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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

Scleroderma is characterized by excessive synthesis and accumulation of matrix proteins in lesional tissues. Transforming growth factor β (TGFβ) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of fibroblasts; however, the underlying mechanisms are poorly understood. We undertook this study to examine the expression and function of SMADs, recently characterized intracellular effectors of TGFβ signaling, in scleroderma fibroblasts.

Methods

Primary dermal fibroblasts obtained from 14 patients with scleroderma and from 4 healthy adult volunteers were studied. Northern analysis was used to determine the expression of endogenous SMAD messenger RNA (mRNA), and Western analysis was used to determine SMAD protein expression. Intracellular compartmentalization of cellular SMAD proteins in the presence and absence of TGFβ was studied by antibody-mediated immunofluorescence confocal microscopy. The effect of TGFβ blockade on SMAD subcellular distribution was determined using anti-TGFβ antibodies as well as a dominant-negative TGFβ receptor type II (TGFβRII) vector to disrupt TGFβ responses. SMAD-regulated luciferase reporter expression was examined to investigate the potential functional significance of activation and nuclear accumulation of endogenous SMADs in scleroderma fibroblasts.

Results

Protein and mRNA levels of SMAD3, but not of SMAD4 or SMAD7, were variably elevated in scleroderma fibroblasts compared with those from healthy controls. In sharp contrast to control fibroblasts, which displayed predominantly cytoplasmic localization of SMAD3/4 in the absence of exogenous TGFβ, in scleroderma fibroblasts SMAD3 and SMAD4 consistently showed elevated nuclear localization. Furthermore, phosphorylated SMAD2/3 levels were elevated and nuclear localization of phosphorylated SMAD2/3 was increased, suggesting activation of the SMAD pathway in scleroderma fibroblasts. Blockade of autocrine TGFβ signaling with antibodies or by expression of dominant-negative TGFβRII failed to normalize SMAD subcellular distribution, suggesting that elevated nuclear SMAD import was due to alterations downstream of the TGFβ receptors. The activity of a SMAD-responsive minimal promoter–reporter construct was enhanced in transiently transfected scleroderma fibroblasts.

Conclusion

This study is the first to demonstrate apparently ligand-independent constitutive activation of the intracellular TGFβ/SMAD signaling axis in scleroderma fibroblasts. SMAD signaling may be a mechanism contributing to the characteristic phenotype of scleroderma fibroblasts and playing a role in the pathogenesis of fibrosis.

Scleroderma is associated with fibrosis of affected tissues due to excessive synthesis and progressive accumulation of connective tissue macromolecules (1). Scleroderma fibroblasts display features of sustained activation in vitro and in vivo. In culture, these cells show elevated synthesis of collagens, fibronectin, proteoglycans, and tissue inhibitors of metalloproteinases, constitutive secretion of interleukin-1α (IL-1α) and IL-6, and reduced production of collagenase (for review, see ref. 2). These phenotypic alterations closely correspond to changes in normal fibroblasts stimulated with transforming growth factor β (TGFβ) and persist during serial passage for a limited number of replications in vitro. The mechanisms responsible for the activated phenotype of scleroderma fibroblasts remain unknown.

TGFβ, the principal mediator of physiologic tissue remodeling, is strongly implicated in the pathogenesis of scleroderma (3). The expression of TGFβ is elevated in scleroderma dermal fibroblasts, as well as in monocyte/macrophages and other infiltrating inflammatory cells, even prior to the appearance of fibrosis (4–11). Levels of TGFβ receptor type I (TGFβRI) and TGFβRII are also elevated on scleroderma fibroblasts (12–14). Tissue expression of TGFβ is prominent in animal models of scleroderma, and blockade of TGFβ signaling with neutralizing antibodies or naturally occurring TGFβ antagonists is effective in reducing collagen accumulation and ameliorating experimental fibrosis (15, 16). Furthermore, TSK mice that have been genetically engineered to be deficient for one TGFβ allele show marked reduction in the thickness of the dermis and accumulation of matrix (17).

