Skip to main content
NCBI home page
The American Journal of Pathology logoLink to The American Journal of Pathology
. 2010 Oct;177(4):1743–1754. doi: 10.2353/ajpath.2010.091183

Transforming Growth Factor-β1 Induces Smad3-Dependent β1 Integrin Gene Expression in Epithelial-to-Mesenchymal Transition during Chronic Tubulointerstitial Fibrosis

Yi-Chun Yeh *, Wei-Chun Wei *, Yang-Kao Wang †, Shih-Chieh Lin *, Junne-Ming Sung ‡, Ming-Jer Tang *,§,*
PMCID: PMC2947271  PMID: 20709799

Abstract

Transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) contributes to the pathophysiological development of kidney fibrosis. Although it was reported that TGF-β1 enhances β1 integrin levels in NMuMG cells, the detailed molecular mechanisms underlying TGF-β1-induced β1 integrin gene expression and the role of β1 integrin during EMT in the renal system are still unclear. In this study, we examined the role of β1 integrin in TGF-β1-induced EMT both in vitro and in vivo. TGF-β1-induced augmentation of β1 integrin expression was required for EMT in several epithelial cell lines, and knockdown of Smad3 inhibited TGF-β1-induced augmentation of β1 integrin. TGF-β1 triggered β1 integrin gene promoter activity as assessed by luciferase activity assay. Both knockdown of Smad3 and mutation of the Smad-binding element to block binding to the β1 integrin promoter markedly reduced TGF-β1-induced β1 integrin promoter activity. Chromatin immunoprecipitation assay showed that TGF-β1 enhanced Smad3 binding to the β1 integrin promoter. Furthermore, induction of unilateral ureteral obstruction triggered increases of β1 integrin in both renal epithelial and interstitial cells. In human kidney with chronic tubulointerstitial fibrosis, we also found a concomitant increase of β1 integrin and α-smooth muscle actin in tubule epithelia. Blockade of β1 integrin signaling dampened the progression of fibrosis. Taken together, β1 integrin mediates EMT and subsequent tubulointerstitutial fibrosis, suggesting that inhibition of β1 integrin is a possible therapeutic target for prevention of renal fibrosis.

Tubulointerstitial fibrosis is a critical event in chronic renal failure.1,2 These processes are characterized by accumulation of interstitial fibroblasts, deposition of extracellular matrix (ECM), and loss of renal tubule epithelial cells, which collectively lead to end-stage renal failure.3 Previous studies indicate that the tubular epithelial cells undergoing epithelial-to-mesenchymal transition (EMT) contribute to one-third of myofibroblast during kidney fibrosis.4,5 Myofibroblasts are fully differentiated fibroblasts which are α-smooth muscle actin (α-SMA)-positive cells and the main source for ECM. Thus, myofibroblasts can be used as prognostic indicators for disease progression.6 In contrast, several studies indicate that such event of EMT in kidney fibrosis could be reversed by growth factors, such as hepatocyte growth factor and bone morphogenetic protein-7.7,8

In addition to organ fibrosis, recent studies indicated the pivotal role of EMT in pathological progression, including cancer metastasis and wound healing.9–11 There are two major cellular processes in EMT; one is the loss of epithelial cell polarity. This phenomenon can be manifested by membrane proteins involved in cell polarization and the reduction of cohesive interactions, which mediated by expression of various adherent molecules, such as E-cadherin. The other process is the acquisition of mesenchymal cell characteristics, such as enhanced cell motility, contractility or even invasive ability, and deposition of ECM.12,13 Many cytokines and growth factors are involved in EMT. Nevertheless, transforming growth factor-β1 (TGF-β1) and its downstream Smad-dependent signaling are the major pathways that trigger EMT both in vitro and in vivo.14,15 On binding of TGF-β1 to its receptor, TGF-β1 induces receptor autophosphorylation, which in turns activates the downstream R-Smad, Smad2, and Smad3. The activated R-Smads form complex with Smad4, and this complex translocates into nucleus and binds to specific promoter sequence.16 Smad2 and Smad3 control different target gene expression in response to TGF-β1. It has been documented that Smad3 mediates most of the gene induction related to TGF-β1-induced EMT, particularly during kidney fibrosis.17,18

ECM signaling mediated by integrins is important in regulating growth factors signaling, including epidermal growth factor, hepatocyte growth factor, and vascular endothelial growth factor.19 Among these integrin family members, β1 integrin is the most critical one, given that β1 integrin can pair with different α subunits that make it become a receptor for many types of ECM component. The expression of β1 integrin is ubiquitous in different tissues.20 Knockout of β1 integrin in mice resulted in embryonic lethal because of the defect in gastrulation and organ formation.21,22 Recent studies have demonstrated that inhibition of β1 integrin signal impairs TGF-β1 downstream signaling and epithelial plasticity in mammary gland epithelial cells.23 Blocking of integrin downstream signaling including integrin-link kinase (ILK) and focal adhesion kinase (FAK) can also reduce TGF-β1-induced up-regulation of mesenchymal markers.24,25 However, the role of β1 integrin in renal fibrosis has not been studied. In addition, although studies have shown that TGF-β1 induces the gene expression of β1 integrin in both mammary and mesangial cells,26,27 the molecular mechanism whereby TGF-β1 up-regulates gene expression of β1 integrin is still unclear.

In this study, we demonstrate that TGF-β1 up-regulates β1 integrin gene expression in different types of epithelial cell through transcriptional regulation, and β1 integrin gene expression is critical for TGF-β1-induced EMT. Furthermore, Smad3 and the Smad-binding element on β1 integrin promoter sequence are responsible for TGF-β1-induced β1 integrin gene expression. The in vivo study also shows the up-regulation of β1 integrin during kidney fibrosis. β1 integrin and coexpressed with α-SMA in both mouse model and human samples. Furthermore, blocking of β1 integrin signal alleviates unilateral ureteral obstruction (UUO)-induced renal fibrosis.

Materials and Methods

Cell Culture and Treatment

LLC-PK1, Madin-Darby canine kidney, HaCaT, and NMuMG cells were cultured in Dulbecco's modified minimal essential medium supplemented with 10% FBS. For experiments, 8 × 105 cells per 10-cm dish were plated and cultured in medium containing 10% FBS for 24 hours. After that, cells were treated recombinant TGF-β1 (PeproTech, London, UK) in serum free medium at the indicated time point. For β1 integrin blocking experiments, cells were pretreated with blocking antibody 4B4 (Beckman Coulter, Fullerton, CA) at the dosage of 10 μg/ml for 30 minutes, followed by incubating with 10 ng/ml TGF-β1 for another 48 hours.

Western Blotting

Western blot was performed according to the established procedure.28,29 The monoclonal antibodies against β1 integrin, E-cadherin, and fibronectin were purchased from BD Biosciences (San Jose, CA). Antibodies against integrins α1, α2, α5, and αv were all purchased from Millipore (Billerica, MA). Polyclonal antibody against p-Smad3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against α-SMA and Smad3 were purchased from Sigma-Aldrich (St. Louis, MO) and Zymed Laboratories (South San Francisco, CA), respectively.

RT-PCR

Total RNA from cultured cells was extracted with the RNeasy Mini kit (Qiagen; Hilden, Germany). Total RNA from each mouse kidney was isolated by using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). For RT-PCR, first-strand cDNA was synthesized from 0.2 μg of total RNA with an oligo-dT primer and the Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). The sequences of PCR primers were as follows: forward primer (5′-CGGGAGAAAATGCTCCAATA-3′) and reverse primer (5′-CACACTCAAACGTCCCATTG-3′) were designed from Sus scrofa β1 integrin (CD29) (NCBI accession number NM_213968), and the resulting PCR product was 227 bp. Forward primer (5′-GCCAGGGCTGGTTATACAGA-3′) and reverse primer (5′-TCACAATGGCACACAGGTTT-3′) were designed from Mus musculus β1 integrin (CD29) (NCBI accession number NM_010578), and the resulting PCR product was 226 bp. Forward primer (5′-ATACGCCTGAGTGGCTGTCt-3′) and reverse primer (5′-TCTCTGTGGAGCTGAAGCAA-3′) were designed from Mus musculus TGF-β1 (Tgfb1) (NCBI accession number NM_011577), and the resulting PCR product was 300 bp.

