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Published ahead of print on April 17, 2003, doi:10.1165/rcmb.2003-0063OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 397-404, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2003-0063OC

Smad3 Mediates Transforming Growth Factor-ß–Induced {alpha}-Smooth Muscle Actin Expression

Biao Hu, Zhe Wu and Sem H. Phan

Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan

Address correspondence to: Dr. Sem H. Phan, Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602. E-mail: shphan{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor-ß (TGF-ß)–induced {alpha}-smooth muscle actin (ASMA) expression is a key indicator of myofibroblast differentiation from fibroblasts. Recent studies suggest that a TGF-ß control element is important in the regulation of the ASMA gene promoter by TGF-ß. In this study, the role of Smad3, a key component of the Smad pathway that mediates TGF-ß signaling in regulation of ASMA gene expression, is investigated. All members of the Smad family were expressed in rat lung fibroblasts, and Smad3 expression was elevated upon TGF-ß1 treatment. Transfection with a Smad3-expressing plasmid markedly increased Smad3 and ASMA protein expression, whereas transfection with an antisense Smad3 plasmid suppressed Smad3 and ASMA expression. Similar effects were noted when the cloned rat ASMA promoter-luciferase reporter gene construct was used to monitor transcriptional activation of the ASMA gene. Electrophoretic mobility shift assays and DNA affinity precipitation indicated Smad3 binding to at least two regions of the promoter containing CAGA motifs, termed Smad3-binding elements (SBEs). Mutation of one of the SBEs decreased promoter activity significantly, indicative of a functional role for this SBE. Taken together, these findings suggest a role for Smad3 in TGF-ß regulation of ASMA gene expression in myofibroblast differentiation.

Abbreviations: {alpha}-smooth muscle actin, ASMA • Dulbecco's modified Eagle's medium, DMEM • electrophoretic mobility shift assay, EMSA • phosphate-buffered saline, PBS • plasma-derived serum, PDS • phenylmethylsulfonyl fluoride, PMSF • Smad3-binding element, SBE • TGF-ß control element, TCE • transforming growth factor-ß, TGF-ß


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
{alpha}-Smooth muscle actin (ASMA), the most abundant protein in smooth muscle cells, is expressed primarily in normal adult smooth muscle cells and transiently in cardiac and skeletal muscle cells during development (1, 2). Expression, however, is not exclusive to smooth muscle cells, and is a prominent feature of other cells such as myofibroblasts (3, 4). Myofibroblasts have a phenotype intermediate between fibroblasts and smooth muscle cells, and their emergence such as in pulmonary fibrosis may be induced by cytokines, especially transforming growth factor-ß (TGF-ß), an important cytokine regulating cell growth, morphogenesis, cell differentiation, and apoptosis (5–8). However, the regulatory mechanism for induction of ASMA gene expression in myofibroblast differentiation from fibroblasts is incompletely understood, and may be important toward understanding the pathogenesis of fibrotic lung diseases wherein myofibroblasts are a key feature as a source of extracellular matrix and fibrogenic cytokines.

Recently, a novel TGF-ß control element (TCE) at -42 to -61 from the transcriptional start site of the ASMA promoter is found to be important for its stimulation by TGF-ß (8, 9). Deletion or mutation of the TCE results in significant reduction of basal and TGF-ß–induced ASMA promoter activity (8–10). BTEB2, GKLF, SP1, and other Krüppel-like factors are reported to bind to the TCE and regulate promoter activity (10–12). In these studies, only the first 125-bp core promoter region of the ASMA gene was investigated because the effect of TGF-ß induction can be demonstrated in the absence of additional upstream sequences. Nevertheless, this core 125-bp promoter construct may not fully confer the overall and cell-specific transcriptional regulation of the ASMA gene. Previous studies have demonstrated that even though the core 125-bp ASMA promoter is highly active in rat aortic smooth muscle cells, it lacks activity in L6 myotubes, a cell type that endogenously expresses the ASMA gene (13). Additionally, this 125-bp promoter/reporter construct exhibits high activity in endothelial cells, a cell type that does not endogenously express the ASMA gene (13). Moreover, this 125-bp ASMA promoter fragment is inactive when tested in transgenic mice (14). Thus, a longer fragment of the ASMA promoter should be studied, and cis-acting elements in loci other than the core promoter region must be considered for a complete picture of the regulation of ASMA gene transcriptional activation. As an example, enhancer activity in intron 1, in addition to the core promoter and upstream regions of the ASMA gene, are found to be important in its transcriptional regulation in activated mesangial cells (15).

