a single well of a 24-well plate at time 0; n = 6 per treatment. Data expressed as means ± SE. * P

EGF receptor stimulation, Akt activation, and modification of the apoptotic/proliferatory responses to TGF- 1. To provide some mechanistic evidence for the relative resistance to apoptosis and increases in proliferation observed in EGF-treated cultures, we examined Akt activation following growth factor treatment and also the effect of inhibiting PI3-kinase, on Akt phosphorylation and later effects of growth factor treatment on apoptotic and proliferatory responses. Western blot analysis demonstrated that EGF but not TGF- 1 treatment led to Akt activation and that TGF- 1 did not prevent EGF from eliciting this effect during cotreatment ( Fig. 7 A ). Preincubation with LY-294002 prevented EGF-induced increases in Akt phosphorylation.8 f% A: A9 Z. V

Fig. 7. Effects of PI3 kinase inhibition on EGF-induced Akt phosphorylation and associated changes in apoptosis and proliferation during EMT. A : HK-2 cells were incubated for 30 min with either dimethylsulphoxide vehicle (VEH) or 20 µM LY-294002 (LY) for 30 min before the addition of TGF- 1 (3 ng/ml) EGF (10 ng/ml) or a combination of TGF- 1 and EGF (3 and 10 ng/ml). A : Western blotting for the Ser 473 phosphorylated and total forms of Akt was carried out on cell extracts taken at 30-min postgrowth factor stimulation. -Actin was used as a loading control. B : at 48 h posttreatment with TGF- 1 (3 ng/ml) or a combination of TGF- 1 and EGF (3 and 10 ng/ml), propidium iodide staining of trypsinized and Triton X-100-solubilized cells was carried out and 5,000 events per sample were analyzed by flow cytometry. Cellular debris was excluded by raising the forward threshold and apoptosis was calculated as the percentage of all events localizing to the sub G 0 region of histograms. * P 4 {/ ?+ r% {& ~  A% f% B: t5 Z
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When cells were treated with TGF- 1 or a TGF- 1 /EGF combination and left in culture for 48 h, preincubation with LY-294002 was shown to increase apoptotic rates vs. VEH-treated cells in both groups. The highest rate was found in the TGF- 1 -treated group ( P 6 [% E4 \8 i' A. g0 n( m: Z8 s
9 m% E4 ~2 B! ~
Increases in proliferation in TGF- 1 and TGF- 1 /EGF groups were lost when pretreated with LY-294002 ( P
% @3 ?, G( P" o3 m$ E* Y/ R% k; ]
DISCUSSION

TGF- 1 is implicated in tubular cell apoptosis and fibrosis during the course of chronic renal injury ( 30 ). Studies in vitro in human kidney cell (HKC) cells have shown that TGF- 1 primes for staurosporine-induced apoptosis in a p38 MAP kinase-dependent manner ( 4 ). This effect was associated with increased caspase cleavage, indicating that TGF- 1 promotion of apoptosis is caspase dependent. We report here that TGF- 1 induces a small but consistent increase in apoptosis in HK-2 cells, and we confirmed its priming effect during staurosporine-induced apoptosis. TGF- 1 is also known to affect an EMT-like change in cultured renal tubular cells from mice ( 37 ). In our experiments, we found that the apoptotic response, but not the EMT-like changes found following TGF- 1 treatment of HK-2 cells, was suppressed by preincubation with a pan-caspase inhibitor. This suggests that EMT occurring in response to TGF- 1 is independent of any caspase-mediated apoptotic response.

This observation led us to hypothesize that EGF could promote EMT by offsetting the proapoptotic effects of TGF- 1 without preventing its EMT-inducing effect, thereby facilitating the improved survival of cells undergoing EMT.