Immunofluorescence

For immunofluorescence studies, cells were fixed with 4% paraformaldehyde in PBS, and cell membrane was permeabilized using 0.5% Triton X-100. These samples were blocked with commercial blocking solution (Thermal Scientific, Rockford, IL) at room temperature and then incubated with primary antibody, followed by incubation with fluorescence-conjugated secondary antibody (Invitrogen-Molecular Probes, Carlsbad, CA).30 Antibodies against fibronectin and E-cadherin were purchased from BD Biosciences. Phalloidin conjugated with tetramethylrhodamine isothiocyanate was purchased from Fluka (Steinheim, Germany). For tissue sections, 4-μm tissue sections from paraffin or Tissue-Tek OCT compound-embedded were used for analyses. Tissue samples were fixed by acetone for 2 minutes followed by antigen retrieval with citrate acid buffer by microwave. Antibodies against β1 integrin (BioLegend, San Diego, CA; clone HMβ1-1, Alexa 488), E-cadherin (BD Biosciences), TGF-β1 (Santa Cruz Biotechnology), p-Smad3 (Santa Cruz Biotechnology), and α-SMA (Sigma-Aldrich) were used. Finally, images were taken by using the confocal microscope (FV-1000; Olympus, Melville, NY).

Short Hairpin RNA Inhibition

To knockdown β1 integrin expression in LLC-PK1 epithelial cells, 19-mer short hairpin RNA (shRNA) against Sus scrofa β1 integrin expressed in pSUPER vector was synthesized. Sequences for β1 integrin are 5-GTGCTCAGCCTTACTGATA-3′ and 5′-TATCAGTAAGGCTGAGCAC-3′. Two mouse Smad3 shRNAs purchased from GenDiscovery Biotechnology (Taipei, Taiwan) (catalog number RMM4431-98765463 and RMM4431-99202964, labeled as numbers 1 and 2, respectively) were used. These clones were expressed in GPIz expression vector (Open Biosystems, Huntsville, AL). One microgram of specific or control shRNA duplexes were transiently transfected into LLC-PK1 cells by using the Arrest-In transfection reagent (Open Biosystems). After transfection, cells were trypsinized and subjected to various experiments.

Plasmid Construction, Transfection, and Reporter Assay

The β1 integrin promoter (nt −1057 to + 77) was cloned from mouse kidney using PCR amplification; the primer sequence for cloning was 5′-TCCCTCCTCAAGTCACACG-3′ and 5′-GCTTCTCGGTTGGTCTCG-3′. The promoter sequence was conjugated into pGL3-basic vector by using the NheI and BglII restriction enzyme sites. The identification of the transcriptional factor binding site of β1 integrin promoter sequence was made by the online software PROMO 3.0 (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3). The GACA core binding element on Smad-binding element (SBE) in β1 integrin promoter from −172 to −181 was mutated using PCR amplification with the following primer: 5′-AAAGACCGAGCTCCTTTGAAAACACCCTGCCCCACCTTCC-3′ and 5′-GGAAGGTGGGGCAGGGTGTTTTCAAAGGAGCTCGGTCGTT-3′. The promoter activity assay was previously described 31 with modification. The underlined nucleotide sequences were made for generating point mutation in SBE. Briefly, NMuMG cells were cotransfected with reporter plasmids and the β-galactosidase plasmid, used as internal control reporter (provided by Dr. Shaw-Jenq Tsai, National Cheng Kung University, Taiwan, People's Republic of China). Luciferase assay was performed using the Dual Luciferase Assay System kit (Promega) per manufacture's protocol. Relative luciferase activity (arbitrary unit) was reported after normalizing with transfection efficiency.

Chromatin Immunoprecipitation

The DNA-protein complexes were fixed using 1% formaldehyde at room temperature for 10 minutes at indicating time period. The ice-cold glycine was performed to block the cross-link reaction. Cell lysates were collected and the chromatin was sheared to 200 to 1000 bp by using sonication. Same amount of DNA was used for immunoprecipitation by incubating with anti-Smad3 antibody (Santa Cruz Biotechnology). After reversion of the cross-linking of DNA and protein, the DNA was subject to PCR amplification using primers specific for amplifying regions corresponding to SBE (nt −172 to −181). The DNA was subject to a first round of PCR amplification using the outer primers (5′- GACTGCGGCTGGGCTTTC-3′ and 5′-AGCGAGTGGGCGGAGACT-3′) for 20 cycles. The cDNA was then diluted (1/1000) with water and subject to second round of amplification using nested primers (5′-GACTGCGGCTGGGCTTTC-3′ and 5′-AGGCGGCAGGAAGAAGGA-3′) for 30 cycles.

UUO Animals and β1 Integrin Antibody Treatment

Procedures involving animal subjects have been proved by the Institutional Animal Care and Use Committee at the National Cheng-Kung University (Tainan, Taiwan). One-month-old male C57BL/6 mice, weighing ∼20 to 25 g, were obtained from the animal center in National Cheng-Kung University. UUO was performed with an established procedure, as described previously.32 After UUO surgery, mice were sacrificed at various time points, and kidneys were removed. Part of kidney was fix by 4% paraformaldehyde and embedded either in paraffin or in Tissue-Tek OCT compound, respectively. The rest parts of kidney were divided and lysed for either protein or RNA analysis.

For α5 or β1 integrin functional blocking experiments, 60 μl of α5 integrin blocking antibody (clone eBioHMa5-1; eBioscience, San Diego, CA) or β1 integrin blocking antibody (3 mg/kg body weight) (clone HMβ1-1, LEAF; BioLegend) was injected into the parenchyma of UUO kidney immediately right after the ureteral obstruction. The same amount of PBS was injected as a control. After 7 days, mice were scarified, and the kidneys were harvested for further analysis.

Human Kidney Specimens

Three delinked human kidney specimens obtained from tissue bank of National Cheng Kung University Hospital were immunohistochemically investigated for the presence of marker for interstitial fibrosis and β1 integrin. All three patients were diagnosed clinically as chronic kidney diseases (their urine protein/creatinine ratios were all <1, and serum creatinine levels were 4.1, 3.5, and 2.4 mg/dl, respectively), and renal biopsies were performed for the definite pathological diagnosis. Two patients were diagnosed as chronic and moderate tubulointerstitial fibrosis where the other patient was diagnosed as moderate tubulointerstitial fibrosis and mild mesangial proliferation with small amount of IgA deposition. The distributions of β1 integrin were stained by anti-human β1 integrin antibody, 4B4, followed by secondary antibody conjugated with Alexa 488.

Statistical analyses

All results were expressed as mean ± SEM and were analyzed by using one-way analysis of variance (analysis of variance) using GraphPad Prism version 3.0 (GraphPad, San Diego, CA). Tukey's procedure was used to test the differences between individual treatment groups. Differences in comparison were considered as statistically significant when P < 0.05.

Results

TGF-β1 Induced Up-Regulation of β1 Integrin before EMT

To explore the mechanism of TGF-β1-induced EMT, we first examined the changes of β1 integrin, fibronectin and E-cadherin in TGF-β1-treated LLC-PK1 cells. On TGF-β1 stimulation, β1 integrin was up-regulated within 4 hours, whereas fibronectin and α-SMA levels were up-regulated and E-cadherin was down-regulated within 24 hours (Figure 1A). TGF-β1 treatment induced an increase not only in premature β1 integrin (∼110 kDa), but also glycosylated β1 integrin (∼130 kDa), consistent with the previous report.33 The quantitative results showed that TGF-β1 markedly augmented β1 integrin levels from 4 to 16 hours and the increase was maintained at 2.5- to 3-fold of control between 24 and 72 hours. In contrast, TGF-β1-triggered alterations of fibronectin, E-cadherin and α-SMA protein levels started at 24 hours (Supplemental Figure 1A, see http://ajp.amjpathol.org). To understand the mechanism of TGF-β1-induced up-regulation of β1 integrin, levels of β1 integrin mRNA were assessed by RT-PCR. The mRNA levels of β1 integrin increased within 4 hours, consistent with protein level changes on TGF-β1 stimulation (Figure 1B and Supplemental Figure 1B, see http://ajp.amjpathol.org). This result suggests that transcriptional regulation could be involved. To test this possibility, LLC-PK1 cells were treated with TGF-β1 and different concentrations of actinomycin D, 0.5 or 1 μg/ml, for 16 hours. As shown in Figure 1C, TGF-β1-induced augmentation of β1 integrin mRNA levels was inhibited by actinomycin D in a dose-dependent manner. These data indicate that TGF-β1 up-regulates β1 integrin through transcriptional regulation.

Figure 1.

