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* Department of Medicine II, University of Giessen Lung Center, Justus Liebig University Giessen, Giessen, Germany; and
Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA
1Correspondence: University of Giessen Lung Center, Department of Medicine II, Aulweg 123, Rm. 6–11, D-35392 Giessen, Germany. E-mail: oliver.eickelberg{at}innere.med.uni-giessen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: alveolar epithelial cell • signal transduction • bleomycin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. The TGF-β superfamily comprises more than 40 members, including activins, inhibins, bone morphogenetic proteins (BMP), growth and differentiation factors (GDF), and TGF-βs themselves (3
, 4)
. To date, three different TGF-β isoforms have been cloned and characterized: TGF-β1, TGF-β2, and TGF-β3. TGF-β1 is the most important isoform in the cardiopulmonary system, and it is ubiquitously expressed and secreted by endothelial, epithelial, and smooth muscle cells, as well as fibroblasts and most cells of the immune system (5)
.
Signal transduction by TGF-β family members is induced by ligand binding to the type II TGF-β receptor. This initiates the formation of a heteromeric complex of type I [activin-like kinase (Alk) 5] and type II TGF-β receptors, and finally, transphosphorylation of the type I receptor by the constitutively active serine/threonine kinase of the type II receptor (3
, 4
, 6)
. Phosphorylation of the type I receptor-specific GS domain transmits specific intracellular signals via Smad proteins. To date, eight different Smads have been cloned and characterized, which have been categorized into three different subgroups: receptor-regulated Smads (R-Smads), common Smads (co-Smads), and inhibitory Smads (I-Smads). Smad2 or Smad3, in combination with the co-Smad Smad4, positively regulate TGF-β-induced effects, while the I-Smads (Smad6 and Smad7) negatively regulate TGF-β signaling (4
, 6)
. Smad2 and Smad3 have been shown to transmit TGF-β signaling via Alk5-dependent serine phosphorylation of their C-terminal SSXS motif on ligand binding, and subsequently undergo nuclear translocation in complex with Smad4. It is reported, however, that only Smad3 is capable of direct DNA binding via its N-terminal mad homology (MH)1 domain. The direct binding of Smad2 to DNA is prevented by insertion of an additional exon encoding for 30 amino acids unique to Smad2 (4)
.
Dysregulated TGF-β signaling has been implicated in a variety of diseases, including cancer, autoimmune diseases, pulmonary hypertension, or tissue fibrosis (2)
. Increased TGF-β signaling is the key pathophysiological mechanism that leads to fibrotic transformation of multiple tissues, including the kidney, liver, or lung (5
, 7)
. Among these, IPF is a devastating disease characterized by an increase in activated (myo)fibroblasts and excessive deposition of extracellular matrix, in large part mediated through enhanced TGF-β signaling (8
9
10
11
12
13)
. No effective therapy for IPF currently exists, reflecting our limited understanding of the basic mechanisms at play in the pathogenesis of this progressive and fatal fibrotic disease. The pathogenesis of IPF includes repetitive epithelial injury and repair processes, migration, epithelial-to-mesenchymal transition (EMT), and growth factor/cytokine-mediated remodeling processes that lead to transient activation and altered gene expression profiles of alveolar epithelial type II (ATII) cells and (myo)fibroblasts, which perpetuate the fibrotic process.
One of the hallmarks of IPF is an increased number of 
-smooth muscle actin-positive, activated (myo)fibroblasts that reside in fibroblast foci, but their origin remains to be elucidated (14)
. Currently, three major theories attempt to explain the accumulation of these (myo)fibroblasts: First, resident pulmonary fibroblasts proliferate in response to fibrogenic cytokines and growth factors (such as TGF-β), thereby increasing the fibroblast pool by local fibroproliferation (15)
. Second, bone marrow-derived circulating fibrocytes cells traffic to the lung during experimental lung fibrosis and may serve as progenitors for interstitial fibroblasts (16)
. Third, ATII cells are capable of undergoing the process of EMT, thus contributing to the increased pool of (myo)fibroblasts (17
18
19)
. EMT represents the phenotypic, reversible switching of epithelial to fibroblast-like cells, which is initiated by an alteration to the transcriptional and proteomic profile of ATII cells. The orchestrated series of events initiating EMT include remodeling of epithelial cell-cell and cell-matrix adhesion contacts, reorganization of the actin cytoskeleton, and induction of mesenchymal gene expression (18
, 20
, 21)
.
