Modulation of Endogenous Smad Expression in Normal Skin Fibroblasts by Transforming Growth Factor-β

Experimental Cell Research 258, 374 –383 (2000) doi:10.1006/excr.2000.4930, available online at http://www.idealibrary.com on

Modulation of Endogenous Smad Expression in Normal Skin Fibroblasts by Transforming Growth Factor-␤ Yasuji Mori, Shu-Jen Chen, and John Varga 1 Section of Rheumatology, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60607-7171

Transforming growth factor-␤ (TGF-␤) stimulation of collagen synthesis plays a fundamental role in physiological tissue repair as well as pathological fibrosis. Members of the SMAD family of intracellular proteins are phosphorylated by TGF-␤ receptors and convey signals to specific TGF-␤-inducible genes. Ligand binding initiates signaling through the SMAD pathway, but it is unknown how signaling is terminated. The expression and regulation of Smads have been characterized mostly in transformed cells using transient expression systems. In this study, we investigated the physiological regulation of endogenous Smads by TGF-␤ in nontransformed normal skin fibroblasts in vitro. Treatment with TGF-␤ resulted in time- and dose-dependent translocation of SMAD3 and SMAD4 from the cytoplasm to the nucleus. The levels of SMAD3 and Smad3 mRNA were profoundly down-regulated by TGF-␤1 or TGF-␤3 in a time-dependent manner, whereas expression of antagonistic Smad7 was rapidly and transiently induced. The stability of Smad3 mRNA transcripts was unaffected by TGF-␤. Cycloheximide prevented the inhibition of Smad3, but not the induction of Smad7, mRNA expression by TGF␤1, identifying Smad7 as an immediate-early gene target of TGF-␤ in fibroblasts. In Smad4-deficient breast cancer cells, TGF-␤ failed to modulate Smad expression, suggesting that SMADs mediate their own regulation in response to ligand. These results demonstrate that TGF-␤ not only triggers functional activation of the SMAD signaling cascade in primary skin fibroblasts, but also simultaneously exerts potent effects on endogenous SMAD expression and intracellular trafficking. Taken together with recent reports implicating ubiquitination in SMAD turnover, these findings indicate the existence of multiple levels of control for modulating SMAD-mediated TGF-␤ signaling in fibroblasts. © 2000 Academic Press Key Words: Smad signaling; skin fibroblasts; TGF-␤; Smad3; Smad7.

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To whom correspondence and reprint requests should be addressed at the Section of Rheumatology, University of Illinois at Chicago College of Medicine, Room 1158 MBRB, 900 S. Ashland Avenue, Chicago, IL 60607-7171. Fax: (312) 413-9271. E-mail: [email protected]. 0014-4827/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

INTRODUCTION

Transforming growth factor-␤ (TGF-␤) 2 plays crucial roles in regulating cellular processes, including growth and differentiation [1]. The diverse responses elicited by TGF-␤ are mediated through transcriptional activation of distinct sets of genes in target cells. Collagen, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) represent TGF-␤-inducible extracellular matrix proteins that are fundamental in physiological tissue repair as well as pathological fibrogenesis [2]. Mutational analysis of the regulatory regions of the genes encoding these proteins have delineated DNA elements that mediate their responsiveness to TGF-␤. For instance, a 200-bp region of the human ␣2(I) procollagen gene (COL1A2) promoter was shown to be necessary for induction of expression by TGF-␤ [3– 6]. Considerable evidence indicates that TGF-␤ is a key mediator responsible for excessive accumulation of collagen in fibrosis [7]. Despite a great deal of functional overlap between the isoforms of TGF-␤ [8], evidence exists indicating that TGF-␤1 and TGF-␤3 display distinct expression patterns in tissues and effects in vitro [9, 10]. Members of the TGF-␤ cytokine family initiate signaling through their interaction with heteromeric type I/type II TGF-␤ receptor complexes distributed on virtually all cell types [11]. The TGF-␤ receptors are transmembrane serine/threonine kinases that are activated by TGF-␤ and propagate signals downstream. SMADs associate with activated TGF-␤ receptors and play a crucial role in TGF-␤ signal transduction. Mutations in SMADs have been found in several types of malignancies, rendering the cancer cells resistant to growth inhibition by TGF-␤ [12]. To date, nine SMADs have been identified in vertebrates. While they share conserved carboxy-terminal Mad homology (MH)-1 and MH-2 domains, the SMADs segregate into three structurally and functionally distinct classes [13]. The highly conserved SMAD2 and SMAD3 are called receptor-associated SMADs because they transiently inter2 Abbreviations used: TGF-␤, transforming growth factor-␤; BMP, bone morphogenetic protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CHX, cycloheximide.