Figure 1

Open in a new tab

TGF-β1 up-regulates β1 integrin expression. A: TGF-β1 induced changes of EMT proteins in LLC-PK1 cells. LLC-PK1 cells were cultured with or without 10 ng/ml TGF-β1 at the indicated times, and protein levels were assessed by Western blot analysis. B: LLC-PK1 cells were treated with or without 10 ng/ml TGF-β1 at the indicated times. Cells were harvested, and the levels of β1 integrin mRNA were analyzed by RT-PCR. C: LLC-PK1 cells were treated with or without 10 ng/ml TGF-β1 in the presence or absence of different doses (0.5 or 1 μg/ml) of actinomycin D (Act. D) for 16 hours. Cells were harvested, and their levels of β1 integrin RNA levels were assessed by RT-PCR. D: LLC-PK1 cells treated with 10 ng/ml TGF-β1 for 24 hours were harvested. Protein levels, including α1, α2, α5, αv, and β1 integrin, were subjected to Western blot analysis. E and F: Epithelial cells, including LLC-PK1, Madin-Darby canine kidney (MDCK), and NMuMG (E), and renal fibroblast cells, NRK49F (F), were incubated with 10 ng/ml TGF-β1 for 24 hours, and protein abundance of β1 integrin was assessed by Western blot.

To examine whether other integrins were regulated by TGF-β1, the protein levels of α1, α2, α5, and αv integrin in LLC-PK1 cells treated with TGF-β1 for 24 hours were assessed. TGF-β1 up-regulated α2 and αv integrins moderately but not α1 and α5 integrin protein levels (Figure 1D). Because the protein level changes of β1 integrin was more significant on TGF-β1 stimulation, we then focused on the regulation and function of β1 integrin. Next, we examined whether TGF-β1-induced up-regulation of β1 integrin could be observed in different epithelial cell lines, including LLC-PK1, Madin-Darby canine kidney, and NMuMG cells, as well as in renal fibroblast cells (NRK49F). The results show that TGF-β1-induced up-regulation of β1 integrin is a general phenomenon in these epithelial and renal fibroblast cells examined (Figure 1, E and F).

β1 Integrin Signal Is Required for TGF-β1-Induced EMT

Although it had been shown that β1 integrin signal is required for TGF-β1-triggered lost of epithelial plasticity in mammary gland epithelial cells, the functions of β1 integrin in renal epithelial cells during EMT has never been reported. Here, we used β1 integrin specific blocking antibody, 4B4, to examine the role of β1 integrin in TGF-β1-triggered EMT in renal epithelial cells. 4B4 treatment alone did not affect the protein abundances of fibronectin, E-cadherin, or α-SMA. However, 4B4 treatment significantly blocked TGF-β1-induced up-regulation of fibronectin and α-SMA and down-regulation of E-cadherin (Figure 2, A and B). Consistent with the results shown in Western blot analysis, 4B4 treatment attenuated TGF-β1-triggered decrease in E-cadherin and increase in fibronectin and α-SMA staining (Figure 2C). Moreover, 4B4 treatment blocked TGF-β1-induced spindle shape and stress fiber formation, which are the characteristics of mesenchymal cells.

Figure 2.

Figure 2

Open in a new tab

Blocking of β1 integrin signal inhibits TGF-β1-induced EMT. A: LLC-PK1 cells were pretreated with or without 10 μg/ml 4B4, β1 integrin-specific blocking antibody, for 30 minutes. Cells were cultured with or without 10 ng/ml TGF-β1 in the presence or absence of 4B4 for another 48 hours, and protein levels were assessed by Western blot. B: Quantitative results show the effects of 4B4 on the inhibition of TGF-β1-induced changes in EMT marker protein abundance, including E-cadherin, α-SMA, and fibronectin. Each bar represents the mean ± SE from three independent experiments. ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001. C: LLC-PK1 cells treated with TGF-β1 in the presence or absence of 4B4 for 48 hours were fixed and immunostained with mouse monoclonal antibodies against E-cadherin, fibronectin, or α-SMA, and the immunocomplex was detected by anti-mouse Alexa-488 (green). F-actin was stained by phalloidin conjugated with tetramethylrhodamine isothiocyanate (red), and nuclei were stained with Hoechst 33258 (blue).

Usually, fibrosis is accompanied with fibril collagen deposition and α2 integrin is the major mediator for collagen signaling. To examine whether α2β1 integrin heterodimer was required in TGF-β1-induced EMT, we used the blocking antibody, 5E8.34 The result showed that blocking of α2β1 integrin prevented TGF-β1-induced up-regulation of fibronectin and α-SMA and down-regulation of E-cadherin (Supplemental Figure 2, see http://ajp.amjpathol.org). These results indicate α2β1 integrin signal is critical for TGF-β1-triggered EMT in renal epithelial cells.

Knockdown of β1 Integrin Prevents TGF-β1-Induced EMT

To examine whether protein abundance of β1 integrin was important in TGF-β1-induced EMT, shRNA against Sus scrofa β1 integrin was performed. RT-PCR result showed that β1 integrin shRNA reduced both mRNA and protein levels of β1 integrin compared with scramble shRNA trascfection group. Knockdown of β1 integrin blocked TGF-β1-induced up-regulation of fibronetin and α-SMA and down-regulation of E-cadherin (Figure 3A). The data suggest that induction of β1 integrin is required for TGF-β1-triggered EMT.

Figure 3.

Figure 3

Open in a new tab

β1 integrin is required for TGF-β1-induced EMT. A: LLC-PK1 cells transiently transfected with scramble or β1 integrin shRNA were incubated with or without 10 ng/ml TGF-β1 for 48 hours. Cells lysates and RNA were harvested and analyzed by Western blot or RT-PCR, respectively. B, left: HaCaT cells were treated with or without 10 ng/ml TGF-β1 for 48 hours, and protein levels were assessed by Western blot analysis. B, right: HaCaT cells were stably transfected with constitutively active β1 integrin (Caβ), and protein levels in vector control cells (mock) and Caβ-overexpressing clones, numbers 1 and 2, were assessed. C: Expression of E-cadherin, fibronectin, and actin cytoskeleton was examined in HaCaT cells treated with or without TGF-β1 for 48 hours or overexpressed with Caβ integrin. Mouse monoclonal antibodies against E-cadherin (upper panels) and fibronectin (lower panels) were detected by anti-mouse Alexa-488 (green). F-actin was stained with phalloidin conjugated with tetramethylrhodamine isothiocyanate (red), and nuclei were stained with Hoechst 33258 (blue).

Next, we examined whether the overexpression of β1 integrin alone triggered EMT. HaCaT cells stably expressed with β1 integrin were used. On TGF-β1 stimulation, β1 integrin protein level increased within 48 hours (Figure 3B), further suggesting that TGF-β1-up-regulated β1 integrin expression is a general phenomenon in epithelial cells. In addition, TGF-β1 treatment resulted in the up-regulation of fibronectin and down-regulation of E-cadherin in HaCaT cells, which could also be observed in cells transfected with constitutively active β1 integrin (Caβ), clone numbers 1 and 2 (Figure 3B). Immunofluorescence study showed that both TGF-β1 treatment and overexpression of Caβ induced mesenchymal cell morphology, enhanced fibronectin expression, and reduced E-cadherin levels (Figure 3C). These results indicate that forced increase in β1 integrin signal alone accelerates EMT progression.

TGF-β1 Induces β1 Integrin Up-Regulation through Smad3-Dependent Pathway

To identify which signaling pathway was involved in TGF-β1-induced β1 integrin up-regulation, phosphorylation levels of Smad3, Erk, Akt, and p38 were assessed on TGF-β1 stimulation. TGF-β1 treatment induced a significant increase of phosphorylation in Smad3, but not Erk, Akt, or p38, within 30 minutes and sustained to 4 hours in both LLC-PK1 and NMuMG cells (data not shown). To further verify the role of Smad3 in TGF-β1-induced β1 integrin gene expression, two different shRNAs against mus musculus Smad3 were used, numbers 1 and 2 (Figure 4A). Knockdown of Smad3 suppressed TGF-β1-induced β1 integrin protein and mRNA up-regulation in a dose-dependent manner (Figure 4, A and B). These data indicate that Smad3 is required for TGF-β1-induced β1 integrin up-regulation.

Figure 4.

Figure 4

Open in a new tab

Knockdown of Smad3 reduces β1 integrin expression induced by TGF-β1. NMuMG cells transiently transfected with scramble or two different Smad3 shRNAs (numbers 1 and 2) were cultured in the presence or absence of 10 ng/ml TGF-β1 for 24 hours. Cells transfected with scramble shRNA were used as a control. A: Protein levels of Smad3 and β1 integrin were assessed by Western blot analysis. B: Cells subjected to RNA isolation were used for β1 integrin mRNA levels analyzed by RT-PCR.