A system comprised of Smad proteins is found to mediate intracellular signaling of the TGF-ß superfamily of ligands in many cells (16–20). For the TGF-ß pathway, the Smad proteins, Smad2 and Smad3, are ligand-responsive (16, 17). They are molecules of relative molecular mass 42–60 kD with two regions of homology at the amino and carboxy terminals, termed Mad-homology domains 1 (MH1) and 2 (MH2), respectively. These regions are connected by a proline-rich linker sequence of variable length that is not as highly conserved (18). The MH1 domain of Smad3 and Smad4, but not of Smad2, mediates direct binding to DNA, and in conjunction with other transcription factors, regulate promoter activity in this manner (18, 19). The MH2 domain mediates interaction with other proteins to form homo- and heteromeric complexes, resulting in either activation or inhibition of gene expression, partly by regulating DNA binding by the MH1 domain (21). Smad4 functions as a cofactor in this signaling pathway, forming complexes with the previous two Smads and translocating to the nucleus to regulate gene expression (17). Upon TGF-ß binding to the TGF-ß type II receptor (TßRII), the active type II receptor kinase phosphorylates and activates the type I receptor (TßRI) (22, 23). The activated TßRI is phosphorylated at its carboxy-terminal and transiently associates with Smad2 and Smad3 via mediation by a membrane-associated FYVE domain–containing protein, Smad anchor for receptor activation or SARA (20). These receptor-activated Smads are phosphorylated, which enable them to form a heteromeric complex with Smad4 and then translocate into the nucleus where target genes are transcriptionally regulated (24). The activation of TGF-ß–induced transcription can be mediated through the binding element of Smad3/Smad4 containing the CAGA box (25–27) or through the cooperation of Smad proteins with other trans-acting factors (28). Despite this well-known role of the Smad pathway in mediating TGF-ß regulation of gene expression, the role of Smad proteins has not been fully elucidated in TGF-ß–induced ASMA expression and myofibroblast differentiation.

To address this issue, the role of Smad3 in TGF-ß–induced lung myofibroblast differentiation in terms of ASMA gene expression was examined. First, its expression along with other Smad proteins was confirmed in isolated rat lung fibroblasts. TGF-ß1 treatment enhances the expression of Smad3 and Smad4. The role of Smad3 in ASMA expression was confirmed by its elevated expression in cells transfected with a Smad3-expressing plasmid, and inhibition by an antisense construct. Cotransfection analysis of a 764-bp rat ASMA promoter construct with Smad3 or anti-Smad3 expression constructs confirmed that Smad3 could stimulate ASMA promoter activity. Sequence analysis identified two potential Smad3-binding elements (SBEs), one of which resides upstream of the core 125-bp ASMA promoter used in previous studies (10–12). The functional importance of one of these SBEs to ASMA gene transcription was suggested by mutational analysis of the 764-bp rat ASMA promoter construct in rat lung fibroblasts. Binding of Smad3 to these sequences was demonstrated by gel shift assay, supershift analysis, and DNA affinity precipitation. These findings taken in their totality suggest that in addition to the Krüppel-like factor–TCE interaction, a Smad3–SBE interaction is also important in mediating TGF-ß1–induced ASMA gene expression in rat lung myofibroblast differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Fibroblasts were isolated from adult rat lungs as described previously (8). Briefly, 4- to 6-wk-old rats were killed, and their lungs were perfused with phosphate-buffered saline (PBS). The lung tissue was digested in trypsin-EDTA solution until the cells were released. The cells were cultured in complete medium composed of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% plasma-derived serum (PDS) (Cocalico Biologicals, Inc., Reamstown, PA), 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml fungizone, 1% ITS (insulin, transferrin, selenium) (Sigma Chemicals, St. Louis, MO), 5 ng/ml PDGF (R&D Systems, Minneapolis, MN), and 10 ng/ml EGF (R&D Systems).

TGF-ß1 Treatment
Cells were cultured overnight in complete medium. They were deprived of serum by rinsing twice in PBS and incubating in DMEM containing 2 mg/ml bovine serum albumin for 24 h. This was followed by addition of 2 ng/ml TGF-ß1 (R&D Systems) for different time as indicated in the text before harvesting.

Construction of ASMA Promoter Constructs and cDNA Clones
The rat ASMA promoter was cloned by polymerase chain reaction (PCR) from rat genomic DNA with primers 5'-ACGGTCCTTAAGCATGATAT-3' and 5'-CTTACCCTGATGGCGACTGGCTGG-3' according to the published sequence (GenBank, S76011). It was inserted into vector pGal3-basic (Promega, Madison, WI) at the smaI site to form the {alpha}-SMAp-luc fusion plasmid pGal3-{alpha}SMAp, which is used as the template for site-directed mutagenesis. Two putative SBEs, identified as SBE1 (-552 to -513) and SBE2 (-5 to +28), were discovered by sequence comparison analysis. According to the sequences at these two sites, the following primers were used for the construction of promoter mutants to confirm functional importance of these two SBEs: A (5'-TACAGACTTCATTGATACTACACAAAGCTTCCAGACTACATAC-3'); B (5'-GTATGTAGTCTGGAAGCTTTGTGTAGTATCAATGAAGTCT-3'); C (5'-CCACCCACCTGCAGTGGAGAAGCCCAGC-3'); D (5'-CTGGGCTTCTCCACTGCAGGTGGGTGGT-3'); E (5'-TGCAAACCATGCCTGCAGATGCTTCATGACACTAGC-3'); and F (5'-GCAAGTGTCATGAAGGTTCTGCAGGCAGGGTTTGCA-3').

They form three primer pairs used in the QuickChange mutagenesis kit (Stratagene, La Jolla, CA) to obtain two SBE mutants and one unrelated mutant as a control. These mutant promoter constructs were designated as follows: {alpha}SMAp-luc-SBEm1 (C-524, A-525, and C-528 changed to T-524, C-525, and A-528, to mutate the SBE1 site), {alpha}SMAp-luc-SBEm2 (C+15, A+16, and G+17 changed to T+15, G+16, and C+17 to mutate the SBE2 site) and {alpha}SMAp-luc-SBEcontrol (C-294, T-295, and G-296 changed to A-294, G-295, and A-296 to mutate a site other than SBE1 and SBE2). A HindIII or a PstI restriction endonuclease site was created in each construct, respectively, for screening.