In fibroblasts, TGFβ stimulates matrix production and induces its own synthesis (autoinduction) as well as that of connective tissue growth factor (CTGF) (18–20). Recent studies have shed light on the intracellular signaling mechanisms that mediate relevant profibrotic TGFβ responses. Binding of TGFβ to the ubiquitous type II serine/threonine kinase receptor (TGFβRII) triggers heterodimerization with and activation of TGFβRI. The signal is then propagated downstream through SMADs, a family of evolutionarily conserved intracellular mediators that convey information from the cell membrane into the nucleus (21). In vertebrates, members of the SMAD family segregate into 3 functionally distinct groups: SMADs 2 and 3 are direct substrates for activated TGFβRI, SMAD4 is the common signaling partner, and SMAD7 is inhibitory.

Upon their phosphorylation and activation by the TGFβ receptor kinase at the cell membrane, SMAD2 and SMAD3 associate with SMAD4. The heteromeric SMAD complex then translocates into the nucleus, where, in conjunction with other DNA binding factors or with transcriptional coactivators or corepressors, it regulates target gene transcription. While SMAD2 and SMAD3 work synergistically and in tandem with SMAD4 in transducing TGFβ signals, the antagonistic SMAD7 binds to the TGFβ receptor complex and prevents SMAD2/3 access, thereby blocking SMAD phosphorylation. Because its expression is rapidly induced by TGFβ (22–24), SMAD7 serves a critical negative feedback function by limiting the amplitude and duration of cellular responses to TGFβ. In addition to inhibitory SMAD7, multiple other intracellular mechanisms to modulate the intensity of SMAD signaling have been characterized (25–28). In normal fibroblasts, SMAD3 and SMAD4 are predominantly cytosolic; upon stimulation, these SMADs reversibly translocate into the nucleus (24). Nuclear import is required for SMAD-dependent transcriptional responses. The relative subcellular compartmentalization of SMAD3 and SMAD4 is thus an important factor in setting the level of TGFβ signaling, but the mechanisms governing SMAD nuclear–cytoplasmic shuttling in physiologic TGFβ responses are poorly understood.

The SMAD pathway plays a fundamental role in regulation of collagen synthesis, and SMADs are necessary to mediate TGFβ-dependent stimulation (29–33). In light of the singular importance of TGFβ in both initiating and sustaining fibroblast activation, and given the pivotal role of SMADs as major intracellular effectors of TGFβ-induced profibrotic responses, we undertook extensive characterization of SMAD signaling in a large panel of scleroderma fibroblasts. The present results indicate that messenger RNA (mRNA) and protein expression of the positive signal mediator SMAD3, but not those of inhibitory SMAD7, are variably elevated in scleroderma fibroblasts. Treatment with TGFβ caused repression of SMAD3 expression and stimulation of SMAD7 expression in both healthy control and scleroderma fibroblasts. In sharp contrast to control fibroblasts, however, scleroderma fibroblasts were characterized by substantial increase in endogenous SMAD2/3 phosphorylation and nuclear accumulation in the absence of TGFβ stimulation. Nuclear redistribution did not appear to be driven by autocrine TGFβ, because blockade of TGFβ signaling failed to diminish the proportion of scleroderma fibroblasts showing nuclear SMAD localization. Collectively, these results demonstrate ligand-independent constitutive activation of the SMAD signaling pathway downstream of the TGFβ receptors in scleroderma fibroblasts, suggesting a novel mechanism that may contribute to the pathogenesis of fibrosis.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Cell culture.

Primary dermal fibroblasts were established from excisional skin biopsy samples using previously described procedures (29). In these experiments, 14 patients with scleroderma were studied. The diagnosis of scleroderma was established according to the classification criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (34). The mean duration of disease at the time of biopsy was 12 months, and 3 patients were receiving disease-modifying therapy (methotrexate, prednisone, or D-penicillamine). Biopsy samples were obtained from the leading edge of clinically apparent “lesional” skin. The characteristics of the patients are shown in Table 1. As a control, skin biopsy samples were obtained from 4 healthy adult volunteers (1 man and 3 women, mean age 45 years). Biopsies were performed with written informed consent, and the protocol was approved by the Institutional Review Board of the University of Illinois.