TGF-β1-Triggered β1 Integrin Promoter Activity Is Smad3 Dependent

Because Smad3 is responsible for most of the genes triggered by TGF-β1,17,18 we were wondering whether there was SBE on β1 integrin promoter. Using bioinformatics analysis, we have discovered one Smad3 and Smad4 binding element at 182 bp upstream of the transcription start site on mouse β1 integrin promoter (Figure 5A). We also analyzed the promoter sequence among different species and found that SBE on the β1 integrin promoter within 1 kbp in several species, including mouse, rat, dog, and porcine. We then cloned β1 integrin promoter sequence of 1134 bp from mouse kidney genomic DNA into pGL3-Luc plasmid and tested whether TGF-β1 up-regulated β1 integrin promoter activity. NMuMG cells transfected with β1 integrin promoter- driven luciferase showed basal activation levels, whereas TGF-β1 treatment increased promoter activity in a dose-dependent manner (Figure 5B). To examine the role of Smad3 in TGF-β1-induced up-regulation of β1 integrin promoter activity, NMuMG cells were cotransfected Smad3 shRNAs, numbers 1 or 2, and β1 integrin promoter. The results showed that knockdown of Smad3 decreased TGF-β1-induced β1 integrin promoter activity (Figure 5C). These results indicate that Smad3 is required for TGF-β1-induced β1 integrin gene expression.

Figure 5.

Figure 5

Open in a new tab

TGF-β1-induced increase of β1 integrin promoter activity is Smad3 dependent. A: Schematic representation of the β1 integrin promoter sequence shows predicted binding sites for Smad3 and Smad4. The 1134-bp β1 integrin promoter sequence was cloned from normal mouse kidney. The Smad-binding element mutant (Mut) with core binding element CAGA mutated to TTTT was generated using a point mutagenesis kit. B: NMuMG cells transfected with control vector (p-GL3 basic) or wild-type β1 integrin promoter (β1 WT) along with a β-galactosidase plasmid were cultured in serum-free media in the presence or absence of different doses of TGF-β1 (1, 5, or 10 ng/ml) for 24 hours. The β1 integrin promoter activities were assessed by luciferase assay. C: NMuMG cells co-transfected with WT-β1 integrin promoter (β1 WT) and different Smad3 shRNAs, number 1 or 2, or scramble shRNA were treated with or without 10 ng/ml TGF-β1 for 24 hours. D: NMuMG cells transfected with wild-type β1 integrin promoter (β1 WT) or the mutant form of the β1 integrin promoter (β1 Mut) were cultured with or without 10 ng/ml TGF-β1 for 24 hours and then subjected to promoter activity assay. The β1 integrin promoter activities were assessed by luciferase assay, and the results were normalized by β-galactosidase activity levels (Luc./β-gal). ***P < 0.001. E: Representative results of chromatin immunoprecipitation (ChIP) assay show that TGF-β1 enhanced the interactions between Smad3 and the β1 integrin promoter sequence in NMuMG cells. NMuMG cells were cultured in the presence or absence of 10 ng/ml TGF-β1 for the indicated times. Cell lysates were immunoprecipitated with monoclonal anti-Smad3 antibody. The immunoprecipitated DNA was subjected to PCR amplification using primers specific for SBE on the β1 integrin promoter sequence. F: Quantitative results of the relative intensity of the PCR product of the β1 integrin promoter as assessed by ChIP in NMuMG cells treated with TGF-β1 for the indicated times. Each bar represents the mean ± SE of three independent experiments. *P < 0.05; **P < 0.01.

Although bioinformatics analysis and above results suggest that SBE is present on β1 integrin promoter sequence, the direct evidence of binding of Smad3 to β1 integrin promoter remains unclear. To delineate whether SBE is responsible for TGF-β1-triggered β1 integrin promoter activity, mutation of SBE by site-directed mutagenesis was made. The results showed that SBE mutant markedly reduced TGF-β1-triggered β1 integrin promoter activity from eight- to threefold of control (Figure 5D), suggesting that the predicted SBE on β1 integrin promoter is critical for TGF-β1-triggered β1 integrin gene regulation. To further examine whether Smad3 directly binds to β1 integrin promoter on TGF-β1 stimulation, we used chromatin immunoprecipitation assay. The results showed that TGF-β1 induced Smad3 binding to the SBE region on β1 integrin promoter within 1 to 8 hours (Figure 5E). Quantitative analysis demonstrated that TGF-β1 augmented the interactions of Smad3 with β1 integrin promoter (Figure 5F). Taken together, we conclude that TGF-β1 stimulation triggers physical interaction of Smad3 with SBE of β1 integrin promoter to enhance gene regulation.

UUO Induces Up-Regulation of β1 Integrin in Renal Tubular Epithelial Cells during Kidney Fibrosis

Although in vitro studies indicate the important role of β1 integrin in TGF-β1-induced EMT, little is known about the role of β1 integrin in the pathogenesis of renal fibrosis. To examine the role of β1 integrin in the development of kidney fibrosis, we used UUO. Results of Western blot analysis showed that β1 integrin protein levels increased by sixfold in UUO kidney within 3 days, accompanied with the up-regulation of α-SMA and down-regulation of E-cadherin at the same time (Figure 6A). Since up-regulation of TGF-β1 plays an important role in initiation of renal fibrosis, we assessed the time course of mRNA levels of TGF-β1 and β1 integrin. We found that there was an increase in both TGF-β1 mRNA and β1 integrin mRNA levels within 1 day of UUO (Figure 6B). Immunofluorescence study was used to detect the alterations of TGF-β1 and β1 integrin in UUO for 7 days. In control kidney, the intensity of TGF-β1 and β1 integrin in tubule epithelium was relatively weak, whereas the staining of β1 integrin was high in glomeruli. After UUO, the intensity of TGF-β1 was markedly enhanced in both glomerulus and tubular epithelial cells, consistent with the previous report.32 The β1 integrin staining was significantly augmented in renal tubular epithelial cells as well as cortical interstitium (Supplemental Figure 3, see http://ajp.amjpathol.org). These results highlight the spatial correlations of TGF-β1 and β1 integrin during kidney fibrosis.

Figure 6.

Figure 6

Open in a new tab

β1 integrin is enhanced in UUO-induced fibrotic kidney. A: Mice with UUO were sacrificed at 3, 7, 14, or 21 days, and the sham group was used as a surgery control. The protein levels of β1 integrin, E-cadherin, and α-SMA were assessed by Western blot analysis of the contralateral kidney (C) or UUO kidney (U). B: Kidney samples from mice with UUO for 1, 3, and 7 days were subjected to RT-PCR analysis. The mRNA levels of TGF-β1 and β1 integrin were analyzed by specific primers. To rule out contamination by genomic DNA, four samples subjected to RT-PCR without addition of reverse transcriptase (MMLV) enzyme (-RT) were used as a negative control. C and D: Kidney samples from mice with UUO for 7 days were subjected to double-staining with p-Smad3 and β1 integrin (C) or α-SMA and β1 integrin (D). Tissue sections were incubated with β1 integrin monoclonal antibody conjugated with Alexa-488 (green) and anti-p-Smad3 antibody, followed by anti-rabbit Alexa-594 (red). Nuclei were stained with Hoechst 33258 (blue). Monoclonal antibody against α-SMA was used followed by anti-mouse Alexa-594 (red). Stars indicated renal tubules with positive β1 integrin and α-SMA staining.

To examine the signaling events triggered by TGF-β1 in UUO, we analyzed the activation levels of Smad3. Interestingly, the up-regulation of both Smad3 protein and p-Smad3 was present in UUO kidney within 1 day, which was associated with the up-regulation of β1 integrin protein levels (Supplemental Figure 4, A and B, see http://ajp.amjpathol.org). To further confirm the signaling cascade of TGF-β1/p-Smad3/β1 integrin gene expression, double-staining of p-Smad3 and β1 integrin was performed. The results indicated in UUO kidney that the fluorescence intensity of p-Smad3 in nuclei was increased and associated with the up-regulation of β1 integrin staining (Figure 6C). These results highlight the importance of Smad3-dependent pathway in TGF-β1-induced up-regulation of β1 integrin during kidney fibrosis.

As shown in Figure 6D, normal renal tubule epithelial cells exhibited cuboid cell shape and little immunostaining of α-SMA, whereas UUO triggered these cells to exhibit mesenchymal cell shape with markedly increased α-SMA staining. In addition, expansion of the interstitium with enhanced α-SMA staining could also observed in UUO kidney (Figure 6D). Interestingly, the tubule epithelial cells exhibiting α-SMA staining also showed strong staining of β1 integrin, supporting the correlation of β1 integrin with EMT in renal epithelium during renal fibrosis.