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act with, and are directly phosphorylated by, the activated type I TGF-␤ receptor. In its native state, SMAD3 is unable to bind to DNA or to other proteins (with the exception of SARA), presumably because both its DNA-binding and protein-binding domains are masked. Upon serine phosphorylation in its carboxyterminal SSXS motif by the type I TGF-␤ receptor, activated SMAD3 is “opened,” allowing it to dimerize with the common signal mediator SMAD4 in the cytoplasm. The heterodimeric SMAD complex translocates into the nucleus in a manner similar to that of STAT1␣. Within the nucleus, the SMAD complex binds to specific sequences of TGF-␤-inducible genes directly, or indirectly by interaction with other nuclear proteins, stimulating transcription through incompletely characterized mechanisms [14]. Ligand-induced nuclear translocation of intracellular signaling proteins is observed in several transduction pathways, suggesting that intracellular compartmentalization of SMADs regulates the activity of the SMAD signaling cascade [15]. Once in the nucleus, activated SMAD2 is rapidly degraded by ubiquitination [16]. SMAD7 displays substantial structural divergence from SMAD2 and SMAD3: it shows only limited conservation of the DNA-binding MH-1 domain and lacks the SSXS phosphorylation motif [17–20]. Like the pathway-restricted SMADs, SMAD7 interacts with the activated type I TGF-␤ receptor [21]. In contrast to SMAD2 and SMAD3, however, SMAD7 forms stable association with the receptor complex and prevents receptor-mediated phosphorylation of pathway-restricted SMADs, resulting in disruption of TGF-␤ signaling [19, 21]. In the Mv1Lu mink lung epithelial cell line, transfected SMAD7 inhibited TGF-␤ induction of early response genes such as jun-B and PAI-1 [22] and stimulation of PAI-1 promoter activity [23]. We have previously shown that in primary fibroblasts, transient overexpression of SMAD7 completely abrogated stimulation of COL1A2 promoter activity by TGF-␤ [24]. These finding suggest that SMAD7 is an antagonistic SMAD which may have an important role as a negative autocrine regulator of TGF-␤ signaling [21]. However, it is still not clear if SMAD7 is a nonspecific negative regulator of all TGF-␤ signals [23]; furthermore, it is not known whether physiologic levels of endogenous SMAD7 in fibroblasts can also abrogate ligand-induced phosphorylation of pathway-restricted SMADs. These observations indicate that SMAD3 and SMAD7 play fundamental and antagonistic roles in TGF-␤ regulation of cellular gene expression. These conclusions are based in large part on studies with immortalized or transformed cell lines and ectopically expressed Smads. The behavior of immortalized or transformed cell lines often bears only limited similarity with, or relevance for, that of nontransformed primary cells obtained directly from normal tissue. In addition, the regulation of signal transduction and tar-