Table 1. Clinical characteristics of patients with scleroderma
Patient/age/sexRace*Disease duration, yearsSkin involved, %
  • *

    B = black; H = Hispanic; O = Oriental; W = white.

  • Interval from first non-Raynaud symptom of scleroderma to time of skin biopsy.

  • Extent determined by clinical examination and recorded utilizing a body surface diagram.

1/56/FB120
2/35/FH110
3/46/FB15
4/59/FO15
5/39/FW150
6/61/MW15
7/50/MH250
8/38/FW136
9/47/FW140
10/43/FW15
11/19/FH0.540
12/19/FB115
13/30/FW110
14/44/MW120

Culture media were from BioWhittaker (Walkersville, MD); all other tissue culture reagents were from Gibco BRL (Grand Island, NY). For these experiments, control and scleroderma fibroblasts were grown simultaneously and studied between passages 4 and 8. Cells were grown at 37°C in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 1% vitamin solution, 100 units/ml penicillin/streptomycin, and 2 mML-glutamine. When the fibroblasts reached confluence, fresh medium with indicated concentrations of TGFβ1 (Amgen, Thousand Oaks, CA) was added. In some experiments, cultures were incubated with panspecific neutralizing antibody against TGFβ (R&D Systems, Minneapolis, MN) or nonimmune rabbit IgG as control, alone or in combination with recombinant latency-associated peptide (LAP), a naturally occurring TGFβ antagonist (R&D Systems), for 0.5 hours prior to addition of fresh medium. In other experiments, scleroderma or control fibroblasts were transduced with adenoviral vector expressing green fluorescent protein (GFP) as control, or with dominant-negative kinase-deficient TGFβRII to inhibit signal transduction from all 3 mammalian TGFβ isoforms (35). Transduction was carried out by incubation of confluent fibroblasts for 48 hours at room temperature with 3 × 109 plaque-forming units/ml of virus. Efficient transduction was confirmed by high levels of GFP expression in all fibroblasts.

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 γ32P-dCTP–labeled complementary DNA (cDNA) probes, as described (29). Following extensive washing of the nitrocellulose membranes, the cDNA–mRNA hybrids were visualized by autoradiography on X-AR5 film (Eastman Kodak, Rochester, NY) exposed for 24–72 hours with intensifying screens. The following human cDNA probes were used for hybridization: a 1.4-kb restriction fragment that included the entire coding region of human SMAD3, a 1.6-kb restriction fragment that included the entire coding region of human SMAD4, a 1.9-kb restriction fragment that included the entire coding region of human SMAD7, GAPDH, and 18S. Signal intensities were quantitated by densitometry, and results were normalized to the levels of GAPDH or 18S mRNA in each sample.

Western analysis of cellular SMADs.

Immunoblot of whole cell lysates was performed as previously described (29). To prevent protein dephosphorylation, a Phosphatase Inhibitor Mix (Sigma, St. Louis, MO) was added. Nuclear extracts were prepared as previously described (31). Protein concentrations in the cytosolic, nuclear, or whole cell fractions were determined by Bradford assay (Bio-Rad, Hercules, CA). Equal aliquots of whole cell lysates (15 μg/lane) or cytosolic or nuclear extracts (20 μg/lane) were separated by reducing electrophoresis in 10% sodium dodecyl sulfate (SDS)–polyacrylamide gels. Proteins in the gels were transferred onto Immobilon-P (polyvinylidene difluoride) membranes (Millipore, Bedford, MA). Following blocking with 5% nonfat dry milk in Tris buffered saline–Tween at room temperature for 1 hour, membranes were incubated for 2 hours with antibodies against SMAD3 (Zymed, South San Francisco, CA) or against SMAD2/3, phospho-SMAD2/3, SMAD4, SMAD7, TGFβRI, histone H3, or actin (all from Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with horseradish peroxidase (HRP)–conjugated secondary antibodies. Antibody specificities were confirmed using blocking peptides supplied by the manufacturer.