Correlation of α-SMA Expression with β1 Integrin Expression in Human Kidney with Chronic Tubulointerstitial Fibrosis

It is well established that there is enhanced expression of α-SMA in patients with chronic tubulointerstitial fibrosis.6 However, the expression pattern of β1 integrin in kidney of chronic tubulointerstitial fibrosis was not clear. Here we examined the expression of β1 integrin and α-SMA in human kidney sample from patients with chronic tubulointerstitial fibrosis. The results showed that in regions with severe interstitial fibrosis, both α-SMA and β1 integrin were highly expressed in interstitial cells, particularly α-SMA. More importantly, dilated tubular epithelial cells also showed strong staining of α-SMA and β1 integrin (Figure 7A). Statistical analysis revealed that there was a significant correlation between expression levels of β1 integrin and α-SMA. This result suggests the correlation of β1 integrin with EMT during renal fibrosis (Figure 7B).

Figure 7.

Figure 7

Open in a new tab

Expression of β1 integrin and α-SMA in human fibrotic kidney. Three human kidney samples with chronic tubulointerstitial fibrosis were coimmuostained with anti-α-SMA antibody conjugated with Cy3 (red) and β1 integrin antibody, followed by secondary antibody conjugated with Alexa-488 (green). Nuclei were stained with Hoechst 33258 (blue). A: Representative immunofluorescence images from three diseased kidneys. B: The fluorescence intensity of β1 integrin and α-SMA was assessed by Olympus FV-1000 software. Five images of each section from each patient were taken for analysis. The correlation coefficient was calculated, R2 = 0.7733 and these data showed significant correlation by using Pearson correlation analysis.

Blocking of β1 Integrin Alleviates Renal Fibrosis

To understand the role of β1 integrin in the pathophysiology of renal fibrosis, we used β1 integrin blocking antibody in UUO mice. After the injection of β1 integrin blocking antibody and concomitant with UUO treatment for 7 days, the protein levels of E-cadherin and α-SMA were assessed. Western blot analysis showed that blocking of β1 integrin signal alleviated down-regulation of E-cadherin and up-regulation of α-SMA induced by UUO within 7 days (Figure 8, A and B). Histological studies showed that the β1 integrin blocking antibody reduced interstitial expansion and tubular dilation (Figure 8C, upper panel). In addition, the Sirius red staining showed that blocking of β1 integrin signal also reduced UUO-induced collagen deposition (Figure 8C, lower panel). These studies demonstrate the critical role of β1 integrin signal in the development of renal fibrosis.

Figure 8.

Figure 8

Open in a new tab

Blocking of β1 integrin signal alleviates renal fibrosis. A: Western blot analysis shows the protein levels of E-cadherin and α-SMA in 7-day UUO kidney in mice treated with or without β1 integrin-blocking antibody (β1 Ab). The contralateral kidneys were used as a control. B: Quantitative results of protein levels of E-cadherin (left) and α-SMA (right) in 7-day UUO kidney with or without β1 Ab are shown. Each bar represents the mean ± SE of four mice. * indicates P < 0.05 and *** indicates P < 0.001, compared with the control group. C: Hematoxylin and eosin (HE) staining (upper panel) and Sirius red staining (lower panel) of 7-day control and UUO kidney treated with or without β1 Ab. Application of β1 Ab significantly reduced collagen deposition in the UUO kidney. Representative immunofluorescence images show the localization of β1 integrin, E-cadherin (D), and α-SMA (E) in 7-day control and UUO kidney treated with or without β1 Ab. Kidney specimens were immunostained with E-cadherin or α-SMA monoclonal antibody, followed by anti-mouse Alexa-594 (red), shown in A and B, respectively, and with β1 Ab conjugated with Alexa-488 (green). Nuclei were stained with Hoechst 33258 (blue).

The immunofluorescence staining was used to further examine the effects of β1 integrin blocking antibody on the prevention of EMT during renal fibrosis. As shown in Figure 8D, E-cadherin staining, localized in the basal-lateral site of tubular epithelial cells in control kidney, was markedly reduced in UUO kidney. Blocking of β1 integrin reverted UUO-triggered down-regulation of E-cadherin in tubule epithelial cells (Figure 8D) and alleviated UUO-triggered augmentation of α-SMA staining in both renal epithelium and interstitium (Figure 8E). To further examine the specificity of β1 integrin blocking antibody, we also tested the effects of α5 integrin blocking antibody, namely CD49e, on UUO kidney. The Western blotting results showed that injection of CD49e did not rescue UUO-induced down-regulation of E-cadherin and up-regulation of α-SMA (Supplemental Figure 5, A and B, see http://ajp.amjpathol.org). The immunofluorescence staining also demonstrated that blocking of α5 integrin signal did not affect UUO-induced interstitial fibrosis, tubular atrophy, down-regulation of E-cadherin staining (Supplemental Figure 5C, see http://ajp.amjpathol.org), or up-regulation of α-SMA staining (Supplemental Figure 5D, see http://ajp.amjpathol.org). In summary, although previous study has implicated the involvement of α5 integrin in TGF-β1 and UUO-induced EMT,35 our studies reported here show that blocking of α5 integrin does not prevent UUO-induced renal fibrosis. Taken these results together, β1 integrin is an important target for alleviation of chronic interstitial fibrosis.

Discussion

TGF-β1-triggered EMT has been a key issue in pathogenesis of tissue or organ fibrosis. In kidney, TGF-β1-induced EMT not only results in accumulation of interstitial fibroblasts but also leads to kidney function impairment. However, the molecular mechanisms whereby TGF-β1 induces EMT have not been fully understood. In this study, we demonstrate that augmented expression of β1 integrin, a very critical ECM receptor, plays important roles in TGF-β1-induced EMT both in in vitro and in vivo. In addition, we also elucidate the molecular mechanisms whereby TGF-β1 up-regulates β1 integrin gene expression. Blocking of β1 integrin signal prevented TGF-β1-induced down-regulation of E-cadherin and up-regulation of fibronectin and α-SMA in renal tubular epithelial cells. Results from bioinformatics search showed a Smad3/4 binding element on β1 integrin promoter sequence and knockdown of Smad3 suppressed TGF-β1-induced β1 integrin up-regulation. This is the first evidence to demonstrate that the Smad-dependent pathway is responsible for TGF-β1-induced β1 integrin gene regulation. These observations indicate that β1 integrin not only acts as a pivotal mediator of TGF-β1-induced EMT but also serves as an accelerator for mesenchymal transition. Results from our in vivo studies support the hypothesis that β1 integrin plays essential role in the development of renal fibrosis. First, the increases of β1 integrin mRNA in kidney appeared as early as 1 day after UUO. Second, β1 integrin levels were enhanced not only in interstitium but also in tubular epithelium along with the augmentation of α-SMA. Finally, the injection of β1 integrin blocking antibody significantly reduced renal fibrosis and EMT, as manifested by interstitial expansion, tubular dilation, collagen deposition, E-cadherin down-regulation, and α-SMA up-regulation.

Recently, a series of studies identified the important role of ILK and FAK in TGF-β1-induced development of EMT. However, the blocking of both ILK and FAK activation could not completely inhibited TGF-β1-induced EMT,24,25 suggesting that signals other than ILK and FAK were involved in TGF-β1-induced EMT. In our study, inhibition of β1 integrin either by blocking antibody or shRNA significantly blocked TGF-β1-induced fibronectin and α-SMA expression and down-regulation and delocalization of E-cadherin. Overexpression of active β1 integrin in epithelial cells not only lost epithelial phenotype but also resulted in higher fibronectin levels even in the absence of TGF-β1. Because β1 integrin blockade exerts a potent effects on inhibition of TGF-β1-induced EMT, our data support the notion that β1 integrin is one of the pivotal membrane proteins to orchestra downstream proteins involved in TGF-β1-induced EMT. There are several downstream proteins mediating β1 integrin signaling other than focal adhesion complex proteins, such as RhoA, and its downstream proteins, Rho kinase (ROCK) and myosin light chain (MLC).36 It has been documented that Rho/ROCK/MLC proteins are important mediators in TGF-β1-induced α-SMA expression in renal tubule epithelial cells.37,38 TGF-β1 triggers activation of Rho/ROCK/MLC within 30 minutes, and this activation of MLC induces nuclear translocation of MRTF, a potent transcriptional coactivator for α-SMA gene regulation, to accelerate TGF-β1-induced α-SMA expression within 24 hours.39 In our study, we found that ROCK and MLC inhibitors decreased not only α-SMA protein levels but also morphological changes induced by TGF-β1. However, these inhibitors did not affect TGF-β1-induced β1 integrin (data not shown). Since ROCK and MLC act at the downstream pathways of β1 integrin in TGF-β1-induced EMT, they may not be better therapeutic targets for the treatment of EMT or renal fibrosis.