TGF-β represents a main inducer of fibrosis and regulator of the ATII cell phenotype, but direct mediators linking these processes remain poorly defined. The objective of our study was therefore to identify novel transcriptional processes induced by TGF-β in ATII cells. We performed a chromatin immunoprecipitation approach and identified transgelin, a cytoskeleton-associated protein with as of yet unknown function, as a novel direct target of Smad3/Alk5-dependent TGF-β signaling in ATII cells. In vivo analysis revealed that transgelin is specifically localized to ATII cells in mice and that it represents a key regulator of ATII cell migration with altered expression in experimental and idiopathic pulmonary fibrosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), e-cadherin (eCad), Smad2/3 (all from BD Biosciences, San Jose, CA, USA), anti-Smad2, anti-phospho-Smad3/1 (both from Cell Signaling, Danvers, MA, USA), anti-Smad2 (MBL International, Woburn, MA, USA), anti-Smad3 (Upstate/Millipore, Billerica, MA, USA), anti-transgelin (Abcam, Cambridge, UK), anti--smooth muscle actin (SMA) (Chemicon International, Temecula, CA), and anti-tight junction protein (Tjp) 1 (Zymed Laboratories, San Francisco, CA). Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). The Alk5 kinase inhibitor SB431542 and the p38 inhibitor SB203580 were purchased from Tocris Cookson (Bristol, UK) and EMD Biosciences (San Diego, CA, USA).
Cell culture
The human lung epithelial cell line A549 (ATCC CCL-185; American Type Culture Collection, Manassas, VA, USA) was maintained in Dulbecco’s modified Eagle’s medium (DMEM, from GIBCO/Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS, from PAA Laboratories, Pasching, Austria). Primary mouse alveolar epithelial type II (ATII) cells were isolated and cultured as described previously (22)
.
Animal and human tissues
All experiments were performed in accordance with the guidelines of the Ethics Committee of University of Giessen School of Medicine and approved by the local authorities. Bleomycin sulfate was dissolved in sterile saline solution and applied by microspray as a single dose of 0.08 mg/mouse in a total volume of 200 µl. Control mice received 200 µl saline. Mice were sacrificed after the indicated times, and lung tissues were excised and snap frozen, or inflated with 4% (m/v) paraformaldehyde in phosphate-buffered saline (PBS; PAA Laboratories) at 21 cm H2O pressure for histological analyses. Human lung tissue biopsies were obtained from 10 patients with IPF [usual interstitial pneumonia (UIP) pattern; mean age 51.3±11.4 yr; 4 females, 6 males] and 10 control subjects (organ donors, mean age 47.5±13.9 yr; 5 females, 5 males). Informed consent was obtained from each subject for the study protocol.
Chromatin immunoprecipitation (ChIP)
ChIP analyses were performed according to the protocol originally described by Weinmann and Farnham (23)
with the following modifications: 1 x 107 A549 cells were treated with TGF-β1 (2 ng/ml) for up to 2 h. Cells were then cross-linked with 1% formaldehyde for 12 min at room temperature (RT), after which glycine (125 mM) was added to quench the formaldehyde. The cells were washed twice with ice-cold PBS and lysed in 500-µl cell lysis buffer [50 mM Tris-HCl, pH 8.0; 1% Triton X-100; 10 mM KCl; supplemented with Complete protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland)]. Nuclei were pelleted at 5000 rpm for 5 min at 4°C, and resuspended in 400 µl of nuclear lysis buffer (50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 0.1% SDS; supplemented with Complete). The samples were sonicated 3 x 10 s to yield sheared DNA fragments between 200 and 700 bp, and lysates were clarified by centrifugation (13,000 rpm, 10 min, 4°C).