In vivo, administration of EGF before the induction of renal injury in neonatal rats has been shown to have a protective effect by rendering tubular cells resistant to apoptosis and promoting the proliferation of the nascent epithelium in areas of damaged tubule ( 2 ). However, conflicting evidence has shown that the expression of a tubule-targeted, dominant-negative mutant of the EGF receptor reduces renal injury and fibrosis following renal mass reduction, prolonged ischemia, and ANG II infusion ( 22, 46 ). Aside from the direct effects of EGF on cell survival and proliferation, EGF is implicated in the development of the invasive phenotype in carcinoma, a phenotype which in some aspects recapitulates what is occurring in transdifferentiating renal tubular cells ( 25 ).
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We demonstrate that EGF can prevent the small but significant increase in apoptosis observed with TGF- 1 treatment in unstressed HK-2 cells. We show that the combination of TGF- 1 and EGF leads to an increased proliferation and net cell accumulation compared with when cells are treated with TGF- 1 only. This was additionally confirmed by examination of the proliferation antigen Ki-67. Increases in nuclear Ki-67 have previously been shown during EMT and stromal invasion of limbal epithelial progenitor cells ( 17 ).0 l" E8 @( V) M$ b2 U
% k4 a; y4 M! ^  L9 ^4 _+ Q
We also noted that 48 h of TGF- 1 only treatment led to an accumulation of cells in the G 2 /M phase of the cell cycle relative to control cultures, suggesting an increase in proliferation. This would be contrary to studies showing that TGF- 1 causes growth arrest of tubular epithelial cells ( 48 ). However, it is possible that once EMT is underway, TGF- 1 plays a mitogenic role in the myofibroblast, as it does in kidney and skin fibroblast culture, and that these cells are in the early phase of a proliferatory response ( 44 ). Another possible explanation for this finding is that these cells are G 2 /M phase growth inhibited. This conclusion is supported by the fact that no complimentary increase in cell number or nuclear Ki-67 expression was observed with TGF- 1 treatment alone, as opposed to when EGF was present. Treatment of podocyte cultures and the HKC cell line with TGF- 1 leads to p38 MAP kinase-dependent apoptosis ( 4, 49 ). In podocytes this has been shown to occur secondary to a G 2 /M arrest ( 49 ). TGF- 1 also induces p38-dependent apoptosis in murine hepatocytes ( 51 ). In this case, it has been shown to occur secondary to smad-dependent increases in GADD45b expression. GADD45b overexpression has been shown to induce a G 2 /M cell cycle arrest by sequestering cyclin B1 outside the nucleus ( 15 ). A similar process may account for TGF- 1 -induced G 2 /M accumulation and apoptosis in our model.
9 \- @1 p9 Z: u: S6 z2 X; A6 g: I
EMT was evident in the TGF- 1 /EGF-treated cultures confirming that EGF, despite its inhibition of the proapoptotic effects of TGF- 1, does not prevent the EMT process. In the case of E-cadherin loss, EGF promoted this change both when administered singly and when administered in combination with TGF- 1. The negative effect of EGF on E-cadherin expression has been dissected in cancer cell lines ( 8, 25 ). The loss of E-cadherin correlates with the development of the invasive phenotype and metastasis in breast cancer ( 38 ). This again draws a parallel between these processes and the appearance of myofibroblasts of tubular origin in the tubulointerstitium of diseased kidneys.
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The induction of fibronectin in tubular epithelial cells during TGF- 1 -induced EMT has recently been shown to occur in a smad-independent JNK-dependent manner ( 34 ), whereas the activation of the 3TP-Lux PAI-1 reporter construct used in our studies is known to be dependent on both active smad transcriptional complexes and the assembly of AP-1 complexes ( 18 ). Our results therefore demonstrate that EGF does not prevent TGF- 1 from exerting either smad-dependent or -independent transcriptional responses during EMT.
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The collagen I gel contraction assay relies on dynamic remodeling and migratory behavior in HK-2 cells, and in a simple way recapitulates aspects of the wound-healing process. TGF- 1 -induced contraction in bovine trabecular meshwork cells has been shown to rely on the activation of Rho kinase, protein kinase C, and the mysoin light chain kinase and the formation of stress fibers ( 33 ). TGF- 1 and EGF have been shown to increase Rho GTPase activity in human lens epithelial cells ( 26 ). EGF was additonally shown to induce Rac GTPase activity and to induce membrane ruffling as opposed to stress fiber formation ( 26 ). We had noted stress fiber formation in TGF- 1 -treated cells, and this is likely to account for their enhanced contraction of collagen gels. A combination of the cytoskeletal reorganizing properties of both growth factors is likely to have caused the enhanced contraction of collagen gels by the TGF- 1 /EGF double treatment, demonstrating that the growth factors can combine to promote a functional, transcription-independent property of transdifferentiation.$ Z$ f2 a5 U  @& Z
* `5 Y4 q% z' b0 B, `+ B3 e% `
Following PI3-K activation by receptor tyrosine kinases, such as that of the EGF receptor, Akt is recruited to the membrane where it is activated by the phosphoinositide-dependent kinase-1 (PDK1). The phosphorylation of Akt is known to be associated with decreases in apoptosis and an increased proliferation rate ( 38 ). We therefore examined whether the decreased apoptotic sensitivity and increased proliferation during TGF- 1 /EGF double treatment were associated with increases in Akt activation. Western blot analysis demonstrated that EGF but not TGF- 1 treatment led to Akt activation. TGF- 1 did not prevent EGF from eliciting this effect during cotreatment. We thus demonstrated that EGF caused the early activation of a well-characterized survival pathway, supporting a role for it as a cytoprotective growth factor during EMT. To examine whether the antiapoptotic/proproliferatory effects of EGF were Akt dependent, we preincubated cells with LY-294002, a PI3-K inhibitor. This caused a dramatic early reduction in EGF induction of Akt phosphorylation and correlated with the later inhibition of the anti-apoptotic and proproliferatory effects of EGF. This supports a link among EGF, Akt, and cytoprotection during EMT and may explain the reduced fibrosis following renal injury in mice carrying an EGF-R dominant-negative transgene ( 22, 46 ).
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The work presented highlights two main pieces of information; first, TGF- 1 can induce EMT independently of its proapoptotic effects. Second, it demonstrates that EGF aids the EMT process in response to TGF- 1, directly via its effect on E-cadherin expression and collagen remodeling, and indirectly via conferring apoptotic resistance and mitogenic potential to cells undergoing EMT. This occurs without compromising key smad-dependent and -independent aspects of EMT. These findings are summarized in a general hypothesis of the effects of EGF receptor activation during EMT in the injured kidney presented pictorially in Fig. 8.