Rat Smad3 cDNA was first cloned in plasmid pCDNA3.0-Rsmad3 as previously described (29). It was cut down with EcoRI and inserted in frame into mammalian expression vector pEGFP-C2 (BD Biosciences Clontech, Palo Alto, CA) at EcoRI site to form smad3 expression plasmid pEGFPC2-RSmad3. The same fragment cloned in vector pEGFPC2 but in reverse direction was named pEGFPC2-RSmad3-Rev, whose transcript is an antisense Smad3 mRNA.

Western Blotting
Cells (7 x 104/well) were plated in 6-well plates and treated as above with TGF-ß1. The cells were harvested by scraping into Laemmli's sample loading buffer. Equal amounts of protein were electrophoresed through 12% SDS polyacrylamide gels. The separated protein bands were transferred onto nitrocellulose membranes. Nonspecific binding was blocked with 10% nonfat milk (Bio-Rad Laboratories, Hercules, CA) in 10 mM Tris-buffered saline containing 0.5% Tween 20. ASMA was detected using anti-ASMA monoclonal antibody (Cymbus Biotechnology Ltd., Hampshire, UK) at a dilution of 1:2,000, an anti-mouse IgG linked to horseradish peroxidase (Amersham Biosciences Co., Piscataway, NJ) and chemiluminescent substrate LumiGLO (New England Biolabs, Beverly, MA). All other antibodies were purchased from Santa Cruz Biotechnology, Inc. Santa Cruz, CA. Specificity of antibodies was confirmed using isoform-matched nonimmune immunoglobulins. The blots were exposed to Hyperfilm ECL film (Amersham Biosciences Co.).

Transfection and Reporter Gene Assay
All transient transfections were performed using the FuGENE6 reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. Supercoiled DNA was isolated with an endotoxin-free Qiagen column kit (Qiagen Inc, Valencia, CA). Unless otherwise indicated, cells were seeded in 6-well plates at a density of 105 per well in DMEM containing 10% PDS, and incubated at 37°C overnight. Routinely, 1 µg of the indicated DNA construct was used for transfection per culture in serum-free medium containing 2 mg/ml bovine serum albumin with or without 2 ng/ml TGF-ß1 treatment. For reporter gene assay, plasmid pSV–ß-galactosidase control vector was co-transfected for normalization. After 24 h the cells were harvested, and luciferase and ß-galactosidase activities were measured using the luciferase assay system and ß-galactosidase assay kit from Promega, respectively. Experiments with each construct were repeated two to four times, and relative activity (fold over promoterless control) was expressed as mean ± SE.

Nuclear Extract Preparation
Nuclear extracts were prepared from untreated and TGF-ß1–treated cultures as previously described (8). Briefly, the cultures were rinsed twice with cold PBS, and then with Dignam's Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], and 4 µg/ml leupeptin). The cells were then scraped into Buffer A and washed once with the same. The cell pellet was finally resuspended in Buffer A and kept on ice for 15 min before brief extraction in 0.6% NP40. The extract was vortexed, centrifuged briefly, and the cytoplasmic extract was then removed. The nuclei were further extracted in Dignam's Buffer C (20 mM HEPES, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, 4 µg/ml leupeptin) for 20 min on ice. The extracts were then centrifuged, and the supernatants stored at -70°C until used. Protein was estimated by the BCA method (Pierce, Rockford, IL).

Electrophoretic Mobility Shift Assay
The sequences of the oligonucleotide probes to detect the binding of Smad3 corresponds to the region between -552 and -513 (SBE1) or -5 and +28 (SBE2) of the ASMA promoter. Single-stranded oligonucleotides in the sense and antisense directions were synthesized. The sequences of the sense strands were: SBE1, 5'-TACAGACTTCATTGATACTACACACAGACTCCAGACTAC-3' and SBE2, 5'-TCCCCAGACACCACCCACCCAGAGTGGAGAAGC-3'. SBE-mutated oligonucleotide probes were also prepared for confirmation of specificity of binding, and the sense sequences of these probes were as follows (mutated bases are in lower case): SBE1 mut, 5'-TACAGACTTCATTGATACTACACAaAGctTCCAGAC-3'; SBE2 mut, 5'-CCCAGACACCACCCACCtgcAGT-3'.

The oligonucleotides were annealed before labeling with T4 polynucleotide kinase and ({gamma}-32P) ATP to detect double stranded DNA-binding proteins. Electrophoretic mobility shift assay (EMSA) reaction mixtures contained 3–5 µg of protein extract, 1.0 µg of Poly dI-dC, 0.1 µg poly-L-lysine, 0.5–1 ng labeled probe (20,000–30,000 cpm), and antibodies as indicated, in a final volume of 15 µl of Dignam's Buffer C. Where indicated, the EMSA reaction mixtures were preincubated with antibodies on ice for 30 min before probe addition and incubation for another 20 min at room temperature. Samples were then analyzed by electrophoresis on 8% nondenaturing polyacrylamide gels at 300 V in 1x TBE. After electrophoresis, the gels were dried and exposed to X-ray film for 24 h.