Studies reported here showed the first evidence on TGF-β1-induced β1 integrin gene expression in renal epithelial cells, which played important roles in pathogenesis of chronic tubulointerstitial fibrosis. Although TGF-β1 binds to its receptor, the binding complex recruits and activates Smad3, which in turn forms complex with Smad4. The complex translocates to the nucleus to control the target gene expression via binding to specific promoter sequence, SBE. In this study, we identified the presence of CAGA box on putative SBE of β1 integrin promoter. Studies on promoter activity by mutation of SBE, located between −181 to approximately −172 bp, showed that this mutant markedly decreased TGF-β1-triggered promoter activity. These results indicate that SBE core binding element plays important roles in TGF-β1-induced β1 integrin gene up-regulation. More importantly, chromatin immunoprecipitation assay demonstrated that TGF-β1 treatment enhanced the interactions between Smad3 and β1 integrin promoter. Taken together, we conclude that Smad3 mediates β1 integrin promoter activity in response to TGF-β1 stimulation.

It has been shown that the ECM signaling mediated by integrins is required for growth factor-regulated cellular response.40,41 In this study, we show the blocking of β1 integrin signal decreased TGF-β1-induced EMT particularly in kidney epithelial cells; however, which α subunit that paired with β1 integrin to mediate TGF-β1-induced EMT is still unclear. We found that TGF-β1 treatment induced levels of α2 subunit and blocking of α2β1 integrin signal completely blocked TGF-β1-induced down-regulation of E-cadherin and up-regulation of fibronectin and α-SMA, which suggests the importance of α2β1 integrin. Previous studies showed that α3β1 integrin was required for TGF-β1-regulated p-Smad2/p-β-catenin complexes formation through the interactions with TGF-βRI and E-cadherin in both lung and kidney epithelial cells.42,43 In α3-null kidney epithelial cells and cells expressing mutant α3 integrin, which was unable to interact with E-cadherin, failed to up-regulate mesenchymal marker proteins such as collagen type I, vimentin, and α-SMA, after TGF-β1 stimulation. They also found the decrease of p-Smad2/p-β-catenin complex in these cells, suggesting the involvement of α3 integrin in the formation of p-Smad2/p-β-catenin complex. Since the blocking of Smad3 or mutant SBE on β1 integrin promoter sequence could not completely block TGF-β1-triggered promoter activity, it is possible that p-Smad2/p-β-catenin may be involved in TGF-β1-regulated β1 integrin gene expression. Previous study also showed that E-cadherin-mediated cell-cell contact regulated integrins expression, including α5 and β1.44 These studies suggest that the cross talk between integrin and E-cadherin-mediated adherent junction is important in regulating TGF-β1-induced EMT.

β1 integrin was shown to be involved in various cellular functions, including adhesion, proliferation, differentiation, and survival.45 The role of β1 integrin in fibroblast migration, proliferation, and monocyte infiltration had also been reported.46–49 Although this study elucidated the mechanism in β1 integrin gene regulation triggered by TGF-β1 in normal epithelial cells, it also implied the role of β1 integrin in fibroblast differentiation from both in vitro and in vivo evidence. Because we not only observed the up-regulation of β1 integrin in response to TGF-β1 stimulation in NRK49F cells, the β1 integrin staining also increased in interstitial cells in both human sample and UUO model. Since the expansion of interstitium and development of EMT in renal tubule are two major events in the progression of renal fibrosis, the role of β1 integrin in renal fibroblasts could be comprehensive which highlight the possibility that β1 integrin could be considered as a therapeutic target for preventing renal fibrosis.

The signaling balance between different cytokines, such as TGF-β1 and bone morphogenetic protein-7, in maintenance of tubule epithelial cell differentiation or development of EMT, plays very important role of pathogenesis of renal fibrosis. Under physiological conditions, the expression of integrins and adherens junctions is important for maintaining epithelium properties. The basal levels of integrin are critical for cell attachment, survival, and proliferation. From our data, the overproduction of cytokines, such as TGF-β1, result in abnormal increase in β1 integrin induction, which triggers expression of its ligands, ie, fibronectin and collagen. The increase in fibronectin and collagen matrix may subsequently trigger β1 integrin activation and results in a positive loop of augmentation of matrix deposition and EMT. In addition, TGF-β1 induces the increase of ILK and PINCH-1, and activation of FAK and Rho/ROCK/MLC pathways, that are all considered the downstream signal proteins of β1 integrin. Thus, TGF-β1-induced increase in β1 integrin is considered the most critical step for TGF-β1-induced EMT. Our studies both in vitro and in vivo strongly support the role of β1 integrin in the pathogenesis of renal fibrosis progression.

Acknowledgements

We gratefully acknowledge the provision of p-GL3 basic plasmid from Dr. Shaw-Jenq Tsai, sh-RNA for β1 integrin knockdown from Dr. Wen-Tsan Chang, 5E8 blocking antibody from Dr. Richard B. Bankert, and the technical assistance by Ms. Tsu-Ling Chen.

Footnotes

Supported by National Health Research Institute grant NHRI-EX98-9840SI and National Science Council grant NSC98-2627-B-006-007.

Supplemental material for this article can be found on http://ajp.amjpathol.org.

Web Extra Material

Figure S1
mmc1.pdf (39.4KB, pdf)

Time course of TGF- β 1-induced changes of β 1 integrin, fibronectin, α -SMA and E-cadherin (A) and β 1 integrin mRNA (B) in LLC-PK1 cells.

(A) TGF- β 1-induced protein level changes in LLC-PK1 cells were assessed by Western blot. The protein levels were normalized with β actin at different time point ad the results were presented by fold induction over control. (B) TGF- β 1 induced changes of β 1 integrin mRNA, which was normalized with GAPDH as assessed by RT-PCR, at indicated time point in LLC-PK1 cells. Each bar represents mean ± SE from three experiments. * indicates p<0.05 and ** indicates p<0.001, compared with control group.

Figure S2
mmc2.pdf (47.4KB, pdf)

α 2 β 1 integrin signal is required for TGF- β 1-triggered EMT.

LLC-PK1 cells were treated with or without TGF- β 1 (10ng/ml) and/or α 2 β 1 neutralizing antibody 5E8 (20mg/ml) for 48 h. Protein levels of fibronectin, E-cadherin and α-SMA were assessed by Western blot analysis.

Figure S3
mmc3.pdf (306.9KB, pdf)

Distributions of TGF- β 1 and β 1 integrin in fibrotic kidney

Immunofluorescence staining showed the localization of TGF- β 1 and β 1 integrin in UUO kidney of 7 d. Kidney specimens were immunostained with rabbit polyclonal antibodies of TGF- β 1, followed by anti-rabbit Alexa-594 (red), and by Alexa-488-conjugated monoclonal β 1 integrin antibody (green) and with Hoechst 33258 for nuclei (blue).

Figure S4
mmc4.pdf (128.9KB, pdf)

Time course of changes of β 1 integrin, p-Smad3 and Smad2/3 in UUO kidney.

(A) Western blot of β 1 integrin protein levels and Smad3 activation levels in control (C) and UUO kidney (U) of 1, 3 and 7 d was shown. (B) Quantitative results of Western blot analysis of β 1 integrin and Smad2/3 protein levels in control (C) and UUO (U) kidney at different time point. Each bar represents mean±SE of 4 mice.*P<0.05 ** P<0.01 and *** P<0.001 versus control.

Figure S5
mmc5.pdf (569KB, pdf)

Blocking of α 5 integrin signal does not affect renal fibrosis.

(A) Western blot analysis shows the protein levels of E-cadherin and α-SMA in UUO kidney in mice treated with or without α 5 integrin blocking antibody (CD49e Ab) for 7 d. The contralateral kidneys were used as control. (B) Quantitative results of protein levels of E-cadherin (left) and α-SMA (right) in UUO kidney with or without treatment of α 5 integrin blocking antibody (CD49e Ab) for 7 d. Each bar represents mean ± SE of 3 mice. ** indicates p<0.01 and *** indicates p<0.001, compared with control group. Representative immunofluorescence pictures showed the localization of β 1 integrin, E-cadherin (C) and α-SMA (D) in control and UUO kidney treated with or without α 5 integrin blocking antibody (CD49e Ab) for 7 d. Kidney specimens from 7 d UUO mice were immunostained with E-cadherin or α-SMA monoclonal antibody, followed by anti-mouse Alexa-594 (red). β 1 integrin were stained by monoclonal antibody conjugated with Alexa-488 (green). Nuclei were stained by Hoechst 33258 (blue).