After washing, immunoblots were developed with chemiluminescence reagents (Pierce, Rockford, IL) according to the manufacturer's protocol. In order to detect phosphorylated TGFβRI or SMAD2/3 in fibroblasts, anti-TGFβRI or anti-SMAD2/3 antibodies with protein G–Sepharose were used for immunoprecipitation. The immunoprecipitates from lysates of fibroblasts left untreated or treated either with TGFβ (500 pM) or with anti-TGFβ antibody were eluted by boiling, electrophoresed through 8% SDS–polyacrylamide gels, and processed as described above. Following overnight blocking, membranes were incubated with antiphosphoserine antibody (Zymed). Signal intensities were quantitated by densitometry, and results were normalized to the intensities of IgG or histone H3 bands in each sample.

Cellular immunofluorescence imaging.

The expression and intracellular localization of endogenous SMAD proteins were studied by immunocytochemistry and fluorescence confocal microscopy. For this purpose, fibroblasts (10,000/well) from healthy controls or patients with scleroderma were seeded into 8-well Lab-Tek II chamber glass slides (Nalge Nunc International, Naperville, IL). The next day, fresh media with 0.1% FCS and indicated concentrations of TGFβ1 were added for 2 hours. Cells were washed and fixed with 100% methanol for labeling of SMADs. Cells were then incubated with antibodies (10 μg/ml) against SMAD2/3 (from Santa Cruz Biotechnology or Zymed; both yielding identical results), SMAD4 or phospho-SMAD2/3 (both from Santa Cruz Biotechnology), or TGFβRII (Santa Cruz Biotechnology) for 1 hour and then washed, followed by incubation with HRP-conjugated secondary antibodies and staining with fluorescein isothiocyanate or rhodamine according to the manufacturer's protocol (Tyramide Signal Amplification; NEN Life Science Products, Boston, MA). To stain the nuclei, chambers were mounted with Vectashield plus 4′,6-diamidino-2-phenylindole (Vector, Burlingame, CA). Nonimmunized IgG was used as negative control.

Following stringent washing of the slides, the pattern and subcellular distribution of fluorescence were evaluated by immunofluorescence or laser scanning confocal microscopy. Each experiment was repeated at least 3 times with consistent results. Quantitative analysis was performed by scoring 100 individual fibroblasts from different microscopic fields as showing a predominantly nuclear or predominantly cytoplasmic distribution of immunofluorescence. The observer was blinded to the identity of each section.

Transient transfection.

Subconfluent cultures of fibroblasts were transfected using Superfect reagent (Qiagen, Valencia, CA), as described previously (35). The plasmid pSBE4-luc (from Dr. B. Vogelstein), containing 4 tandem repeats of an 8-bp palindromic consensus SMAD binding element (SBE) that is specifically recognized by SMAD2 or SMAD3 (36), was used to measure SMAD-mediated transcriptional responses. pGL3-Luc, the empty vector of pSBE4-luc and pRL-TK (both from Promega, Madison, WI) were used as internal controls to correct for variations in transfection efficiency between the samples. Following their recovery overnight in DMEM with 10% FCS, fibroblasts were incubated in fresh media containing 0.1% FCS. After 48 hours, cells were harvested and lysates were prepared using passive lysis buffer (Promega). Luciferase activity in equal aliquots was determined by Dual-Luciferase Reporter Assay System (Promega). All experiments were performed in triplicate and repeated at least 3 times. Significance of differences between experimental groups was determined by Mann-Whitney U test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Expression and regulation of endogenous SMAD mRNA in scleroderma fibroblasts.