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get gene expression by TGF-␤ is highly cell lineagespecific. Therefore, we were interested in characterizing endogenous Smad signaling and regulation in primary dermal fibroblasts established directly from normal skin and studied at low passage. We have previously shown that in normal skin fibroblasts, expression of recombinant SMAD3, with or without coexpressed SMAD4, mimicked the effect of TGF-␤ and caused transactivation of the COL1A2 promoter in the absence of ligand [24]. TGF-␤ failed to stimulate collagen transcription in Smad-deficient cancer cell lines or in fibroblasts in which endogenous SMAD3 expression was down-regulated by antisense oligonucleotides or antisense cDNA [6], indicating the crucial role of SMAD3 in mediating transcriptional TGF-␤ responses. The present results demonstrate that TGF-␤ treatment causes a dramatic decrease in Smad3 mRNA expression and SMAD3 protein levels in these cells. In marked contrast, the levels of antagonistic Smad7 were increased in TGF-␤-stimulated fibroblasts. Inhibition of Smad3 gene expression by TGF-␤ was delayed and protein synthesis-dependent, whereas stimulation of Smad7 was rapid and protein synthesis-independent. However, both of the Smad responses appeared to require intact cellular SMAD signaling. We also show that endogenous SMAD3 and SMAD4 rapidly translocated from the cytoplasm into the nucleus upon stimulation of fibroblasts with TGF-␤. SMAD7 appeared to be localized predominantly within the nucleus. Taken together, these results provide novel insight into the modulation of cellular Smad signaling in primary skin cells. The findings indicate the existence of multiple levels for regulating SMAD signaling activity in fibroblasts, not surprising in light of the fundamental role of this signaling pathway in cellular responses. Impairment of Smad regulatory mechanisms could contribute to aberrant TGF-␤ responses underlying pathological fibrosis. MATERIALS AND METHODS Cell cultures. Primary cell cultures were established from biopsies of adult skin and neonatal foreskin by previously described explant techniques [25]. Media were obtained from BioWhittaker (Walkersville, MD); all other tissue culture reagents were from Gibco BRL (Grand Island, NY). Cells were grown at 37°C in a 5% CO 2 atmosphere in modified Eagle’s medium supplemented with 10% fetal calf serum (FCS), 1% vitamins, 100 U/ml penicillin/streptomycin, and 2 mM L-glutamine and studied between passages 4 and 8. The MDA-MB468 human breast adenocarcinoma cell line, which has a homologous deletion of the Smad4 coding region [26], was obtained from the American Type Culture Collection (Rockville, MD) and grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS, 100 U/ml penicillin/streptomycin, and 2 mM L-glutamine When the cells reached confluence, fresh medium with the indicated concentration of FCS containing TGF-␤1 (Amgen, Thousand Oaks, CA), TGF-␤3 (Oncogene Science, Inc., Uniondale, NY), activin A (Ajinomoto, Inc., Kawasaki, Japan), or BMP-2 (Genetics Institute, Inc., Cambridge, MA) was added for the indicated period. In some experiments, cultures were incubated with 10 ␮g/ml cycloheximide

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for 2 h or 5 ␮g/ml actinomycin D (both from Sigma, St. Louis, MO), for 1 h prior to addition of TGF-␤1. Extraction and analysis of RNA. At the end of each experiment, total RNA was isolated from fibroblasts with TRIZOL Reagent (Gibco BRL). Relative levels of mRNA were examined by Northern analysis using [␣- 32P]dCTP-labeled cDNA probes. Following washing of the nitrocellulose membranes, the cDNA–mRNA hybrids were visualized by autoradiography on Kodak X-AR5 films exposed for 24 –72 h with intensifying screens. The following cDNA probes were used: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [27], a 1.4-kb restriction fragment that included the entire coding region of human Smad3 [28], a 1.6-kb restriction fragment that included the entire coding region of Smad4 [28], a 1.8-kb restriction fragment that included the entire coding region of Smad7 [29], PAI-1 [30], and fibronectin [31]. Western analysis. At the end of the incubation period, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed on ice in RIPA buffer (50 mM Tris/HCl, pH 7.5; 150 mM NaCl; 1% Nonidet-P40; 0.5% deoxycholate; 0.1% sodium dodecyl sulfate) containing protease inhibitor mix (complete; Boehringer Mannheim, Indianapolis, IN) and 1 mM phenylmethylsulfonyl fluoride. The whole-cell lysates were centrifuged at 13,000g for 20 min at 4°C and the supernatant was collected. The protein content was determined by Bradford protein assay (Bio-Rad, Hercules, CA). The lysates were analyzed by electrophoresis in 10% sodium dodecyl sulfate–polyacrylamide gels. All samples were prepared in Laemmli reducing buffer (final concentration 50 mM Tris/HCl, pH 6.8; 2% sodium dodecyl sulfate; 10% glycerol; 0.1% bromophenol blue; 100 mM dithiothreitol), and equal aliquots (15 ␮g) were boiled for 5 min before loading. Gels were blotted onto Immobilon-P membranes (Millipore, Bedford, MA). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS)–Tween 20 (0.1%) for 1 h at room temperature, followed by incubation with primary antibodies (rabbit polyclonal anti-Smad3, Zymed, South San Francisco, CA; mouse monoclonal anti-Smad4 and goat polyclonal anti-Smad7, Santa Cruz, Santa Cruz, CA) for 2 h at room temperature. The blots were then washed in TBS–Tween three times, followed by incubation with the secondary antibody (anti-mouse, anti-rabbit, and anti-goat horseradish peroxidase-conjugated antibody; Santa Cruz) for 1 h at room temperature. After washing, blots were developed with chemiluminescence reagents according to the manufacturer’s protocol (SuperSignal chemiluminescence HRP substrate; Pierce, Rockford, IL), and intensity of signals was quantitated by densitometry. To determine the specificity of the bands, the primary antibody was incubated with blocking peptide for 30 min at 37°C and used as primary antibody in a separate lane. Cellular immunofluorescence imaging. The expression and intracellular localization of endogenous SMADs in the presence or absence of TGF-␤ were studied by immunocytochemistry. For this purpose, fibroblasts (10,000 cells/well) were seeded into eight-well Lab-Tek II chamber glass slides (Nalge Nunc International, Naperville, IL) with EMEM with 10% FCS. The next day, fresh media with 0.1% FCS were added. Following incubation with the indicated concentrations of TGF-␤1 for 2 h, cells were chilled on ice 5 min, washed with ice-cold PBS, fixed with 100% methanol at ⫺20°C, and washed twice at room temperature with PBS. The cells were then incubated with 10 ␮g/ml primary antibodies (rabbit anti-Smad3, Zymed; mouse anti-Smad1, mouse anti-Smad4, and goat anti-Smad7, Santa Cruz) in TNB buffer (0.1 M Tris/HCl, pH 7.5, 0.15 M NaCl, 0.5% Blocking Reagent) for 1 h. Following three washes in TNT buffer (0.1 M Tris/HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20), cells were incubated for 30 min with horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat secondary antibodies (Santa Cruz) in TNB buffer. After three washes in TNT buffer, the cells were stained according to the manufacturer’s protocol (Tyramide Signal Amplification; NEN Lifescience Products, Boston, MA). Cellular localization of fluorescence was examined by fluorescence or confocal microscopy. Quantitation was performed in a blinded fashion by scoring 100