Fig. 8. Mechanisms by which EGF receptor activation may occur during renal disease and act to enhance the EMT response. The illustration links the proposed mechanisms of EGF receptor activation in stressed renal tubular epithelial cells to an EMT-enhancing effect. HB-EGF, heparin-binding epidermal growth factor.) O7 K) D+ [( q

Although early inhibition of apoptosis by either direct (caspase inhibition) ( 3 ) or indirect (EGF administration) ( 2 ) means prevents progressive renal injury, these results demonstrate that growth factor-mediated inhibition of apoptosis in established renal disease may not always have beneficial effects on the fibrotic outcome. The link between the inhibition of apoptosis and prevention of later fibrosis using such mediators is contingent on their deployment early after acute injuries. In this case, they have the effect of preventing inflammatory infiltration and subsequent fibrosis occurring in response to signals originating from cells dying by apoptosis. Growth factors such as EGF, administered later after the onset of renal injury, may worsen fibrosis, and this work presents evidence of how EGF-R activation during EMT may contribute to this.: N8 ^8 P& w* D" O- h" V0 o

GRANTS

This work was supported by the Irish Health Research Board (#RP/2002/126). D. A. Healy is a postdoctoral fellow of The Punchestown Kidney Fund. M. Murphy is in receipt of a grant from the Wellcome Trust.% X. n- z( {1 o. j) R! K8 b! p4 \1 W

ACKNOWLEDGMENTS
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The authors thank Dr. C. Moss for excellent technical assistance with real-time RT-PCR.$ L. J# B( N2 P
      【参考文献】
Bohle A, Muller GA, Wehrmann M, and Xiao JC. Pathogenesis of chronic renal failure in the primary glomerulopathies, renal vasculopathies, and chronic interstitial nephritides. Kidney Int Suppl 54: S2-S9, 1996.: \1 b. k8 a( ~6 A9 F& M

4 i$ C: \# P: x
Chevalier RL, Goyal S, Wolstenholme JT, and Thornhill BA. Obstructive nephropathy in the neonatal rat is attenuated by epidermal growth factor. Kidney Int 54: 38-47, 1998.