References

  • 1.Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest. 2003;112:1776–1784. doi: 10.1172/JCI20530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Zeisberg M, Kalluri R. The role of epithelial-to-mesenchymal transition in renal fibrosis. J Mol Med. 2004;82:175–181. doi: 10.1007/s00109-003-0517-9. [DOI] [PubMed] [Google Scholar]
  • 3.Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention. J Am Soc Nephrol. 2004;15:1–12. doi: 10.1097/01.asn.0000106015.29070.e7. [DOI] [PubMed] [Google Scholar]
  • 4.Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest. 2002;110:341–350. doi: 10.1172/JCI15518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Strutz F, Okada H, Lo CW, Danoff T, Carone RL, Tomaszewski JE, Neilson EG. Identification and characterization of a fibroblast marker: FSP1. J Cell Biol. 1995;130:393–405. doi: 10.1083/jcb.130.2.393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Roberts IS, Burrows C, Shanks JH, Venning M, McWillian LJ. Interstitial myofibroblasts: predictors of progression in membranous nephropathy. J Clin Pathol. 1997;50:123–127. doi: 10.1136/jcp.50.2.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Yang J, Lui Y. Blockage of tubular epithelial to myofibroblasst transition by hepatocyte growth factor prevents renal interstitial fibrosis. J Am Soc Nephrol. 2002;13:96–107. doi: 10.1681/ASN.V13196. [DOI] [PubMed] [Google Scholar]
  • 8.Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F, Kalluri R. BMP-7 counteracts TGF-β1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med. 2003;9:964–968. doi: 10.1038/nm888. [DOI] [PubMed] [Google Scholar]
  • 9.Thiery JP. Epithelial-mesenchymal transition in development and pathologies. Curr Opin Cell Biol. 2003;15:740–746. doi: 10.1016/j.ceb.2003.10.006. [DOI] [PubMed] [Google Scholar]
  • 10.Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer. 2002;2:442–454. doi: 10.1038/nrc822. [DOI] [PubMed] [Google Scholar]
  • 11.Radisky DC, Kenny PA, Bissell MJ. Fibrosis and cancer: do myofibroblasts come also from epithelial cells via EMT? J Cell Biochem. 2007;101:830–839. doi: 10.1002/jcb.21186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Zavadil J, Bottinger EP. TGF-β and epithelial-to-mesenchymal transitions. Oncogene. 2005;24:5764–5774. doi: 10.1038/sj.onc.1208927. [DOI] [PubMed] [Google Scholar]
  • 13.Fan JM, Ng YY, Hill PA, Paterson-Nikolic DJ, Mu W, Atkins RC, Lan HY. Transforming growth factor-β regulates tubular epithelial-myofibroblast transdifferentiation in vitro. Kidney Int. 1999;56:1455–1467. doi: 10.1046/j.1523-1755.1999.00656.x. [DOI] [PubMed] [Google Scholar]
  • 14.Bottinger EP, Bitzer M. TGF-β signaling in renal disease. J Am Soc Nephrol. 2002;13:2600–2610. doi: 10.1097/01.asn.0000033611.79556.ae. [DOI] [PubMed] [Google Scholar]
  • 15.Border WA, Noble NA. Transforming growth factor β in tissue fibrosis. N Engl J Med. 1994;331:1286–1292. doi: 10.1056/NEJM199411103311907. [DOI] [PubMed] [Google Scholar]
  • 16.Feng XH, Derynck R. Specificity and versatility in TGF-β signaling through Smads. Annu Rev Cell Dev Biol. 2005;21:659–693. doi: 10.1146/annurev.cellbio.21.022404.142018. [DOI] [PubMed] [Google Scholar]
  • 17.Flanders KC. Smad3 as a mediator of the fibrotic response. Int J Exp Path. 2004;85:47–64. doi: 10.1111/j.0959-9673.2004.00377.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Roberts AB, Tian F, Byfield SD, Stuelten C, Ooshima A, Saika S, Flanders KC. Smad3 is key to TGF-β-mediated epithelial-to-mesenchymal transition, fibrosis, tumor suppression and metastasis. Cytokine Growth Factor Rev. 2006;17:19–27. doi: 10.1016/j.cytogfr.2005.09.008. [DOI] [PubMed] [Google Scholar]
  • 19.Streuli CH, Akhtar N. Signal co-operation between integrins and other receptor systems. Biochem J. 2009;418:491–506. doi: 10.1042/BJ20081948. [DOI] [PubMed] [Google Scholar]
  • 20.Al-Jamal R, Harrison DJ. β1 integrin in tissue remodelling and repair: from phenomena to concepts. Pharmacol Ther. 2008;120:81–101. doi: 10.1016/j.pharmthera.2008.07.002. [DOI] [PubMed] [Google Scholar]
  • 21.Stephens LE, Sutherland AE, Klimanskaya IV, Andrieux A, Meneses J, Pedersen RA, Damsky CH. Deletion of β1 integrins in mice results in inner cell mass failure and peri-implantation lethality. Genes Dev. 1995;9:1883–1895. doi: 10.1101/gad.9.15.1883. [DOI] [PubMed] [Google Scholar]
  • 22.Fässler R, Meyer M. Consequences of lack of β1 integrin gene expression in mice. Genes Dev. 1995;9:1896–1908. doi: 10.1101/gad.9.15.1896. [DOI] [PubMed] [Google Scholar]
  • 23.Bhowmick NA, Zent R, Ghiassi M, McDonnell M, Moses HL. Integrin β1 signaling is necessary for transforming growth factor-β activation of p38MAPK and epithelial plasticity. J Biol Chem. 2001;276:46707–46713. doi: 10.1074/jbc.M106176200. [DOI] [PubMed] [Google Scholar]
  • 24.Li Y, Yang J, Dai C, Wu C, Liu Y. Role for integrin-linked kinase in mediating tubular epithelial to mesenchymal transition and renal interstitial fibrogenesis. J Clin Invest. 2003;112:503–516. doi: 10.1172/JCI17913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Cicchini C, Laudadio I, Citarella F, Corazzari M, Steindler C, Conigliaro A, Fantoni A, Amicone L, Tripodi M. TGFβ-induced EMT requires focal adhesion kinase (FAK) signaling. Exp Cell Res. 2008;314:143–152. doi: 10.1016/j.yexcr.2007.09.005. [DOI] [PubMed] [Google Scholar]
  • 26.Xie L, Law BK, Aakre ME, Edgerton M, Shyr Y, Bhowmick NA, Moses HL. Transforming growth factor β-regulated gene expression in a mouse mammary gland epithelial cell line. Breast Cancer Res. 2003;5:187–198. doi: 10.1186/bcr640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Kagami S, Kuhara T, Yasuromo K, Okada K, Loster K, Reutter W, Kuroda Y. Transforming growth factor-β (TGF-β) stimulates the expression of β1 integrins and adhesion by rat mesangial cells. Exp Cell Res. 1996;229:1–6. doi: 10.1006/excr.1996.0336. [DOI] [PubMed] [Google Scholar]
  • 28.Yeh YC, Wang CZ, Tang MJ. Discoidin domain receptor 1 activation suppresses alpha(2)beta(1) integrin-dependent cell spreading through inhibition of Cdc42 activity. J Cell Physiol. 2009;218:146–156. doi: 10.1002/jcp.21578. [DOI] [PubMed] [Google Scholar]
  • 29.Wei WC, Lin HH, Shen MR, Tang MJ. Mechanosensing machinery for cells under low substratum rigidity. Am J Physiol Cell Physiol. 2008;295:1579–1589. doi: 10.1152/ajpcell.00223.2008. [DOI] [PubMed] [Google Scholar]
  • 30.Wang CZ, Yeh YC, Tang MJ. DDR1/E-cadherin complex regulates the activation of DDR1 and cell spreading. Am J Physiol Cell Physiol. 2009;297:419–429. doi: 10.1152/ajpcell.00101.2009. [DOI] [PubMed] [Google Scholar]
  • 31.Su HW, Yeh HH, Wang SW, Shen MR, Chen TL, Kiela PR, Ghishan FK, Tang MJ. Cell confluence-induced activation of signal transducer and activator of transcription-3 (Stat3) triggers epithelial dome formation via augmentation of sodium hydrogen exchanger-3 (NHE3) expression. J Biol Chem. 2007;282:9883–9894. doi: 10.1074/jbc.M606754200. [DOI] [PubMed] [Google Scholar]
  • 32.Wu MJ, Wen MC, Chiu YT, Chiou YY, Shu KH, Tang MJ. Rapamycin attenuates unilateral ureteral obstruction-induced renal fibrosis. Kidney Int. 2006;69:2029–2036. doi: 10.1038/sj.ki.5000161. [DOI] [PubMed] [Google Scholar]
  • 33.Bellis SL, Newman E, Friedman EA. Steps in integrin β1-chain glycosylation mediated by TGFβ1 signaling through Ras. J Cell Physiol. 1999;181:33–44. doi: 10.1002/(SICI)1097-4652(199910)181:1<33::AID-JCP4>3.0.CO;2-#. [DOI] [PubMed] [Google Scholar]
  • 34.Chen FA, Repasky EA, Bankert RB. Human lung tumor-associated antigen identified as an extracellular matrix adhesion molecule. J Exp Med. 1991;173:1111–1119. doi: 10.1084/jem.173.5.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.White LR, Blanchette JB, Ren L, Awn A, Trpkov K, Muruve DA. The characterization of α5-integrin expression on tubular epithelium during renal injury. Am J Physiol Renal Physiol. 2007;292:567–576. doi: 10.1152/ajprenal.00212.2006. [DOI] [PubMed] [Google Scholar]
  • 36.Giannone G, Dubin-Thaler BJ, Dobereiner HG, Kieffer N, Bresnick AR, Sheetz MP. Periodic lamellipodial contractions correlate with rearward actin waves. Cell. 2004;116:431–443. doi: 10.1016/s0092-8674(04)00058-3. [DOI] [PubMed] [Google Scholar]
  • 37.Masszi A, Ciano CD, Sirokmany G, Arthur WT, Rotstein OD, Wang J, McCulloch CA, Rosivall L, Mucsi I, Kapus A. Central role for Rho in TGF-β1-induced α-smooth muscle actin expression during epithelial-mesenchymal transition. Am J Physiol Renal Physiol. 2003;284:911–924. doi: 10.1152/ajprenal.00183.2002. [DOI] [PubMed] [Google Scholar]
  • 38.Szaszi K, Sirokmany G, Ciano-Oliveira CD, Rotstein OD, Kapus A. Depolarization induces Rho-Rho kinase-mediated myosin light chain phosphorylation in kidney tubular cells. Am J Physiol Cell Physiol. 2005;289:673–685. doi: 10.1152/ajpcell.00481.2004. [DOI] [PubMed] [Google Scholar]
  • 39.Fan L, Sebe A, Peterfi Z, Masszi A, Thirone AC, Rotstein OD, Nakano H, McCulloch CA, Szaszi K, Mucsi I, Kapus A. Cell contact-dependent regulation of epithelial-myofibroblast transition via the Rho-Rho kinase-phospho-myosin pathway. Mol Biol Cell. 2007;18:1083–1097. doi: 10.1091/mbc.E06-07-0602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Wang CZ, Su HW, Hsu YC, Shen MR, Tang MJ. A discoidin domain receptor 1/SHP-2 signaling complex inhibits α2β1-integrin-mediated signal transducers and activators of transcription 1/3 activation and cell migration. Mol Biol Cell. 2006;17:2839–2852. doi: 10.1091/mbc.E05-11-1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Edick MJ, Tesfay L, Lamb LE, Knudsen BS, Miranti CK. Inhibition of integrin-mediated crosstalk with epidermal growth factor receptor/Erk or Src signaling pathways in autophagic prostate epithelial cells induces caspase-independent death. Mol Biol Cell. 2007;18:2481–2490. doi: 10.1091/mbc.E06-04-0261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Kim KK, Wei Y, Szekeres C, Kugler MC, Wolters PJ, Hill ML, Frank JA, Brumwell AN, Wheeler SE, Kreidberg JA, Chapman HA. Epithelial cell α3β1 integrin links β-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosis. J Clin Invest. 2009;119:213–224. doi: 10.1172/JCI36940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Kim Y, Kugler MC, Wei Y, Kim KK, Li X, Brumwell AN, Chapman HA. Integrin α3β1-dependent β-catenin phosphorylation links epithelial Smad signaling to cell contacts. J Cell Biol. 2009;184:309–322. doi: 10.1083/jcb.200806067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Wu H, Liang YL, Li Z, Jin J, Zhang W, Duan L, Zha X. Positive expression of E-cadherin suppresses cell adhesion to fibronectin via reduction of α5β1 integrin in human breast carcinoma cells. J Cancer Res Clin Oncol. 2006;132:795–803. doi: 10.1007/s00432-006-0128-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Gilcrease MZ. Integrin signaling in epithelial cells. Cancer Lett. 2007;247:1–25. doi: 10.1016/j.canlet.2006.03.031. [DOI] [PubMed] [Google Scholar]
  • 46.Norman JT, Fine LG. Progressive renal disease: fibroblasts, extracellular matrix, and integrins. Exp Nephrol. 1999;7:167–177. doi: 10.1159/000020597. [DOI] [PubMed] [Google Scholar]
  • 47.Lundberg S, Lindholm J, Lindbom L, Hellstrom PM, Werr J. Integrin α2β1 regulates neutrophil recruitment and inflammatory activity in experimental colitis in mice. Inflamm Bowel Dis. 2006;12:172–177. doi: 10.1097/01.MIB.0000217765.96604.83. [DOI] [PubMed] [Google Scholar]
  • 48.Issekutx TB. In vivo blood monocyte migration to acute inflammatory reactions. IL-1α, TNF-α, IFN-γ, and C5a utilizes LFA-1, Mac-1, and VLA-4. J Immunol. 1995;154:6533–6540. [PubMed] [Google Scholar]
  • 49.Hu K, Wu C, Mars WM, Liu Y. Tissue-type plasminogen activator promotes murine myofibroblast activation through LDL receptor-related protein 1-mediated integrin signaling. J Clin Invest. 2007;117:3821–3832. doi: 10.1172/JCI32301. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1
mmc1.pdf (39.4KB, pdf)