DNA Affinity Precipitation
To confirm Smad3 binding to SBE1 and SBE2, DNA affinity precipitation was undertaken using biotinylated oligonucleotides essentially as described (30). Briefly, equal amounts of cell lysate protein (100 µg) prepared in 1% Nonidet P-40, 150 mM NaCl, 1 mM PMSF, 1.5% aprotinin, and 20 mM Tris-HCl (pH 7.5) were precleared with streptavidin-agarose beads (Sigma Chemical Co.) and then incubated with 200 ng of biotinylated double-stranded oligonucleotides in the presence of 2 µg of poly(dI-dC) at 4°C for 1 h. DNA-bound proteins were mixed with streptavidin-agarose for 1 h with end-over-end rotation, washed extensively with cell lysis buffer, and analyzed by SDS-PAGE and immunoblotting using anti-Smad3 antibodies. The sense strand oligonucleotide sequences were: SBE1, 5'-ACAGACTTCATTGATACTACACACAGACTCCAGACTAC-3'; SBE2, 5'-CCCCAGACACCACCCACCCAGAGTGGAGAAGC-3'; SBE1 mut, 5'-TACAGACTTCATTGATACTACACAaAGctTCCAGAC-3'; SBE2 mut, 5'-CCCAGACACCACCCACCtgcAGT-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Smads in Rat Fibroblast
TGF-ß plays a key role in tissue repair and fibrosis, partly due to its capacity to induce myofibroblast differentiation. Part of this induction includes ASMA gene expression, wherein a novel TCE at -42 to -61 from transcriptional start site is reported to be important for the activation of the 125-bp core ASMA promoter by TGF-ß (8, 9). However, the core promoter may not fully confer the overall and cell-specific transcriptional regulation of the ASMA gene, as its expression level varies in different cell types (13, 14). Thus, a longer fragment of the ASMA promoter should be studied, and cis-acting elements in loci other than the core promoter region must be considered for a more complete picture of how ASMA gene expression is regulated.

In a search for other possible regulatory elements involved, the role of the Smad proteins associated with TGF-ß signaling was investigated. Because this pathway involves the cooperation of the effector Smads proteins, including Smad3 and Smad4, and their binding to an SBE or to a core "CAGA" sequence (or SBE-like sequences) within the regulatory region of the target genes, the ASMA promoter was first analyzed by sequence comparison to search for potential SBE sites. The results of this analysis showed at least two potential SBEs, one at -552 to -513 (SBE1) of the ASMA promoter containing three CAGA motifs, and another at -5 to +28 with two CAGA motifs (Figure 1). Thus, these findings support the possible importance of Smad proteins in regulation of ASMA gene expression in TGF-ß–induced myofibroblast differentiation.



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  		Figure 1. Nucleotide sequence of rat ASMA promoter. The SBE1 and SBE2 are boxed, with CAGA motifs in bold. The TCE is double-underlined, whereas the TATA box is underlined. "+1" represents the transcription initiation site and is indicated by the arrow.

 
As a first step in elucidating the potential role of the Smad pathway, expression of this protein family was examined in rat lung fibroblasts by Western blotting analysis at 48 h after TGF-ß treatment, when the induction of ASMA reached the highest level (data not shown). The results indicated that all members of the Smad family were detected in untreated cells (Figure 2). Upon treatment with TGF-ß, the level of expression was not appreciably changed, with the exception of Smad3, Smad4, and Smad8. The expression of Smad3 and Smad4 increased, whereas the expression level of Smad8 decreased with TGF-ß treatment, consistent with a role for at least Smad3 and Smad4 in regulation of gene expression. Additionally, TGF-ß caused increased Smad3 nuclear localization, which correlated with increased ASMA expression.



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  		Figure 2. Expression of Smads and ASMA in rat lung fibroblasts. Expression of the indicated proteins by untreated (-) or TGF-ß1–treated (+) rat lung fibroblasts was analyzed by Western blotting after 48 h of incubation. All samples were whole-cell lysates, except for "Nuclear Smad3," wherein nuclear extracts were analyzed. The numbers beneath each protein band indicate the relative level of expression (as % of untreated control) determined by densitometric scanning, with the respective untreated control set to 100%.

 
Effects of Smad3-Expressing Construct and Antisense Oligonucleotides on ASMA Expression
To further examine the possible role of Smad3 in the regulation of ASMA gene expression, a Smad3-expressing plasmid and an antisense Smad3 mRNA expression plasmid were transfected into rat lung fibroblasts, and the effects on ASMA expression were analyzed by Western blotting. As shown in Figure 3, treatment with TGF-ß stimulated both ASMA and Smad3 expression. In addition, both the basal and TGF-ß–induced Smad3 and ASMA expression in these cells was inhibited by transfection with the antisense Smad3 plasmid, but stimulated by transfection with the (sense) Smad3 expression construct, when compared with the levels due to transfection of empty vector controls (Figure 3). Thus the levels of ASMA expression correlated with the levels of Smad 3 expression in these cells, suggesting a role for Smad3 in the regulation of ASMA expression. The expression of GAPDH was not affected by either TGF-ß treatment or any of the transfections.



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  		Figure 3. Effect of Smad3 on ASMA expression. Plasmids expressing (sense) "Smad3" (pEGFPC2-RSmad3), "Antisense Smad3" (pEGFPC2-RSmad3-Rev) or "Empty vector" only were transfected into lung fibroblasts. The cells were treated with buffer only ("None") or with TGF-ß1. They were then lysed and the cell extracts analyzed by Western blotting for Smad3, ASMA, or GAPDH protein expression. Equal amounts of protein were loaded for gel electrophoresis before blotting. Duplicate samples were analyzed in each experiment, which was repeated twice with similar results.