' W+ a1 n4 c# E* {  z# G3 E9 C

Daemen MA, van't Veer C, Denecker G, Heemskerk VH, Wolfs TG, Clauss M, Vandenabeele P, and Buurman WA. Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation. J Clin Invest 104: 541-549, 1999.7 @% i% U" W7 G

Dai C, Yang J, and Liu Y. Transforming growth factor- 1 potentiates renal tubular epithelial cell death by a mechanism independent of Smad signaling. J Biol Chem 278: 12537-12545, 2003.
* M: y, N0 Y) ?3 h. k

Dooley S, Hamzavi J, Breitkopf K, Wiercinska E, Said HM, Lorenzen J, Ten Dijke P, and Gressner AM. Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats. Gastroenterology 125: 178-191, 2003.6 d& C& [; X- W: o& t

Falanga V, Qian SW, Danielpour D, Katz MH, Roberts AB, and Sporn MB. Hypoxia upregulates the synthesis of TGF- 1 by human dermal fibroblasts. J Invest Dermatol 97: 634-637, 1991.
8 F$ I3 n: u/ ^6 W( z
9 ^- R+ w& K9 G* g; r

Falanga V, Zhou L, and Yufit T. Low oxygen tension stimulates collagen synthesis and COL1A1 transcription through the action of TGF- 1. J Cell Physiol 191: 42-50, 2002.9 M4 S$ \% D/ x. S

Fujii K, Furukawa F, and Matsuyoshi N. Ligand activation of overexpressed epidermal growth factor receptor results in colony dissociation and disturbed E-cadherin function in HSC-1 human cutaneous squamous carcinoma cells. Exp Cell Res 223: 50-62, 1996.9 @7 M/ [/ n' o" C/ e9 W! }) g

4 `; l; Y  ~* Q
  X' A% U: h4 D. @: P9 T* z( c
Gao X, Mae H, Ayabe N, Takai T, Oshima K, Hattori M, Ueki T, Fujimoto J, and Tanizawa T. Hepatocyte growth factor gene therapy retards the progression of chronic obstructive nephropathy. Kidney Int 62: 1238-1248, 2002.3 a( k* L# N& @4 {
( G: g% a2 [/ ]3 a
) t$ W; B' t" z

Gibson S, Tu S, Oyer R, Anderson SM, and Johnson GL. Epidermal growth factor protects epithelial cells against Fas-induced apoptosis. Requirement for Akt activation. J Biol Chem 274: 17612-17618, 1999.

Gotzmann J, Mikula M, Eger A, Schulte-Hermann R, Foisner R, Beug H, and Mikulits W. Molecular aspects of epithelial cell plasticity: implications for local tumor invasion and metastasis. Mutat Res 566: 9-20, 2004.0 r# g" ^7 B& W/ F: {3 O9 [
* ~  a2 k; z9 q
  I. R0 ]( i' P; B9 Z( Y  a: ~. a6 s! h
; Y4 X6 Q5 G. W9 }' F
Haber PS, Keogh GW, Apte MV, Moran CS, Stewart NL, Crawford DH, Pirola RC, McCaughan GW, Ramm GA, and Wilson JS. Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis. Am J Pathol 155: 1087-1095, 1999.

2 ]! {: p( y' R/ O$ I$ [! V! U
! J7 b8 r' l8 F+ @' }2 c9 s0 E
Hoffmann M, Schmidt M, and Wels W. Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-. Cancer Immunol Immunother 47: 167-175, 1998.
) D2 K+ U. T6 b$ ~

/ p! ?1 p6 w$ X( X7 `) A" X
Inazaki K, Kanamaru Y, Kojima Y, Sueyoshi N, Okumura K, Kaneko K, Yamashiro Y, Ogawa H, and Nakao A. Smad3 deficiency attenuates renal fibrosis, inflammation, and apoptosis after unilateral ureteral obstruction. Kidney Int 66: 597-604, 2004.7 ~0 y+ M; A$ E$ ~3 Y4 }

& L( R3 P1 ]( V$ y
0 d* V/ t' t$ V' q! k
Jin S, Tong T, Fan W, Fan F, Antinore MJ, Zhu X, Mazzacurati L, Li X, Petrik KL, Rajasekaran B, Wu M, and Zhan Q. GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity. Oncogene 21: 8696-8704, 2002.

8 q9 ~0 V1 O' K8 k2 S# E/ H

Kalluri R and Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest 112: 1776-1784, 2003.

7 ?1 w, ]2 c: m# v- C0 [' g
! _% h+ a. P3 S$ B9 s) s
Kawakita T, Espana EM, He H, Li W, Liu CY, and Tseng SC. Intrastromal invasion by limbal epithelial cells is mediated by epithelial-mesenchymal transition activated by air exposure. Am J Pathol 167: 381-393, 2005.

- K$ @. U- \( U
( z; T$ S1 {2 H+ ~8 o" |" O' t: v
Keeton MR, Curriden SA, van Zonneveld AJ, and Loskutoff DJ. Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor. J Biol Chem 266: 23048-23052, 1991.