We previously demonstrated that in normal skin fibroblasts, SMAD3 and SMAD4 played fundamental roles in mediating TGFβ responses, including stimulation of collagen synthesis (29, 30). To begin to elucidate their role in the pathogenesis of fibrosis, we examined the expression of endogenous SMADs in scleroderma fibroblasts. Total RNA was isolated from low-passage confluent fibroblasts and examined by Northern analysis. The results showed that control and scleroderma fibroblasts similarly expressed two specific SMAD3 mRNA transcripts of 7.0 kb and 3.0 kb (Figure 1A). Levels of SMAD3 mRNA were moderately elevated in scleroderma fibroblasts (mean ± SEM 167 ± 51% of those in control fibroblasts), although the difference did not reach significance; in 11 of 14 scleroderma fibroblast lines, SMAD3 mRNA levels were >1 SEM higher than the mean in controls (Figure 1B).

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Figure 1. SMAD mRNA expression in fibroblasts. Fibroblasts derived from lesional skin of 14 patients with scleroderma (S) or 4 healthy controls (N) were cultured in parallel. At early confluence, cells were harvested and total RNA was isolated. Levels of SMAD3, SMAD7, 18S, and GAPDH mRNA were determined by Northern blot analysis. A, Representative Northern blot. Asterisks indicate the positions of 18S and 28S ribosomal RNA. B, SMAD3 and SMAD7 mRNA levels were quantitated by densitometric scanning of the autoradiographs. Results in the upper panel, corrected for the levels of GAPDH mRNA in each sample, are expressed in arbitrary units. Each data point represents the mean of several independent experiments for each individual scleroderma (solid bars) and control (open bars) fibroblast line. Lower panel shows the ratio of SMAD3 mRNA level:SMAD7 mRNA level calculated for each fibroblast line. Middle and upper/lower lines represent the mean ± SEM.

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In contrast to SMAD3, the expression of SMAD4 mRNA (9.5 kb and 3.9 kb) and SMAD7 mRNA (4.6 kb) showed no consistent differences between control and scleroderma fibroblasts, although SMAD7 mRNA levels in 5 scleroderma fibroblast lines were >1 SEM lower than the mean in controls (Figure 1B). The ratio of the mRNA levels of positive (SMAD3) and negative (SMAD7) mediators of SMAD signaling (SMAD3 mRNA:SMAD7 mRNA ratio) was increased in scleroderma fibroblasts (mean ± SEM 160 ± 46% of the ratio in control fibroblasts), although the difference was not statistically significant. These results were reproducible in several independent experiments, and they were consistent when individual fibroblast lines were examined at different passages (passages 4 and 7).

In contrast to catalytic intracellular second messenger systems, the SMADs are devoid of intrinsic enzymatic activity, and therefore the SMAD pathway has no potential for amplifying input signals. Consequently, TGFβ-induced cellular responses are sensitive to small changes in SMAD steady-state levels (37). We therefore examined the regulation of cellular SMAD mRNA expression by TGFβ. The results showed that treatment with TGFβ (500 pM) caused a selective reduction in SMAD3 mRNA expression in normal fibroblasts. After 48 hours of treatment, the SMAD3 mRNA steady-state levels were decreased to 38 ± 6% (mean ± SEM) of those in untreated fibroblasts. Failure to suppress SMAD3 expression could potentially result in uncontrolled TGFβ signaling due to a relative increase in SMAD3 levels and persistent availability of SMAD3 for activation by the TGFβ receptor kinase. We therefore examined whether the regulation of SMAD3 mRNA expression by TGFβ was altered in scleroderma fibroblasts. As shown in a representative Northern blot (Figure 2A), SMAD3 steady-state mRNA levels in scleroderma fibroblasts were markedly reduced by TGFβ (to 34 ± 9% of the levels in untreated fibroblasts). An essentially identical magnitude of TGFβ-induced decrease in SMAD3 mRNA was observed in 4 individual scleroderma fibroblast lines examined.