 
To confirm that Smad3 could stimulate ASMA expression at the transcriptional level, a 764-bp rat ASMA promoter was fused with the luciferase reporter gene to form {alpha}-SMAp-luc fusion construct, and then was cotransfected with either the Smad3 or antisense Smad3 expression plasmid into fibroblasts. Overexpression of Smad3 using this approach stimulated ASMA promoter activity, which was dramatically enhanced by TGF-ß1 treatment (Figure 4). The antisense Smad3 plasmid inhibited basal and TGF-ß–induced ASMA promoter activity, whereas the empty vector did not significantly affect promoter activity (Figure 4). These results confirmed that Smad3 could regulate transcription of the ASMA gene, and that TGF-ß–induced ASMA promoter activation was mediated by Smad3.



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  		Figure 4. Effect of Smad3 on ASMA promoter activity. Fibroblasts were cotransfected with the ASMA promoter construct (pGal3-{alpha}-SMAp-luc, also abbreviated as {alpha}-SMAp-luc) and the indicated individual Smad3 construct or expression vector only. "Smad3" indicates the Smad3 expressing construct, pEGFPC2-Smad3, whereas "anti-sense Smad3" refers to the reverse sequence Smad3 construct, pEGFPC2-Smad3-Rev. The effects of the expression vector only were used as controls (indicated as "vector"). The cells were then treated with buffer only (open bars) or with TGF-ß1 (filled bars), and then the cell extracts were harvested and assayed for luciferase and ß-galactosidase activities. The luciferase activity was normalized for each construct to its respective ß-galactosidase activity, and the results shown as fold increase over the promoterless activity. Data represent means ± SE of triplicate samples.

 
Mutational Analysis of SBEs
Regulation of promoter activity by Smad3 involves its binding to an SBE or to a core "CAGA" sequence (or SBE-like sequences) within the promoter itself (25–27). As determined by sequence analysis (Figure 1), the ASMA promoter contained at least two potential SBEs in the ASMA promoter, one at -552 to -513 (SBE1) from the transcriptional start site containing three CAGA motifs, and the other at -5 to +28 (SBE2) with two CAGA motifs. To test if these SBEs were functionally important for the regulation of ASMA promoter activity by Smad3, a series of {alpha}-SMAp-luc fusion plasmid with mutated SBEs ({alpha}-SMAp-luc-SBEm1 and {alpha}-SMAp-luc-SBEm2) were constructed, along with a control ({alpha}-SMAp-luc-SBEcontrol) plasmid with a mutation outside the SBEs (Figure 5A). They were then used together with the wild-type {alpha}-SMAp-luc fusion plasmid to assess the impact of mutations in the SBE sequences on promoter activity. Upon transfection of such constructs into fibroblasts, the activity of wild-type {alpha}-SMAp-luc was significantly stimulated by TGF-ß1 treatment as expected (Figure 5B). In comparison with the wild-type construct, stimulation of the mutated SBE1 ASMA promoter construct by TGF-ß1 was significantly reduced, whereas mutation of the SBE2 and mutation at a site other than the SBEs did not significantly impair the TGF-ß–induced promoter activity. These results indicated that SBE1 is important for the regulation of ASMA by Smad3.



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  		Figure 5. Effects of SBE mutations on ASMA promoter activity. A representation of maps of the ASMA promoter, its wild-type and mutant constructs is shown in A. The CAGA-binding motifs (boxed) in SBE1 and SBE2 are shown for the wild-type construct ({alpha}-SMAp-luc). Mutated bases in the mutant and control constructs are italicized and underlined. These wild-type and mutant constructs were transfected into fibroblasts, and the cells were treated with buffer only (open bars) or with TGF-ß1 (filled bars). Cell extracts were then analyzed for luciferase activity and normalized to their respective control ß-galactosidase activity. The normalized luciferase activities of the indicated constructs are shown in B. Results were expressed as fold increase over the promoterless control mean value, and shown as means ± SE of triplicate samples.

 
Binding of Smad3 to the SBE Sites
As the effect of Smad3 on a target gene usually depends on formation of a Smad–SBE complex (25–27), Smad3 may recognize and bind to the SBE1 and/or SBE2 in the ASMA promoter. This was indeed suggested by the results of mutational analysis of the SBEs (Figure 5B). To examine such a possibility, single-stranded sense and antisense oligonucleotides corresponding to the sequence of SBE1 or SBE2 were synthesized and annealed to be used as probes in an EMSA. The results showed that a complex was formed using either the SBE1 or SBE2 probe, when incubated with nuclear extracts from either untreated or TGF-ß1–treated fibroblasts (Figure 6). Treatment with TGF-ß1 enhanced the binding of trans-acting factor to the SBE1 probe. The DNA–protein complex was supershifted by the addition of anti-Smad3 or anti-Smad4 antibodies. The binding was specific because the complex was competed off with a 200-fold excess of corresponding unlabeled probe (Figure 6). To confirm the specificity of this binding, probes with mutated SBE sequences were used for the EMSA. Mutation of either of the SBE mutated sequences eliminated the formation of the DNA–protein complex (data not shown).



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  		Figure 6. Binding of Smad3 to SBE1 and SBE2 by EMSA. Nuclear extracts from untreated ("Control") or TGFß1 treated ("+TGFß1") cells were incubated with either SBE1 or SBE2 32P-labeled oligonucleotide probes, and then subjected to polyacrylamide gel electrophoresis under nondenaturing conditions. Protein binding to either probe results in the shifted band indicated by a solid arrow. Selected samples were pretreated with anti-Smad3 or anti-Smad4 antibodies as indicated before addition of radiolabeled probe, and the location of supershifted bands is indicated by an open arrow. Addition of 200x molar excess of unlabeled probe ("Cold Probe") was used to confirm specificity of any protein binding.