fibroblasts in different fields as showing predominantly nuclear or cytoplasmic immunofluorescence.

RESULTS

Expression of Endogenous Smad3 and Smad7 mRNA in Fibroblasts Modulated by TGF-␤ In order to examine the effect of TGF-␤ on endogenous Smad mRNA expression in primary human cells, total RNA was isolated from low-passage confluent foreskin fibroblasts cultured in 0.1% FCS and examined by Northern analysis. As shown in Fig. 1A, expression of Smad3 mRNA with the characteristic 3and 7-kb transcripts was readily detectable in unstimulated fibroblasts. TGF-␤ caused a marked decrease in Smad3 mRNA levels. Inhibition by TGF-␤ was dose- and time-dependent, with maximal reduction by 48 h. In contrast to Smad3, Smad7 mRNA was expressed only at relatively low levels in untreated fibroblasts, but was markedly up-regulated by TGF-␤1 (Fig. 1A). The induction of Smad7 mRNA was rapid, with maximal increase noted 120 min after TGF-␤1 was added. The stimulation, however, was transient, and by 24 h, Smad7 mRNA expression in the TGF-␤treated fibroblasts returned to control levels. The mRNA levels for the common Smad signaling partner Smad4 did not show significant change in response to TGF-␤1 (data not shown). These results were consistent in several independent experiments and with fibroblasts derived from three separate individuals and examined at different passages. To determine if ligand-induced modulation of Smad3 and Smad7 gene expression in the fibroblasts was specific to TGF-␤1, other members of the TGF-␤ cytokine superfamily were examined. The TGF-␤3 isoform is differentially expressed and induces certain responses on the extracellular matrix that are distinct from those triggered by TGF-␤1. For instance TGF–␤3 prevented scarring in a murine wound model, opposite the effect of TGF-␤1 [32]. In order to examine if differential modulation of endogenous Smad expression in fibroblasts could account for the functional differences between the two TGF-␤ isotypes, they were added at the indicated concentrations to confluent fibroblasts. The results shown in Fig. 1B demonstrate that TGF-␤1 and TGF-␤3 caused an identical pattern of dose-dependent inhibition of Smad3 and stimulation of Smad7 mRNA expression. It was noted that modulation of Smad3 and Smad7 mRNA expression by TGF-␤ showed distinct dose responses: 5 nM TGF-␤ was sufficient to induce near-maximal stimulation of Smad7, whereas inhibition of Smad3 mRNA required ⬃100-fold greater concentration of TGF-␤. Next, the effects of TGF-␤ were compared to those of other members of the TGF-␤ superfamily. In contrast to TGF-␤, activin A, which signals through Smad2 and Smad3, and BMP-2, which

TGF-␤ REGULATES ENDOGENOUS SMAD EXPRESSION

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TABLE 1 The Effects of TGF-␤1, Activin A, and BMP-2 on Smad mRNA Expression

a

Smad3 Smad7 b

TGF-␤1

Activin A

BMP-2

6 238

160 134

174 134

Note. Fibroblasts were incubated with TGF-␤1, activin A, or BMP-2 for the indicated periods. At the end of incubation, cells were harvested and mRNA levels were determined by Northern analysis and quantitated by phosphoimage analysis. The results are shown as relative mRNA levels in ligand-stimulated compared to unstimulated fibroblasts (arbitrarily set at 100) and are normalized for GAPDH in the same samples. a mRNA levels were determined after 24 h incubation with 500 pM indicated ligand. b mRNA levels were determined after 90 min incubation with 50 pM indicated ligand.