Khaliq A, Patel B, Jarvis-Evans J, Moriarty P, McLeod D, and Boulton M. Oxygen modulates production of bFGF and TGF- by retinal cells in vitro. Exp Eye Res 60: 415-423, 1995.; V1 m0 N& q/ b

6 G3 l) S; {/ R5 S
, E- m9 x" B8 f' a" e
Kiley SC, Thornhill BA, Tang SS, Ingelfinger JR, and Chevalier RL. Growth factor-mediated phosphorylation of proapoptotic BAD reduces tubule cell death in vitro and in vivo. Kidney Int 63: 33-42, 2003.0 z3 q# g" {5 ~/ k
; t1 v5 a% d. R5 Y. ~( ~
- g8 p. h6 P, L" `! {' X

Lallemand F, Mazars A, Prunier C, Bertrand F, Kornprost M, Gallea S, Roman-Roman S, Cherqui G, and Atfi A. Smad7 inhibits the survival nuclear factor B and potentiates apoptosis in epithelial cells. Oncogene 20: 879-884, 2001.
/ l3 A4 @! d3 t$ l' M
# h( L8 p  |) W9 P$ K+ _
7 `& X$ k- @9 d8 o5 e: G9 o7 e8 v
Lautrette A, Li S, Alili R, Sunnarborg SW, Burtin M, Lee DC, Friedlander G, and Terzi F. Angiotensin II and EGF receptor cross-talk in chronic kidney diseases: a new therapeutic approach. Nat Med 11: 867-874, 2005.( ~8 @/ A4 A0 l6 b. _
+ l& |" Y% f/ z! \: c( \

Li JH, Wang W, Huang XR, Oldfield M, Schmidt AM, Cooper ME, and Lan HY. Advanced glycation end products induce tubular epithelial-myofibroblast transition through the RAGE-ERK1/2 MAP kinase signaling pathway. Am J Pathol 164: 1389-1397, 2004.( J0 w3 d, s) o0 e+ @6 X$ G( B
4 n9 I  g) p. |5 }2 V6 }! ~* _( u

Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention. J Am Soc Nephrol 15: 1-12, 2004.

Lu Z, Ghosh S, Wang Z, and Hunter T. Downregulation of caveolin-1 function by EGF leads to the loss of E-cadherin, increased transcriptional activity of -catenin, and enhanced tumor cell invasion. Cancer Cell 4: 499-515, 2003./ ]! @% I- ?% {" I! o1 B4 O6 @

! b& u: G8 {# \$ v& P% u

Maddala R, Reddy VN, Epstein DL, and Rao V. Growth factor-induced activation of Rho and Rac GTPases and actin cytoskeletal reorganization in human lens epithelial cells. Mol Vis 9: 329-336, 2003.
" R% p8 {/ [9 u/ z5 |7 [( u" p
' F) `2 Q. l3 c

Manotham K, Tanaka T, Matsumoto M, Ohse T, Inagi R, Miyata T, Kurokawa K, Fujita T, Ingelfinger JR, and Nangaku M. Transdifferentiation of culture tubular cells induced by hypoxia. Kidney Int 65: 871-880, 2004.+ p( j- D; F  d  o9 i' o
% D, Q1 ]1 M5 r" n9 v  @' {- n
7 H/ }9 O! m5 p
! a! P: c$ u3 K4 B, ?6 M2 q
McMorrow T, Gaffney MM, Slattery C, Campbell E, and Ryan MP. Cyclosporine A-induced epithelial-mesenchymal transition in human renal proximal tubular epithelial cells. Nephrol Dial Transplant 20: 2215-2225, 2005.
/ n  p( ^) |# \) h" w
  K8 p, Z. Z0 J1 Z
. g8 Z- J' J) q" r& {" K
Mithani SK, Balch GC, Shiou SR, Whitehead RH, Datta PK, and Beauchamp RD. Smad3 has a critical role in TGF- -mediated growth inhibition and apoptosis in colonic epithelial cells. J Surg Res 117: 296-305, 2004.
! q% x- {+ ~( Z
4 o0 }6 k9 @6 d6 Z8 S
% }; U. }) C0 F1 |% j% U; F
Miyajima A, Chen J, Lawrence C, Ledbetter S, Soslow RA, Stern J, Jha S, Pigato J, Lemer ML, Poppas DP, Vaughan ED, and Felsen D. Antibody to transforming growth factor- ameliorates tubular apoptosis in unilateral ureteral obstruction. Kidney Int 58: 2301-2313, 2000.& v7 f7 I* E5 z& K' v/ Q

Miyoshi E, Higashiyama S, Nakagawa T, Hayashi N, and aniguchi N. Membrane-anchored heparin-binding epidermal growth factor-like growth factor acts as a tumor survival factor in a hepatoma cell line. J Biol Chem 272: 14349-14355, 1997.