Samples were then incubated with 25 µg of anti-Smad3 antibody or control IgG (both from Upstate/Millipore) for 1 h at 4°C. To reduce nonspecific association, 30 µg of sonicated salmon sperm competitor DNA and 50 µg of BSA (both from Promega, Madison, WI, USA) were added to each sample. Immunoprecipitation was carried out using 50 µl of 50% (v/v) Protein A/G PLUS-Agarose beads (Santa Cruz) at 4°C overnight. The immune complexes were washed as follows: 3x with low-salt wash buffer (10 mM Tris-HCl, pH 8.0; 0.1% SDS; 0.1% sodium deoxycholate; 1% Triton X-100; 1 mM EDTA; 140 mM NaCl), 3x with high-salt buffer (same as low-salt wash buffer, but with 500 mM NaCl), 2x with LiCl wash buffer (10 mM Tris-HCl, pH 8.0; 250 mM LiCl; 1% Nonidet P-40; 1% sodium deoxycholate; 1 mM EDTA), and 2x with TE buffer (20 mM Tris-HCl, pH 8.0; 1 mM EDTA). Elution was performed twice at 65°C for 15 min, first with 200 µl of 1.5% SDS solution, and then with 250 µl of 0.5% SDS solution. Immunoprecipitated DNA-protein complexes were then reverse cross-linked at 65°C overnight and purified by phenol-chloroform extraction and ethanol precipitation with 30 µg glycogen (Roche Diagnostics). The purified DNA was dissolved in 20 µl of water. For downstream polymerase chain reaction (PCR) or real-time PCR analyses, 1 µl of purified DNA was used as a template, with the primers depicted in Table 1
.
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Preparation of RNA and reverse transcription-PCR (RT-PCR)
Total RNA was extracted from A549 cells, primary ATII cells, or frozen lung samples with RNeasy columns (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Purified RNA was reverse-transcribed using M-MLV reverse transcriptase (Promega) and Oligo(dT)15 primers (Promega). PCR amplification was performed with the gene-specific primers listed in Table 1
, and PCR products were analyzed by agarose gel electrophoresis.
Real-time RT-PCR
Real-time RT-PCR was performed using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) and the Sequence Detection System 7900 (PE Applied Biosystems, Foster City, CA, USA). To evaluate relative mRNA expression of transgelin, the ubiquitously and equally expressed hydroxymethylbilane synthase (hmbs) gene was used as a reference gene (primers are given in Table 1
). Relative transcript abundance of transgelin is expressed in 
Ct values (Ct=Ctreference–Cttarget). Relative changes in transcript levels compared to controls are expressed as Ct values (Ct=Cttreated–Ctcontrol) (22)
. All Ct values correspond approximately to the binary logarithm of the fold change. Real-time RT-PCR was also performed to analyze the abundance of Smad3-bound promoter sequences in ChIP-purified DNA samples, using primers against the human hspa8 promoter as a normalization sequence (Table 1)
. Relative changes of Smad3-bound promoter DNA levels are represented as 2Ct calculations (Ct=Cttreated–Ctcontrol).
Laser-assisted microdissection
In brief, 10-µm cryosections were mounted on glass slides, stained with hemalaun for 45 s, immersed in 70% and 96% ethanol, and stored in 100% ethanol until use. Alveolar septae were selected and microdissected with a sterile 30 G needle under optical control using the Laser Microbeam System (PALM, Bernried, Germany). Microdissected tissues were transferred into reaction tubes containing 200-µl RNA lysis buffer and samples were processed for RNA analysis as described above.
Transfection and luciferase assays
The human tagln promoter construct Tagln-luc (GenBank accession number EF153019) contains nucleotides –1032 to +108 relative to the tagln transcriptional start site. This fragment was PCR-amplified (Table 1)
, introducing a XhoI and a MluI site at the 5'- and 3'-ends of the PCR products, respectively. The XhoI/MluI fragment, including the tagln promoter sequence was then introduced into the luciferase reporter vector pGL3-basic (Promega) to yield Tagln-luc. The TGF-β-responsive p(CAGA)12-luc was kindly provided by Peter ten Dijke (Leiden University Medical Center, Leiden, The Netherlands). The expression plasmids for wild-type and constitutively active Alk5 were a kind gift of Carl-Hendrik Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden). Transient transfection was performed with Lipofectamine 2000 Reagent (Invitrogen) using 150 ng of reporter constructs along with 150 ng of Alk5 expression plasmids or empty vector, as indicated. TGF-β1 treatment (2 ng/ml) was performed 4 h after transfection. Luciferase activities were determined using the Dual Luciferase Assay System (Promega) on a Fusion luminometer (Perkin Elmer, Waltham, MA, USA).