Expression of SMAD3 and SMAD4 was detectable in all the fibroblast strains examined. The levels of SMAD3 were reduced by ⬃70% in whole lysates of fibroblasts exposed to TGF-␤1, whereas SMAD4 expression remained unchanged under these conditions (Fig. 2). SMAD7 expression, difficult to detect in untreated fibroblasts, was increased by ⬃60% upon incubation with TGF-␤; stimulation peaked relatively early (4 h) and was short-lived. As a control for specificity, antisera were incubated with the peptides to which the antiserum was originally raised. FIG. 1. Regulation of endogenous Smad mRNA expression by TGF-␤ in primary human fibroblasts. (A) Confluent fibroblasts were incubated without or with TGF-␤1 (500 pM) in 0.1% FCS EMEM. At the end of the indicated incubation periods, total RNA was isolated and analyzed by Northern hybridization with Smad3 (left) or Smad7 cDNA (right) and GAPDH cDNA. Representative autoradiograms are shown. Arrowheads indicate Smad mRNA transcripts. Bottom: The relative levels of mRNA (means from several independent experiments) were determined by densitometric scanning of the autoradiograms and normalized by GAPDH. (B) Comparison of TGF-␤1 and TGF-␤3 modulation of Smad expression. mRNA levels were determined following incubation with the indicated TGF-␤ isoform for 90 min (left) or 24 h (right). Representative autoradiograms are shown above. Bottom: The relative ratios normalized by GAPDH are shown.

Mechanisms Regulating Smad3 and Smad7 mRNA Expression by TGF-␤ The pathways controlling SMAD synthesis are poorly understood. In order to begin to investigate the

utilizes Smad1 and Smad5, failed to inhibit Smad3 or stimulate Smad7 mRNA expression in the fibroblasts (Table 1). Endogenous SMAD3 and SMAD7 Protein Expression Regulated by TGF-␤ Regulation of SMAD expression was previously studied in cells transfected with exogenous SMADs or in tumor-derived immortalized cell lines [33, 34]. Here, we examined the effects of TGF-␤1 on SMAD expression in primary skin fibroblasts by immunoblotting.

FIG. 2. SMAD expression in fibroblasts treated with TGF-␤. Foreskin fibroblasts were incubated with TGF-␤1 (500 pM) for the indicated periods, and protein expression was analyzed by immunoblotting of whole-cell lysates with anti-SMAD3, anti-SMAD4, antiSMAD7, or anti-actin antibodies. Bottom shows the relative levels of SMAD3 (open bars) or SMAD7 (closed bars), determined by densitometric scanning.

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mechanism underlying TGF-␤ regulation of endogenous Smad expression in fibroblasts, CHX was used. Inhibition of de novo protein synthesis caused a modest increase in Smad3 mRNA levels; however, delayed down-regulation by TGF-␤ was completely prevented (Fig. 3A). In contrast, TGF-␤1 induction of Smad7 mRNA in these cells was unaffected by pretreatment with CHX. These results, consistent with previous findings with regulation of anti-Smad mRNA in Mv1Lu cells [21, 29], indicate that rapid and transient stimulation of Smad7 mRNA did not require de novo protein synthesis, suggesting that in fibroblasts, the Smad7 gene is a direct target of receptor-activated SMAD signals. The possibility that inhibition of Smad3 mRNA expression by TGF-␤ was due to decreased stability of Smad3 mRNA transcripts was examined. Results of Northern analysis indicated that upon treatment with actinomycin D, the levels of Smad3 mRNA declined with comparable rates in untreated and TGF␤-treated fibroblasts (Fig. 3B). To further characterize regulation of Smad gene expression by TGF-␤1, we utilized MDA-MB468 human breast adenocarcinoma cells, which have a homologous deletion of the Smad4 coding region and therefore lack Smad4 expression [26]. These cells show defective TGF-␤ responses, including stimulation of COL1A2 promoter [6]. Both Smad3 and Smad7 mRNAs were detectable in MDA-MB468 cells in the absence of TGF-␤ (Fig. 3C). In striking contrast to fibroblasts, however, TGF-␤ failed to induce delayed down-regulation of Smad3, or rapid up-regulation of Smad7, mRNA expression in this cell line. PAI-1 and fibronectin are target genes for TGF-␤ stimulation in fibroblasts. Expression of PAI-1 mRNA was undetectable in MDAMB468 cells without or with TGF-␤ stimulation (data not shown), consistent with previous reports [29]. Remarkably, the expression of fibronectin mRNA in the Smad4-deficient cells showed an increase after 24 h treatment with TGF-␤. These findings indicate that