/ z6 @, |6 h/ @; w
! R. V0 S) W- d$ C" a
Moriguchi Y, Matsubara H, Mori Y, Murasawa S, Masaki H, Maruyama K, Tsutsumi Y, Shibasaki Y, Tanaka Y, Nakajima T, Oda K, and Iwasaka T. Angiotensin II-induced transactivation of epidermal growth factor receptor regulates fibronectin and transforming growth factor- synthesis via transcriptional and posttranscriptional mechanisms. Circ Res 84: 1073-1084, 1999.

4 c" Z5 l9 P4 L" I

Nakamura Y, Hirano S, Suzuki K, Seki K, Sagara T, and Nishida T. Signaling mechanism of TGF- 1 -induced collagen contraction mediated by bovine trabecular meshwork cells. Invest Ophthalmol Vis Sci 43: 3465-3472, 2002.

Niculescu-Duvaz I, Colville-Nash P, and Dockrell M. TGF- 1 -induced fibronectin production in human proximal tubular cells is regulated by p38MAP kinase and is SMAD independent (Abstract). J Am Soc Nephrol 16: 610A, 2005.

Nightingale J, Patel S, Suzuki N, Buxton R, Takagi KI, Suzuki J, Sumi Y, Imaizumi A, Mason RM, and Zhang Z. Oncostatin M, a cytokine released by activated mononuclear cells, induces epithelial cell-myofibroblast transdifferentiation via Jak/Stat pathway activation. J Am Soc Nephrol 15: 21-32, 2004./ O3 N* Z5 [6 |& f# N. W1 q: V( N

+ g! X' T: u! t  M( V- O; H
7 o2 D4 h( q' }5 _' V! K) i
Ohgushi M, Kuroki S, Fukamachi H, O'Reilly LA, Kuida K, Strasser A, and Yonehara S. Transforming growth factor -dependent sequential activation of Smad, Bim, and caspase-9 mediates physiological apoptosis in gastric epithelial cells. Mol Cell Biol 25: 10017-10028, 2005.
' I3 t5 _. a3 k+ ]( M. w& D: s' P
5 c9 t8 s0 Q% K- s4 z
0 i  N2 x/ X) A6 L- a
Okada H, Danoff TM, Kalluri R, and Neilson EG. Early role of Fsp1 in epithelial-mesenchymal transformation. Am J Physiol Renal Physiol 273: F563-F574, 1997.

Osaki M, Oshimura M, and Ito H. PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis 9: 667-676, 2004./ N4 [% B* |& x8 V  s
3 K. C$ b; p" O, J* y! ^

! U7 i" ^  d+ u% [) Q3 y
Rhyu DY, Yang Y, Ha H, Lee GT, Song JS, Uh ST, and Lee HB. Role of reactive oxygen species in TGF- 1 -induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells. J Am Soc Nephrol 16: 667-675, 2005.
! F9 Q. F* E0 ~
; M4 {: |+ e# k
9 R( ^& @/ z. K; L: b' B: H6 Y, V2 o
Sato M, Muragaki Y, Saika S, Roberts AB, and Ooshima A. Targeted disruption of TGF- 1 /Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction. J Clin Invest 112: 1486-1494, 2003.* J! ^1 N5 p' |* F- i5 r
# {" n0 U" I8 ~

Shah BH, Farshori MP, and Catt KJ. Neuropeptide-induced transactivation of a neuronal epidermal growth factor receptor is mediated by metalloprotease-dependent formation of heparin-binding epidermal growth factor. J Biol Chem 279: 414-420, 2004.
! I4 m* J/ @' E3 x6 Q

* c3 Y1 L' V) C5 B
Shook D and Keller R. Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mech Dev 120: 1351-1383, 2003.
% k9 b5 g# O0 q% B9 f0 w