 
To further confirm that Smad3 does bind to the SBEs in the ASMA promoter, DNA affinity precipitation analysis was undertaken. The results show that Smad3 could be pulled down by both biotinylated SBE1 and SBE2 probes from extracts of both control and TGF-ß1–stimulated cells, with stronger signals noted in the extracts from treated cells (Figure 7). Mutation of the CAGA motif in these probes (SBE1 mut and SBE2 mut) essentially abolished this capability. These results indicated the ability of Smad3 to bind to both SBE1 and SBE2 in the ASMA promoter, which was stimulated by treating cells with TGF-ß.



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  		Figure 7. Binding of Smad3 to SBE1 and SBE2 by DNA affinity precipitation. Equal amounts of lysate protein from control and TGF-ß1–treated cells were incubated with biotinylated wild-type SBE1 or SBE2 double-stranded oligonucleotides as indicated, followed by incubation with streptavidin–agarose. After extensive washing, the precipated oligonucleotide–protein complexes were separated by gel electrophoresis followed by immunoblotting using anti-Smad3 antibody. Specificity of binding to the probe was determined using mutant double-stranded oligonucleotide probes, SBE1 mut or SBE2 mut, instead of the corresponding wild-type probes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The emergence and accumulation of myofibroblasts are key characteristics of pulmonary fibrosis, and appear to arise by differentiation from fibroblasts (3–8). Expression of ASMA is one of the key markers of fibroblast to myofibroblast differentiation (6). When fibroblasts differentiate to myofibroblasts, they produce high amounts of TGF-ß, which in turn could induce more neighboring fibroblasts to differentiate (8, 9, 31). The goal of the present study was to investigate the molecular mechanisms whereby TGF-ß upregulates ASMA expression as a means of providing additional insight to the mechanism of myofibroblast differentiation induced by this cytokine. Previous investigation using the first 125 bp of the ASMA promoter showed that a TCE, located at -42 to -61 from the transcriptional start site of the ASMA promoter, mediates this effect in smooth muscle cells (8, 9). However, more evidence is accumulating to show that the regulation of ASMA gene expression is complex, and likely to be tissue- and cell type–specific (13, 14). The core 125-bp promoter construct may not fully confer the overall and cell-specific transcriptional activation of the ASMA gene, despite the fact that the maximal effect of TGF-ß induction can be seen in this short promoter. This possibility is supported by the observation that the first 125-bp promoter region lacks activity in L6 myotubes, a cell type that constitutively expresses the ASMA gene (13). In contrast to L6 myotubes, the same 125-bp promoter exhibits high activity in endothelial cells, a cell type that does not normally express the ASMA gene (13). Furthermore, when promoter constructs of varying lengths were transiently transfected into skeletal myoblasts that were then induced to differentiate into myotubes, the promoter activities differed from those seen in other cell types. Addition of the region between -125 and -699, which contains the SBE1 reported in this study (Figure 1), results in higher promoter activity compared with that of the first -125-bp core promoter in skeletal myotubes, but causes decreased promoter activity in skeletal myoblasts, endothelial cells, and smooth muscle cells (13). Therefore, cis-acting elements in loci other than the core promoter region must be considered for a complete picture of the transcriptional regulation of the ASMA gene (32).

A system comprised of Smad proteins is found to mediate intracellular signaling of the TGF-ß superfamily of ligands in many cells (16–20). An in vivo study shows that loss of Smad3 greatly attenuated morphologic evidence of fibrosis in bleomycin-treated mice, thus implicating Smad3 in the pathogenesis of pulmonary fibrosis (33). However, the Smad pathway and its possible role in mediating TGF-ß–induced myofibroblast differentiation, and specifically ASMA gene expression, have not been determined. In this study, expression of all members of the Smad family by rat lung fibroblasts was documented. The initial survey also indicated that Smad3 and Smad4 expression were elevated in TGF-ß1–stimulated fibroblasts. Interestingly, in lung type II alveolar epithelial cells that do not express ASMA, Smad3 was undetectable (data not shown). Based on this suggestion of a potential role for the Smad pathway, and especially Smad3, the role of Smad3 in ASMA expression was first tested by transient transfection with Smad3 or antisense Smad3 constructs into rat lung fibroblasts. The importance of Smad3 was suggested by elevated ASMA expression in cells transfected with a Smad3-expressing plasmid, and inhibition of ASMA expression by the antisense construct. Furthermore, analysis of the rat ASMA promoter sequence identified two potential SBEs. The functional importance of one of these SBEs, namely the more upstream SBE1, in regulating ASMA gene expression was demonstrated by mutational analysis of a 764-bp rat ASMA promoter construct. Thus only the construct mutated at the SBE1 site showed significantly reduced promoter activity. The function of SBE2, if any, remains to be determined. Binding of Smad3 to these SBE sequences was shown by gel shift assay combined with supershift analysis, and DNA affinity precipitation. These findings taken in their totality indicate that besides the TCE, Smad3 regulation via binding to SBE1 may also be an important factor in TGF-ß1–induced ASMA gene expression. The relative importance of SBE1 vis-à-vis TCE has not been directly addressed, although a TCE decoy oligonucleotide has been shown to inhibit TGF-ß1–induced ASMA expression (8), indicating that the TCE is essential for this induction. Mutation of the TCE has only been undertaken in the truncated promoter constructs that do not contain the SBE1 sequence (8, 9), thus making it impossible to directly assess the relative contribution of the former versus the latter. In the current study, mutation of SBE1 significantly inhibited promoter (with intact TCE) activity to a similar extent as that seen in the truncated TCE mutant promoter as well as by the decoy TCE oligonucleotide (8). This circumstantial evidence suggests that both SBE1 and TCE may be equally important in regulating the intact promoter, perhaps due to interaction of transcription factors bound to these elements; but that the truncated promoter (without SBE1 sequence) requires only the TCE for activity. Further study is required to confirm or refute such a possibility.