FIG. 3. Mechanism of Smad regulation by TGF-␤. (A) Inhibition of de novo protein synthesis by cycloheximide abrogated TGF-␤ suppression of Smad3, but not induction of Smad7, mRNA. Fibroblasts were incubated with 10 ␮g/ml cycloheximide for 2 h prior to

incubation of TGF-␤1 (500 pM). Levels of Smad7 mRNA were determined at 90 min, and Smad3 mRNA at 24 h, by Northern hybridization. The results, shown as mRNA levels normalized by GAPDH, represent the means ⫾ SD of three separate determinations. *P ⬍ 0.05 TGF-␤ vs TGF-␤ plus CHX; **P ⬍ 0.05 TGF-␤ or TGF-␤ plus CHX vs control. (B) TGF-␤ had no effect on the stability of Smad3 mRNA transcript half-lives. Fibroblasts at confluence were incubated with actinomycin D (5 ␮g/ml) for 1 h prior to TGF-␤. RNA was isolated at the indicated time points after addition of actinomycin D and subjected to Northern analysis. mRNA was quantitated by phosphoimage analysis. (C) TGF-␤ failed to regulate Smad mRNA expression in Smad4-deficient MDA-MB468 cells. Confluent cultures of MDA-MB468 cells were incubated with or without TGF-␤1 (500 pM), and Smad3, Smad7, and fibronectin mRNA levels were determined after 90 min (lanes 1 and 2) or 24 h (lanes 3 and 4) of incubation. Bottom: Relative mRNA levels (Smad7 at 90 min, Smad3 and fibronectin at 24 h) determined by densitometric scanning and normalized by GAPDH. Open bars, untreated fibroblasts; closed bars, TGF-␤1-treated fibroblasts.

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TGF-␤1-induced modulation of endogenous Smad expression is mediated via SMAD signaling, whereas other TGF-␤-responsive genes are regulated by SMADindependent signaling pathways.

adult dermal fibroblast strains derived from several individuals.

TGF-␤ Induces Nuclear Accumulation of Endogenous Receptor-Regulated SMADs

TGF-␤ signaling in mammalian cells is initiated by ligand-induced heteromerization of the two TGF-␤ receptors. Recent studies have established that SMADs play a fundamental role in transmitting TGF-␤ signals to target genes, including collagen and PAI-1, and thus are important participants in physiological and pathological processes of tissue remodeling [24, 35–37]. Ligation of its receptor by TGF-␤ causes rapid phosphorylation of SMAD3 and its interaction with SMAD4 in the cytoplasm, followed by translocation of the heteromeric SMAD complex into the nucleus. Thus, in common with interferon-␥ and other cytokine-triggered cellular responses, TGF-␤ activates its intracellular signaling cascade through posttranscriptional modification and compartmentalization of preexisting signal transducers. This mechanism allows for the very rapid and efficient transmission of information from the cell surface to the nucleus. The results presented here indicate that TGF-␤ caused a marked inhibition of Smad3 gene expression in primary fibroblasts. The mechanisms underlying down-regulation of Smad3 expression are unknown. Examples of genes whose expression is inhibited by TGF-␤ are relatively few and generally involve lineagespecific transcriptional repression such as in the case of collagenase-1 [38], collagenase-3 [39], the Class II transactivator [40], cyclin A [41], ICAM-1 [42], VEGF [43], or c-Myc [44]. Indeed, the present results indicate that down-regulation of Smad3 mRNA expression in TGF-␤-treated fibroblasts was not due to decreased transcript stability, suggesting a transcriptional effect of TGF-␤. Inhibition of Smad3 expression was delayed and required de novo protein synthesis, most likely representing a secondary TGF-␤ response involving a transcriptional repressor. Inhibition induced by TGF-␤ could result from the competition between SMAD3 and an unidentified transcription factor for limiting amounts of nuclear coactivators that interact with both. If basal expression of Smad3 in fibroblasts involves a coactivator such as CBP/p300, than sequestration of this coactivator by SMAD3 could result in inhibition of Smad3 transcription upon ligand stimulation. In light of the reported similarities between activin and TGF-␤-induced signaling and biological responses, the apparent inability of activin to modulate Smad mRNA expression in fibroblasts was surprising and may indicate cell-lineage-specific differences. Indeed, it has been shown in HaCaT cells that Smad7 mRNA induction by activin was substantially reduced, and displayed different kinetics, in comparison to that induced by TGF-␤ [29]. In Smad4-deficient MDA-MB468 cells, TGF-␤ failed to induce Smad3 repression,