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Figure 2. Regulation of SMAD mRNA expression by transforming growth factor β (TGFβ). Fibroblast lines obtained from lesional skin of scleroderma patients or from skin of healthy controls were cultured in parallel. When fibroblasts reached confluence, TGFβ1 (500 pM) was added to the cultures. After the indicated period of incubation, cultures were harvested and relative mRNA levels were determined by Northern analysis. A, SMAD3 and SMAD7 mRNA levels determined in fibroblasts incubated (solid bars in lower panel) or not incubated (open bars in lower panel) with TGFβ for 24 hours (SMAD3) or 90 minutes (SMAD7). An autoradiograph representative of multiple independent experiments is shown in the upper panel. Levels of mRNA were quantitated by densitometric scanning of the autoradiographs (lower panel). Results represent the mean and SEM from 4 pairs of scleroderma (S) and normal (N) fibroblast lines corrected for the levels of GAPDH mRNA in each sample. B, Kinetics of SMAD7 mRNA induction by TGFβ in control (left panel) and scleroderma (right panel) fibroblasts. Note the difference in scales between the two panels. Results for 7 individual scleroderma fibroblast lines are shown.

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Inhibitory SMAD7 competes with SMAD3 for access to the activated TGFβ receptor, thereby preventing SMAD3 phosphorylation and blocking TGFβ-induced responses in fibroblasts (29). Negative feedback inhibition of TGFβ/SMAD signaling mediated through endogenous SMAD7 limits the magnitude and/or duration of cellular responses to TGFβ, and rapid induction of SMAD7 is therefore essential for preventing excessive TGFβ stimulation. Defective induction of cellular SMAD7 results in unopposed SMAD signaling, leading to exaggerated or sustained TGFβ responses. We showed previously that transient disruption of endogenous SMAD7 function using antisense oligonucleotides resulted in increased TGFβ stimulation of COL1A2 transcription in normal fibroblasts (38). We therefore examined the possibility that defective SMAD7 induction could account for the activated phenotype of scleroderma fibroblasts.

As shown in Figure 2A, TGFβ induced a significant early rise in SMAD7 mRNA levels. Quantitation of results from several independent experiments showed that in scleroderma and control fibroblast lines treated with TGFβ for 90 minutes, SMAD7 mRNA levels were increased to 261 ± 42% and 256 ± 35% (mean ± SEM), respectively, of those in untreated fibroblasts. Next, in a separate set of experiments, we carefully compared the kinetics of SMAD7 induction in control (n = 4) and scleroderma (n = 7) fibroblast lines in parallel. As shown in Figure 2B, there was a similar time course of SMAD7 induction, with maximal TGFβ response at 120 minutes both in control and in scleroderma fibroblast lines. Significantly, none of the examined scleroderma fibroblast lines failed to respond to TGFβ with a >2-fold increase in SMAD7 mRNA expression. Taken together, these results clearly indicate that control and scleroderma fibroblasts are characterized by comparable sensitivity to TGFβ, with similar magnitude and kinetics of delayed down-regulation of SMAD3 mRNA expression and rapid up-regulation of SMAD7 mRNA expression.

SMAD protein expression and activation in scleroderma fibroblasts.

The cellular steady-state levels of SMAD proteins are determined by the rates of transcription of the corresponding genes, as well as by posttranscriptional regulatory mechanisms. SMADs are subject to intracellular proteolysis mediated through the ubiquitin ligase proteosome pathways in both ligand-dependent and ligand-independent manners (28). Therefore, it was important to examine the levels of SMAD proteins in control and scleroderma fibroblasts. To this end, whole cell lysates were prepared from confluent control and scleroderma fibroblasts in parallel and subjected to immunoblot analysis.

In Figure 3A, a Western blot from a representative experiment demonstrates bands at ∼52 kd (the expected size of SMAD3) and at ∼45 kd (the expected size of SMAD7). Equal loading and transfer of protein in each lane of the Western blots was confirmed using antiactin antibody. Densitometric analysis of the results demonstrated that 5 of 11 separate scleroderma fibroblast lines consistently displayed elevated SMAD3 protein levels compared with control fibroblasts. Generally, a good correlation between relative levels of SMAD3 protein and mRNA expression was found in individual fibroblast lines. In contrast to SMAD3, the levels of SMAD4 and SMAD7 proteins were similar in control and scleroderma fibroblasts (Figure 3A and results not shown). TGFβ treatment resulted in a marked decrease of SMAD3 protein at 48 hours and in an elevation of SMAD7 protein at 4 hours both in control and in scleroderma fibroblasts, whereas SMAD4 expression remained unchanged. These changes in protein closely paralleled the TGFβ-induced changes in SMAD mRNA levels shown in Figure 2.