In summary, the results of this study extend current understanding of ASMA gene regulation by TGF-ß to more upstream regions of the ASMA promoter, which include SBE1 located beyond the first 125-bp core promoter sequence. The findings and the results of previous studies strongly suggest that binding of both Smad3 to SBE1 and Krüppel-like factors to TCE is essential for the regulation of ASMA gene expression by TGF-ß. Future study will focus on the potential cross talk between these and other transcription factors, as has been reported in the regulation of {alpha}2(I) procollagen (COL1A2) and p15(Ink4B) gene transcription by TGF-ß (33, 34). In those studies, Smad3 co-operates with one of the TCE-binding factors, Sp1, to regulate TGF-ß–induced promoter activity. Similar synergistic interactions may also be important in the regulation of ASMA gene expression.


   Acknowledgments
 
The authors are grateful for the generous gift of rat Smad3 plasmid from Dr. Wylie Vale, Salk Institute. The expert technical assistance of Matt Ullenbruch, Bridget McGarry, and Hong Jin is also gratefully acknowledged. This work is supported in part by NIH grants HL52285, HL28737, and HL31963.

Received in original form February 26, 2003

Received in final form April 11, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Campbell, J. H., O. Kocher, O. Skalli, G. Gabbiani, and G. R. Campbell. 1989. Cytodifferentiation and expression of alpha-smooth muscle actin mRNA and protein during primary culture of aortic smooth muscle cells: correlation with cell density and proliferative state. Arteriosclerosis 9:633–643.[Abstract/Free Full Text]
  2. Woodcock-Mitxhell, J., J. J. Mitchell, R. B. Low, M. Kieny, P. Sengel, P. Rubbia, O. Skalli, B. Jackson, and G. Gabbiani. 1988. Alpha-smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles. Differentiation 39:161–166.[CrossRef][Medline]
  3. Darby, I., Q. Skalli, and G. Gabbiani. 1990. Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab. Invest. 63:21–29.[Medline]
  4. Skalli, O., W. Schurch, T. Seemayer, R. Lagace, D. Montandon, B. Pittet, and G. Gabbiani. 1989. Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins. Lab. Invest. 60:275–285.[Medline]
  5. Desmouliere, A. 1995. Factors influencing myofibroblast differentiation during wound healing and fibrosis. Cell Biol. Int. 19:471–476.[CrossRef][Medline]
  6. Desmouliere, A., A. Geinoz, F. Gabbiani, and G. Gabbiani. 1993. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J. Cell Biol. 122:103–111.[Abstract/Free Full Text]
  7. Ronnov-Jessen, L., and O. W. Pertersen. 1993. Induction of alpha-smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts: implications for myofibroblast generation in breast neoplasia. Lab. Invest. 68:696–707.[Medline]
  8. Roy, S. G., Y. Nozaki, and S. H. Phan. 2001. Regulation of alpha-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts. Int. J. Biochem. Cell Biol. 33:723–734.[CrossRef][Medline]
  9. Hautmann, M. B., C. S. Madsen, and G. K. Owens. 1997. A transforming growth factor beta (TGFbeta) control element drives TGFbeta-induced stimulation of smooth muscle alpha-actin gene expression in concert with two CArG elements. J. Biol. Chem. 272:10948–10956.[Abstract/Free Full Text]
  10. Adam, P. J., C. P. Regan, M. B. Hautmann, and G. K. Owens. 2000. Positive- and negative-acting Kruppel-like transcription factors bind a transforming growth factor beta control element required for expression of the smooth muscle cell differentiation marker SM22alpha in vivo. J. Biol. Chem. 275:37798–37806.[Abstract/Free Full Text]
  11. Brembeck, F. H., and A. K. Rustgi. 2000. The tissue-dependent keratin 19 gene transcription is regulated by GKLF/KLF4 and Sp1. J. Biol. Chem. 275:28230–28239.[Abstract/Free Full Text]
  12. Black, A. R., J. D. Black, and J. Azizkhan-Clifford. 2001. Sp1 and kruppel-like factor family of transcription factors in cell growth regulation and cancer. J. Cell. Physiol. 188:143–160.[CrossRef][Medline]
  13. Corjay, M. H., M. M. Thompson, K. R. Lynch, and G. K. Owens. 1989. Differential effect of platelet-derived growth factor- versus serum-induced growth on smooth muscle alpha-actin and nonmuscle beta-actin mRNA expression in cultured rat aortic smooth muscle cells. J. Biol. Chem. 264:10501–10506.[Abstract/Free Full Text]
  14. Mack, C. P., and G. K. Owens. 1999. Regulation of smooth muscle alpha-actin expression in vivo is dependent on CArG elements within the 5' and first intron promoter regions. Circ. Res. 84:852–861.[Abstract/Free Full Text]