Translocation of SMAD3 and SMAD4 from the cytoplasm into the nucleus is considered to play a central role in SMAD-mediated transcriptional regulation [32, 34]. Because endogenous cellular SMADs have proven difficult to detect immunocytochemically [33], previous reports utilized transient ectopic overexpression of epitope-tagged SMADs in order to characterize their ligand-induced intracellular trafficking. In order to examine SMAD localization in a more physiologic system, low-passage primary skin fibroblasts were used. Fibroblasts were incubated with TGF-␤1 for indicated periods, and distribution of endogenous SMADs was examined by confocal microscopy using antibody-mediated immunofluorescence. Relatively high levels of endogenous SMAD3 were detectable in untreated fibroblasts (Fig. 4A, top). SMAD3 immunofluorescence was localized primarily in the cytoplasm, with only a very low level of nuclear signal. Upon TGF-␤ exposure, however, translocation of SMAD3-associated fluorescence into the nucleus was noted. The relative ratio of predominantly nuclear versus predominantly cytoplasmic fluorescence showed TGF-␤ dose-dependent increase, reaching a maximum of ⬃2.8 compared to untreated fibroblasts (Fig. 4A, bottom). Examination of endogenous SMAD4 localization showed a similar pattern of dose-dependent nuclear accumulation upon TGF-␤ treatment (Fig. 4B, top). The relative ratio of predominantly nuclear to predominantly cytoplasmic SMAD4 fluorescence increased ⬎6-fold with TGF-␤. Translocation of the receptor-activated SMADs from the cytoplasm into the nucleus was time-dependent and rapid (maximal at 120 min), and the intensity of SMAD3 and SMAD4 immunofluorescence markedly decreased by 24 h (data not shown). Consistent with earlier reports [23], SMAD7 was detected predominantly within the nucleus, and only at low levels, in untreated fibroblasts. Treatment with TGF-␤ for 120 min induced a marked increase in the intensity of SMAD7-specific immunofluorescence (Fig. 4C, middle). However, in contrast to SMAD3 or SMAD4, no significant change in the intracellular distribution of SMAD7 was noted. SMAD1, which is activated by the BMP receptors [13], was detectable only in the cytoplasm in untreated fibroblasts and showed no consistent change in its cellular distribution upon TGF-␤ treatment (Fig. 4C, bottom). These results were reproducible in several independent experiments, with neonatal foreskin-derived fibroblasts as well as with