Immunofluorescence staining and immunohistochemistry
Primary ATII cells were plated on 8-well chamber slides (BD Biosciences), incubated in the absence or presence of TGF-β1 (2 ng/ml) for 24 h, and fixed with ice-cold methanol. After blocking nonspecific binding with 5% FBS in PBS, cells were incubated with primary antibodies at 4°C overnight, washed 3x in PBS, and incubated with incubation with FITC-/ or Alexa 555-conjugated secondary antibodies (Zymed and Molecular Probes, Eugene, OR, USA, respectively). Nuclei were visualized by 4,6-diamidino-2-phenylindole staining (DAPI; Roche Diagnostics). The staining was analyzed by deconvolution fluorescence microscopy using the Leica AS-MDW system (Leica Microsystems, Bensheim, Germany). Immunohistochemistry of human or mouse lung sections was performed as described before, with negative controls (species-matched preimmune serum) performed in each run.
Small interfering RNA (siRNA) transfection
The siRNA duplexes targeting mouse and human tagln mRNA (Table 2
) were obtained from Dharmacon Inc. (Lafayette, CO, USA). The siRNAs were transiently transfected into A549 or primary ATII cells using Lipofectamine 2000 reagent (Invitrogen) at an siRNA:Lipofectamine ratio of 1:2 (µg:µl). To optimize tagln silencing conditions, cells were transfected with 100 nM siRNA either once or twice, with an interval of 24 h. Cells were harvested and lysed 48 h after the first transfection, and analyzed by RT-PCR or Western blot analysis, as indicated.
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Proliferation assay
A549 or primary ATII cells were grown in 48-well plates, transfected with 50 nM of tagln or nonspecific siRNA, stimulated with TGF-β1 (2 ng/ml), and cultured for another 24 or 48 h, as indicated. Cells were then labeled with [3H]-thymidine (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) for 4 h, washed 4x with PBS, and lysed in 0.5 M NaOH. The incorporation of [3H]-thymidine was measured by liquid scintillation, as described previously (22)
.
Migration assay
Cell migration was determined using a Boyden chamber assay (ThinCerts Tissue Culture Inserts, 24 wells, pore size 3.0 µm, from Greiner Bio-One, Kremsmünster, Austria). A549 or primary ATII cells were transfected with tagln or nonspecific siRNA, detached, and 5 x 104 cells were seeded into a Boyden chamber insert. Cells were cultured for 8 h to allow their attachment to the membrane, and migration was induced by adding TGF-β1 (2 ng/ml) to the media in the lower wells. After 24 h, cells were fixed and stained using crystal violet solution (Sigma-Aldrich) and nonmigrated cells were removed by cotton swabbing. The membranes were carefully separated from the insert wall, and optical densities of migrated cells were measured with a GS-800 Calibrated Densitometer and analyzed with the Quantity One software (both from Bio-Rad).
Detection of apoptosis
After trypsinization from culture wells, A549 cells were incubated in 50% (v/v) fetal calf serum for 15 min to restore membrane integrity and then centrifuged for 5 min at 1,200 rpm. Apoptotic cells were identified by detection of annexin-V binding using FACScan, according to the protocol provided by Boehringer Mannheim (Mannheim, Germany). Staurosporine was used as a positive control. To exclude necrotic cells, cells were double-stained with 5 µg/ml propidium iodide (22)
.