  15. Kawada, N., T. Moriyama, A. Ando, T. Koyama, M. Hori, T. Miwa, and E. Imai. 1999. Role of intron 1 in smooth muscle alpha-actin transcriptional regulation in activated mesangial cells in vivo. Kidney Int. 55:2338–2348.[CrossRef][Medline]
  16. Heldin, C. H., K. Miyazono, and P. ten Dijke. 1997. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390:465–471.[CrossRef][Medline]
  17. Massague, J., A. Hata, and F. Liu. 1997. TGF-ß signalling through the Smad pathway. Trends Cell Biol. 17:187–192.
  18. Derynck, R., Y. Zhang and X. H. Feng. 1998. Smads: transcriptional activators of TGF-beta responses. Cell 95:737–740.[CrossRef][Medline]
  19. Zhang, Y., X. Feng, R. We, and R. Derynck. 1996. Receptor-associated Mad homologues synergize as effectors of the TGF-beta response. Nature 383:168–172.[CrossRef][Medline]
  20. Massague, J. 1998. TGF-beta signal transduction. Annu. Rev. Biochem. 67:753–791.[CrossRef][Medline]
  21. Wu, J. W., M. Hu, J. Chai, J. Seoane, M. Huse, C. Li, D. J. Rigotti, S. Kyin, T. W. Muir, R. Fairman, J. Massague, and Y. Shi. 2001. Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-beta signaling. Mol. Cell 8:1277–1289.[CrossRef][Medline]
  22. Chen, F., and R. A. Weinberg. 1995. Biochemical evidence for the autophosphorylation and transphosphorylation of transforming growth factor beta receptor kinases. Proc. Natl. Acad. Sci. USA 92:1565–1569.[Abstract/Free Full Text]
  23. Wrana, J. L., L. Attisano, R. Wieser, F. Ventura, and J. Massague. 1994. Mechanism of activation of the TGF-beta receptor. Nature 370:341–347.[CrossRef][Medline]
  24. Zhang, Y., T. Musci, and R. Derynck. 1997. The tumor suppressor Smad4/DPC 4 as a central mediator of Smad function. Curr. Biol. 7:270–276.[CrossRef][Medline]
  25. Dennler, S., S. Itoh, D. Vivien, P. ten Dijke, S. Huet, and J. M. Gauthier. 1998. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J. 17:3091–3100.[CrossRef][Medline]
  26. Jonk, L. J., S. Itoh, C. H. Heldin, P. ten Dijke, and W. Kruijer. 1998. Identification and functional characterization of a Smad binding element (SBE) in the JunB promoter that acts as a transforming growth factor-beta, activin, and bone morphogenetic protein-inducible enhancer. J. Biol. Chem. 273:21145–21152.[Abstract/Free Full Text]
  27. Zawel, L., J. L. Dai, and B. Vogelstein. 1998. Human Smad3 and Smad4 are sequence-specific transcription activators. Cell 1:611–617.
  28. Wong, C., E. M. Rougier-Chapman, J. P. Frederick, M. B. Datto, N. T. Liberati, J. M. Li, and X. F. Wang. 1999. Smad3-Smad4 and AP-1 complexes synergize in transcriptional activation of the c-Jun promoter by transforming growth factor beta. Mol. Cell. Biol. 19:1821–1830.[Abstract/Free Full Text]
  29. Chen, Y., J. J. Lebrun, and W. Vale. 1996. Regulation of transforming growth factor beta- and activin-induced transcription by mammalian Mad proteins. Proc. Natl. Acad. Sci. USA 93:12992–12997.[Abstract/Free Full Text]
  30. Yagi, K., M. Furuhashi, H. Aoki, D. Goto, H. Kuwano, K. Sugamura, K. Miyazono, and K. Mitsuyasu. 2002. c-myc is a downstream target of the Smad pathway. J. Biol. Chem. 277:854–861.[Abstract/Free Full Text]
  31. Gharaee-Kermani, M., E. M. Denholm, and S. H. Phan. 1996. Costimulation of fibroblast collagen and transforming growth factor beta1 gene expression by monocyte chemoattractant protein-1 via specific receptors. J. Biol. Chem. 271:17779–17784.[Abstract/Free Full Text]
  32. Shimizu, R. T., R. S. Blank, R. Jervis, S. C. Lawrenz-Smith, and G. K. Owens. 1995. The smooth muscle alpha-actin gene promoter is differentially regulated in smooth muscle versus non-smooth muscle cells. J. Biol. Chem. 270:7631–7643.[Abstract/Free Full Text]
  33. Zhao, J., W. Shi, Y. L. Wang, H. Chen, P. Bringas, Jr., M. B. Datto, J. P. Frederick, X. F. Wang, and D. Warburton. 2002. Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 282:L585–L593.[Abstract/Free Full Text]
  34. Feng, X. H., X. Lin, and R. Derynck. 2000. Smad2, Smad3 and Smad4 cooperate with Sp1 to induce p15(Ink4B) transcription in response to TGF-beta. EMBO J. 19:5178–5193.[CrossRef][Medline]
  35. Zhang, W., J. Ou, Y. Inagaki, P. Greenwel, and F. Ramirez. 2000. Synergistic cooperation between Sp1 and Smad3/Smad4 mediates transforming growth factor beta1 stimulation of alpha 2(I)-collagen (COL1A2) transcription. J. Biol. Chem. 275:39237–39245.[Abstract/Free Full Text]



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