DISCUSSION

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TGF-␤ REGULATES ENDOGENOUS SMAD EXPRESSION

whereas stimulation of fibronectin mRNA was undisturbed. The latter findings suggest that stimulation of fibronectin by TGF-␤ involves SMAD-independent signaling pathways, as recently shown by others [45]. The failure of TGF-␤ to down-regulate Smad3 mRNA in Smad4-deficient cells, in which activated SMAD3 is unable to translocate into the nucleus, indicates that Smad3 regulation is mediated through the endogenous SMAD pathway. However, a full understanding of the mechanism underlying the regulation of Smad3 mRNA expression will require analysis of Smad3 promoter structure and function. Importantly, ligand-induced down-regulation of its own intracellular signal transducer by TGF-␤, also noted in mesangial cells and epithelial cell lines [46, 47] may represent an important autoregulatory mechanism to terminate signal flow through the SMAD cascade. Failure to down-regulate cellular Smad3 expression following TGF-␤ stimulation could result in continued SMAD translocation into the nucleus and stimulation of TGF-␤-responsive genes, with pathological accumulation of TGF-␤-inducible gene products. In contrast to Smad3, Smad7 mRNA expression in the fibroblasts was markedly induced by TGF-␤ in a protein synthesis-independent manner. These finding are consistent with reports describing endogenous Smad7 induction by TGF-␤ in Mv1Lu cells and HaCaT cells [21]. Thus, Smad7 is an immediate-early gene target of TGF-␤. The mechanisms regulating Smad7 gene expression in human cells are only incompletely understood [48]. Our results showed that Smad7 could not be induced in Smad4-deficient MDA-MB468 cells, indicating a requisite role for endogenous Smad signaling in this response. Consistent with these findings, ligand-induced activation of Smad3 has been implicated in TGF-␤ stimulation of Smad7 gene expression in hepatoma cells [48]. Although it has been suggested that failure of TGF-␤ to induce Smad7 in Smad4-deficient cells was due to loss of Smad7 [29], this cannot be implicated as the explanation for the observed lack of induction in MDA-MB468 cells, which clearly show detectable Smad7 mRNA expression in the absence of TGF-␤ (Fig. 3B). The intracellular localization of endogenous SMAD7 has not been examined to date. In previous studies using inducible ectopic expression systems, recombinant SMAD7 was found to be localized in the nucleus in COS-1 cells [22] or in the cytoplasm in mink lung epithelial cells [23]. Our results indicate that endogenous SMAD7 was detectable, al-

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beit in low levels, in the fibroblasts in the absence of TGF-␤ treatment and was localized primarily in the nucleus. Apparent differences in subcellular distribution of SMAD7 in vitro may relate to cell type, the behavior of endogenous versus recombinant protein, or even the nature of the surface on which the cells are grown [23]. In fact, the subcellular compartmentalization of SMAD7 did not appear to correlate with its inhibitory function [23]. Previous studies attempting to characterize the subcellular compartmentalization of SMAD3 generally utilized epitope-tagged recombinant SMADs that were ectopically expressed in immortalized cell lines [34, 36] and initiated signaling by ectopic expression of constitutively active TGF-␤ receptor mutants [48]. Observations derived from these experimental systems may bear only limited relevance to the behavior of endogenous SMADs in nontransformed cells stimulated by their ligand under physiologic conditions. Therefore, we sought to examine the regulation of endogenous SMADs in early passage human fibroblasts, which express both types of TGF-␤ receptors and show inducible transcription of collagen genes upon TGF-␤ stimulation [49]. The results of the present studies indicated that endogenous SMAD3 was readily detectable and was localized predominantly in the cytoplasm. Translocation into the nucleus occurred relatively rapidly upon TGF-␤ treatment and was followed by a marked decrease in SMAD3 levels. Because SMAD3 phosphorylation in TGF-␤-stimulated fibroblasts occurs in ⬍15 min [46], the kinetics of ligand-induced SMAD3 nuclear accumulation suggest that phosphorylation is a prerequisite for nuclear localization. As the progressive decline in nuclear SMAD3 following TGF-␤ stimulation, demonstrated by immunoblotting and by immunocytochemistry, preceded the decrease in mRNA expression, it may be due to ligand-induced proteasomal degradation, as described for SMAD2 [16]. SMAD4 showed an identical pattern of TGF-␤-stimulated nuclear translocation, presumably via the heteromeric SMAD3–SMAD4 complex. Once in the nucleus, the activated SMAD complex physically interacts with DNA-binding transcription factors such as FAST-1 [51], FAST-2 [52], or TFE3 [53], or coactivators such as p300/CBP [54], and bind directly to DNA [14]. Sitespecific binding by the SMAD– coactivator complex induces the transcription of target genes that mediate cellular responses to TGF-␤. Taken together, the present results indicate that the

FIG. 4. Nuclear accumulation of endogenous SMADs. Fibroblasts were incubated with indicated concentrations of TGF-␤1. After 2 h, anti-Smad3 (A) or anti-Smad4 (B) primary antibodies were added, followed by appropriate secondary antibodies as described under Materials and Methods. Immunofluorescence was examined by confocal microscopy. Bottom: The relative ratios of predominantly nuclear vs cytoplasmic localization of SMAD3- and SMAD4-associated immunofluorescence, determined by scoring 100 cells, are shown. (a) Control; (b– d) 5, 50, or 500 pM TGF-␤1. (C) Time course of SMAD4 (top), SMAD7 (middle), or SMAD1 (bottom) nuclear import induced by TGF-␤1 (500 pM). Relative ratios of predominantly nuclear vs cytoplasmic localization of SMAD4-associated immunofluorescence, determined by scoring 100 cells, are shown below.

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