, X" H0 a- @+ [+ j5 U+ l+ Y5 M
Storch S, Saggi S, Megyesi J, Price PM, and Safirstein R. Ureteral obstruction decreases renal prepro-epidermal growth factor and Tamm-Horsfall expression. Kidney Int 42: 89-94, 1992.
# t. p; Y  l: c' g; S) B

* B. A+ }  Q1 k( f1 L2 {3 x
Strutz F, Zeisberg M, Renziehausen A, Raschke B, Becker V, van Kooten C, and Muller G. TGF- 1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor (FGF-2). Kidney Int 59: 579-592, 2001.2 S8 h/ X  l9 J0 e# |. i

5 i( s8 Y  K" m" w) T' C2 ~0 b
Suzuki S, Dobashi Y, Sakurai H, Nishikawa K, Hanawa M, and Ooi A. Protein overexpression and gene amplification of epidermal growth factor receptor in nonsmall cell lung carcinomas. An immunohistochemical and fluorescence in situ hybridization study. Cancer 103: 1265-1273, 2005.! S5 {. A8 f; A5 [  a( A+ i

3 b* |# Y+ o! h0 B
& J& ^) c" P  I8 h& k, Z& h
Terzi F, Burtin M, Hekmati M, Federici P, Grimber G, Briand P, and Friedlander G. Targeted expression of a dominant-negative EGF-R in the kidney reduces tubulo-interstitial lesions after renal injury. J Clin Invest 106: 225-234, 2000.

( _/ B4 F* P; t/ E0 e
Vesey DA, Cheung CW, Cuttle L, Endre ZA, Gobe G, and Johnson DW. Interleukin-1 induces human proximal tubule cell injury, -smooth muscle actin expression and fibronectin production. Kidney Int 62: 31-40, 2002.
! t0 X) @) s0 [" ^( f4 O4 E

Wolf G, Thaiss F, and Stahl RA. Cyclosporine stimulates expression of transforming growth factor- in renal cells. Possible mechanism of cyclosporines antiproliferative effects. Transplantation 60: 237-241, 1995.5 Z/ a( Q7 m% j; N3 r

Wu DT, Bitzer M, Ju W, Mundel P, and Bottinger EP. TGF- concentration specifies differential signaling profiles of growth arrest/differentiation and apoptosis in podocytes. J Am Soc Nephrol 16: 3211-3221, 2005.

: F4 }- v% R$ B$ y; n* N6 _- _! J
Yang YA, Tang B, Robinson G, Hennighausen L, Brodie SG, Deng CX, and Wakefield LM. Smad3 in the mammary epithelium has a nonredundant role in the induction of apoptosis, but not in the regulation of proliferation or differentiation by transforming growth factor-. Cell Growth Differ 13: 123-130, 2002.
8 N- E$ B. g4 a
: q, r0 j) S4 z3 {8 \
* O9 L. i3 B5 b: ~. |
Yoo J, Ghiassi M, Jirmanova L, Balliet AG, Hoffman B, Fornace AJ Jr, Liebermann DA, Bottinger EP, and Roberts AB. Transforming growth factor- -induced apoptosis is mediated by Smad-dependent expression of GADD45b through p38 activation. J Biol Chem 278: 43001-43007, 2003.
/ R# Y5 p$ {) q# r" O
6 ?9 O- g8 K( y0 [+ B9 }
) S/ Q) K3 |- `
Zavadil J and Bottinger EP. TGF- and epithelial-to-mesenchymal transitions. Oncogene 24: 5764-5774, 2005.
7 K1 `% {6 F- M# N; m: f
$ v0 U  B; L" k/ {# w1 t- u) ?

Zeisberg M, Bonner G, Maeshima Y, Colorado P, Muller GA, Strutz F, and Kalluri R. Renal fibrosis: collagen composition and assembly regulates epithelial-mesenchymal transdifferentiation. Am J Pathol 159: 1313-1321, 2001." M$ Q" X2 b* H4 U. D0 C  }) [

, g  k% S4 v/ e$ \2 R$ a

Zeisberg M and Kalluri R. The role of epithelial-to-mesenchymal transition in renal fibrosis. J Mol Med 82: 175-181, 2004.6 a0 @) T  M% A, W( I( ?; D
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Zhuang S and Schnellmann RG. H 2 O 2 -induced transactivation of EGF receptor requires Src and mediates ERK1/2, but not Akt, activation in renal cells. Am J Physiol Renal Physiol 286: F858-F865, 2004.

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