Time course of TGF- β 1-induced changes of β 1 integrin, fibronectin, α -SMA and E-cadherin (A) and β 1 integrin mRNA (B) in LLC-PK1 cells.

(A) TGF- β 1-induced protein level changes in LLC-PK1 cells were assessed by Western blot. The protein levels were normalized with β actin at different time point ad the results were presented by fold induction over control. (B) TGF- β 1 induced changes of β 1 integrin mRNA, which was normalized with GAPDH as assessed by RT-PCR, at indicated time point in LLC-PK1 cells. Each bar represents mean ± SE from three experiments. * indicates p<0.05 and ** indicates p<0.001, compared with control group.

Figure S2
mmc2.pdf (47.4KB, pdf)

α 2 β 1 integrin signal is required for TGF- β 1-triggered EMT.

LLC-PK1 cells were treated with or without TGF- β 1 (10ng/ml) and/or α 2 β 1 neutralizing antibody 5E8 (20mg/ml) for 48 h. Protein levels of fibronectin, E-cadherin and α-SMA were assessed by Western blot analysis.

Figure S3
mmc3.pdf (306.9KB, pdf)

Distributions of TGF- β 1 and β 1 integrin in fibrotic kidney

Immunofluorescence staining showed the localization of TGF- β 1 and β 1 integrin in UUO kidney of 7 d. Kidney specimens were immunostained with rabbit polyclonal antibodies of TGF- β 1, followed by anti-rabbit Alexa-594 (red), and by Alexa-488-conjugated monoclonal β 1 integrin antibody (green) and with Hoechst 33258 for nuclei (blue).

Figure S4
mmc4.pdf (128.9KB, pdf)

Time course of changes of β 1 integrin, p-Smad3 and Smad2/3 in UUO kidney.

(A) Western blot of β 1 integrin protein levels and Smad3 activation levels in control (C) and UUO kidney (U) of 1, 3 and 7 d was shown. (B) Quantitative results of Western blot analysis of β 1 integrin and Smad2/3 protein levels in control (C) and UUO (U) kidney at different time point. Each bar represents mean±SE of 4 mice.*P<0.05 ** P<0.01 and *** P<0.001 versus control.

Figure S5
mmc5.pdf (569KB, pdf)

Blocking of α 5 integrin signal does not affect renal fibrosis.

(A) Western blot analysis shows the protein levels of E-cadherin and α-SMA in UUO kidney in mice treated with or without α 5 integrin blocking antibody (CD49e Ab) for 7 d. The contralateral kidneys were used as control. (B) Quantitative results of protein levels of E-cadherin (left) and α-SMA (right) in UUO kidney with or without treatment of α 5 integrin blocking antibody (CD49e Ab) for 7 d. Each bar represents mean ± SE of 3 mice. ** indicates p<0.01 and *** indicates p<0.001, compared with control group. Representative immunofluorescence pictures showed the localization of β 1 integrin, E-cadherin (C) and α-SMA (D) in control and UUO kidney treated with or without α 5 integrin blocking antibody (CD49e Ab) for 7 d. Kidney specimens from 7 d UUO mice were immunostained with E-cadherin or α-SMA monoclonal antibody, followed by anti-mouse Alexa-594 (red). β 1 integrin were stained by monoclonal antibody conjugated with Alexa-488 (green). Nuclei were stained by Hoechst 33258 (blue).

Articles from The American Journal of Pathology are provided here courtesy of American Society for Investigative Pathology

RESOURCES