Statistical analysis of data
Values are presented as mean ± SE or mean ± SD, as indicated in the legend. The means of indicated groups were compared using 2-tailed Student’s t test, or a 1-way analysis of variance (ANOVA) with Tukey HSD post hoc test for studies with more than 2 groups. A level of P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, only two antibodies clearly discriminated between Smad2 and 3: an -Smad2 antibody obtained from BML, and an -Smad3 antibody from Upstate. The latter was used in all ChIP experiments in this study. Next, a time course of Smad3-DNA binding was carried out in A549 cells treated with TGF-β using ChIP-PCR primers amplifying fragments of the smad7 and serpine1 promoters, two well-characterized TGF-β target genes that contain Smad-binding elements (SBEs) within their promoter sequences. As shown in Fig. 1B
, binding of Smad3 to the smad7 or serpine1 promoter was significantly induced by TGF-β1, with the highest binding observed 30 min after TGF-β treatment. No binding was observed in unstimulated cells. Next, we examined whether the ChIP protocol could also spatially discriminate Smad3 binding within the same promoter. To do so, a 123 bp fragment 1000 bp downstream of the SBEs in the smad7 promoter (Fig. 1C
, Smad7 DS) was amplified, but yielded no product, further demonstrating specificity of the Smad3-ChIP.
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Smad3 directly binds to and transactivates the tagln promoter
Three different clones that were sequenced from a Smad3 ChIP screen were homologous to the transgelin (tagln) promoter. To confirm this, we performed amplification of the tagln promoter using specific primers (positions are indicated in Fig. 2
C and Table 1
). Smad3 binding to the tagln promoter was induced 30 min after TGF-β1 treatment compared with untreated controls, as assessed by semiquantitative and quantitative real-time RT-PCR (Fig. 2A, B
). The RT-PCR revealed that TGF-β1 stimulation for 30 min led to a 3.8 ± 0.8-fold and a 7.2 ± 0.5-fold induction of Smad3 binding to the tagln and smad7 promoter, respectively (Fig. 2B
). Amplification of the hspa8 promoter sequence (a negative control used for assessment of nonspecific associations) was similar in TGF-β-treated and control cells, supporting the specific binding of Smad3 to the tagln promoter fragment (Fig. 2A
).
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Next, we sought to assess whether TGF-β treatment also altered tagln promoter activity in a luciferase reporter assay. To do so, a fragment of the tagln promoter (from –1031 to+108, relative to the transcriptional start site, Fig. 2C
) was cloned upstream of firefly luciferase (designated Tagln-luc) and transfected into A549 cells. TGF-β1 treatment resulted in a 2.8 ± 0.2-fold induction of Tagln-luc compared with untreated cells (Fig. 2D
). Cotransfection of a vector constitutively expressing the wild-type TGF-β type I receptor Alk5 led to a 4 ± 0.9-fold induction, while cotransfection of constitutively active Alk5 led to a 12 ± 0.3-fold induction of Tagln-luc. Similar inductions were observed for the widely used Smad3 reporter construct p(CAGA)12-luc, which was tested in parallel as a positive control (Fig. 2D
).
TGF-β1 induces tagln mRNA as well as protein expression in lung epithelial cells
We next investigated the effect of TGF-β1 stimulation on endogenous tagln expression. Levels of tagln, but not tagln2, mRNA were up-regulated after TGF-β1 treatment in A549 cells (Fig. 3
A). Inhibition of Alk5 kinase activity using the specific inhibitor SB431542 fully blocked tagln induction, whereas p38 MAP kinase inhibition (using SB203580) had no influence on tagln expression (Fig. 3B
). Tagln protein levels were also upregulated by TGF-β1 (Fig. 3C
), demonstrating that Tagln is a direct target of TGF-β1/Smad3 signaling in A549 cells. To eliminate the possibility that the induction of Tagln expression is an A549 (cell line)-specific effect, we next analyzed primary mouse ATII cells. As depicted in Fig. 3D, E
, both tagln mRNA and protein levels were increased by TGF-β1, as shown by RT-PCR and immunofluorescence analysis, respectively. Taken together, these results show that tagln mRNA and protein expression is reproducibly induced by TGF-β1/Alk5/Smad3 signaling in lung epithelial cells.
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Tagln expression in experimental and idiopathic pulmonary fibrosis
TGF-β signaling induces EMT (17)
, a mechanism recently shown to contribute to the pathogenesis of lung fibrosis (17
, 19)
. We therefore sought to characterize tagln expression in experimental and idiopathic pulmonary fibrosis and analyzed whether TGF-β1-induced tagln expression modified ATII cell migration and EMT in primary ATII cells. As shown in Fig. 4
A, tagln mRNA expression in freshly isolated ATII cells from control mice or mice subjected to bleomycin treatment demonstrated a significant up-regulation of tagln mRNA 7 and 14 days after bleomycin application. Immunohistochemical staining localized Tagln protein expression to ATII cells of normal and fibrotic lungs (Fig. 4B
). We then asked whether Tagln expression was also increased in the lungs of patients with IPF. As depicted in Fig. 5
, tagln mRNA expression was significantly increased in lung specimen obtained from IPF patients (Fig. 5A
), as well as in microdissected septae obtained of IPF lungs (Fig. 5B
). Of note, immunohistochemical analysis revealed a different localization of Tagln protein expression in human lungs compared with mice. In human lungs, Tagln protein localized to fibroblast foci, smooth muscle, and ATII cells obtained from donors or IPF patients (Fig. 5C
). These results strongly suggest that Tagln plays an important role in pulmonary fibrosis, and that it is expressed in a species-specific manner in the lung.
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Tagln is required for ATII cell proliferation and migration
We finally sought to analyze Tagln function in ATII cells, and its possible role during TGF-β-induced migration and EMT. To modify tagln expression, we characterized tagln mRNA knockdown efficiency using four different small interfering (si)RNAs. Western blot analysis revealed efficient knockdown of Tagln protein expression using at least two different siRNAs (Fig. 6
A; siRNA #2 and #4), independent of the number of siRNA applications. Proliferation assays revealed that TGF-β treatment led to a strong inhibition of A549 proliferation (78±14 and 50±8% of control cells 24 and 48 h after TGF-β treatment, respectively), which was further augmented by tagln knockdown (Fig. 6B
). Even in the absence of TGF-β1, silencing of Tagln reduced A549 proliferation to 68 ± 13% of control cells 48 h post-transfection, suggesting that Tagln is required for A549 proliferation. Importantly, the inhibition of proliferation was not dependent on the induction of apoptosis, as neither TGF-β nor knockdown of tagln induced apoptosis in A549 cells (Fig. 6C
).
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Next, we investigated whether Tagln was involved in TGF-β-induced migration of ATII cells, a key feature of EMT (20
, 21)
. To do so, A549 cells were transfected with nonspecific and tagln siRNA, and cell migration was assessed in a modified Boyden chamber assay. As depicted in Fig. 6D
, TGF-β1 treatment led to a 4 ± 0.9-fold induction of cell migration. Tagln knockdown using siRNAs significantly reduced TGF-β1-dependent migration, indicating that Tagln is required for migration of A549 cells.
We next corroborated the above described results obtained in A549 cells using primary mouse ATII cells. As depicted in Fig. 7
A, B, tagln knockdown resulted in similar effects on the proliferation and migration of primary ATII cells. TGF-β treatment resulted in a less-pronounced, but significant decrease of ATII cell proliferation, but this was not additionally modified by tagln knockdown (Fig. 7A
). Furthermore, TGF-β induced a significant increase in ATII cell migration, which was fully attenuated by tagln knockdown (Fig. 7B
). Finally, the effects of tagln and TGF-β on EMT of primary mouse ATII cells were assessed by determining the mRNA expression of the epithelial marker genes occludin, tight junction protein 1, or e-cadherin, as well as the mesenchymal marker genes smooth muscle actin or fibronectin 1, using real-time RT-PCR. While efficient knockdown of tagln was also obtained in primary mouse ATII cells, the effect of TGF-β on the expression of epithelial marker genes occludin and e-cadherin was not affected by tagln knockdown (Fig. 7C
). Similarly, no significant differences in mesenchymal marker gene expression were observed comparing scrambled and tagln siRNA-treated cells (Fig. 7D
). Similar data were also obtained in A549 cells in the presence of siRNAs targeting tagln expression (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Of all genes identified as Smad3 targets by our ChIP screen, we focused on tagln, as multiple clones sequenced from the ChIP screen entailed tagln promoter fragments. In addition, tagln expression was found to be increased in ATII cells that were isolated from bleomycin-treated mouse lungs compared with controls in an independent microarray screen (unpublished results). Tagln, also called smooth muscle-specific protein (SM)-22, is a transformation and shape change-sensitive actin cross-linking/gelling protein initially described in fibroblasts and smooth muscle cells, but its exact functional role remains unclear (24
25
26
27)
. It has been reported that Tagln represents one of the earliest smooth muscle cell markers during smooth muscle differentiation, but it has also been previously described to be expressed in intestinal and breast epithelial cells (28)
. Three members of this protein family, Tagln1–3, have thus far been cloned and characterized, but Tagln3 (also called NP-22 or -25) has thus far only been described in neuronal cells.
One of the most surprising results of our study was the distinct localization and expression of Tagln in ATII cells, in vitro in A549 and primary mouse ATII cells, and in vivo in the mouse lung, a finding that has not been reported before. Tagln was initially isolated, purified, and characterized as an abundant 22-kDa protein in chicken gizzard smooth muscle (26
, 27)
. Subsequently, Tagln expression was also reported in smooth muscle tissues from various other species and organs; its highest expression levels were found in aorta, intestine, lung, stomach, and uterus (29)
. In humans, the Tagln gene is located on chromosome 11q23.2, contains 5 exons, and encodes a 22-kDa protein consisting of 201 amino acids (24)
. Tagln expression was initially reported to be restricted to smooth muscle cells, largely because of the finding that heterozygous Tagln+/–LacZ mice demonstrated smooth muscle-specific activity of β-galactosidase (30)
. In smooth muscle cells, TGF-β increases Tagln expression via a Smad3-dependent process (31)
. Tagln knockout mice demonstrate normal vascular development, showed no developmental abnormalities, and were viable and fertile (30)
.
Recent reports, however, have described extramuscular localization of Tagln in fibroblasts (32)
, endothelial cells (33)
, embryonic and neuronal stem cells (34)
, B-1 B cells (35)
, or intestinal epithelial cells (28)
, as assessed by genomic and proteomic analysis. In this respect, it is of interest that an increase in Tagln expression was detected in fibroblast-to-myofibroblast transdifferentiation (32)
, as well as in embryoid bodies transforming into smooth muscle cells (34)
. Tagln expression was also increased in endothelial cells subjected to cyclic strain, which is a process leading to endothelial-to-smooth muscle cell transdifferentiation (33)
. Taken together, these data highly suggest that Tagln expression coincides with cellular plasticity, which is further documented by the data presented in this manuscript.
Using siRNA knockdown technology, we were able to show that Tagln is required for TGF-β-dependent epithelial cell migration and proliferation. While a lack of Tagln expression slightly affected proliferation, its effect on migration was much more pronounced, both in A549 and primary ATII cells (Figs. 6
and 7)
. A lack of Tagln completely abrogated the positive effect of TGF-β on ATII cell migration and reduced TGF-β-induced changes on epithelial cell proliferation. These data suggested to us that Tagln played a role in TGF-β-dependent EMT. To this end, we performed detailed RT-PCR analysis of epithelial and mesenchymal marker gene expression in A549 and primary mouse ATII cells (Fig. 7C, D
). While we observed changes in marker gene expression in cells depleted of tagln, we could not confirm a role for tagln in EMT using the latter assays. Recently, A549, as well as rat and mouse primary ATII cells, has been described to undergo EMT in response to TGF-β (17
, 19
, 37)
. In this respect, our data support a role for Tagln in ATII cell migration, but do not support a role for Tagln in EMT using the current assays.
Taken together, the available in vivo and in vitro data on Tagln indicate that increased TGF-β activity, which is known to drive fibrotic tissue transformation (5)
, transactivates Tagln expression in ATII cells via direct binding of Smad3 to the SBEs within the Tagln promoter. Subsequent Tagln protein expression alters and rearranges the epithelial cytoskeleton and facilitates epithelial cell migration, which itself perpetuates the fibrotic process in the lung. Modification of Tagln expression modulates the ATII cell phenotype, targeting this pathway may therefore represent a valuable option for maintaining physiological ATII cell function in lung fibrosis.
Received for publication May 29, 2007. Accepted for publication December 23, 2007.
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