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Figure 3. SMAD protein expression in fibroblasts. Fibroblasts from normal control or lesional scleroderma skin were cultured in parallel. At early confluence, cells were harvested and identical amounts of whole cell lysates were electrophoresed and subjected to immunoblotting. A, A representative Western blot. Antibodies against SMAD2/3, SMAD7, and actin were used. Numbers at the bottom indicate control (N) or scleroderma (S) fibroblast lines. B, Increased SMAD phosphorylation in scleroderma fibroblasts. Whole cell lysates were prepared from confluent control (lanes 1–3 [N3] and 4 [N1]) or scleroderma (lanes 5 [S3], 6 [S5], 7 [S11], and 8 [S12]) fibroblasts in parallel. In lanes 2 and 3, fibroblasts were incubated with transforming growth factor β for 30 minutes and 60 minutes, respectively. Proteins were immunoprecipitated with antibody against SMAD2/3. The complexes were electrophoresed through sodium dodecyl sulfate–polyacrylamide gel, followed by immunoblotting with antiphosphoserine antibody (p-Serine). Whole cell lysates were analyzed by immunoblotting for IgG. A representative blot is shown. Numbers at the bottom indicate the ratio of phospho-SMAD2/3 to total SMAD2/3 in each lane, determined by quantitating the signal intensities by densitometry and normalizing results to IgG to correct for small variations in protein loading and transfer.

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Upon TGFβ stimulation, cellular SMAD2 and SMAD3 become rapidly phosphorylated by the activated TGFβ receptor serine/threonine kinase. The phosphorylation state of endogenous SMAD2/3 thus serves as a marker for the activation of the TGFβ/SMAD pathway. We therefore sought to determine the relative phosphorylation state of receptor-activated SMADs in scleroderma fibroblasts. For this purpose, unstimulated quiescent control and scleroderma fibroblasts in parallel were lysed, and equal aliquots were immunoprecipitated with anti-SMAD2/3 antibody, followed by electrophoresis and immunoblotting with antiphosphoserine antibody.

As shown in a representative immunoblot in Figure 3B, only low levels of SMAD2/3 phosphorylation could be detected in untreated control fibroblasts. Treatment of the fibroblasts with TGFβ rapidly induced phosphorylation of SMAD2/3, as expected. The expression levels of total SMAD2/3 proteins were unaffected by treatment with TGFβ for 60 minutes, as indicated by immunoblotting with the anti-SMAD2/3 antibody. In each of the scleroderma fibroblast lines studied, phosphorylated SMAD2/3 could be clearly detected in the absence of TGFβ stimulation (Figure 3B). Quantitation of the immunoblot results from several independent experiments indicated that the ratio of phosphorylated SMAD2/3 to total SMAD2/3 in lysates from scleroderma fibroblasts (n = 4) was 1.62 ± 0.47, compared with 0.19 ± 0.05 in control fibroblasts (n = 2) (Figure 3B and data not shown).

Nuclear accumulation of SMAD3 and SMAD4.

Because SMAD-mediated TGFβ signal transduction is critically dependent on translocation of SMAD3 and SMAD4 from the cytoplasm into the nucleus, the activity of the SMAD signaling pathway can be regulated by the spatial compartmentalization of endogenous SMADs. In order to examine subcellular SMAD distribution in scleroderma, SMAD3/4 nuclear accumulation was analyzed using antibody-mediated immunofluorescence with confocal microscopy.

As shown in Figure 4A, SMAD3 showed predominantly nuclear distribution in <10% of control fibroblasts in the absence of exogenous TGFβ. Treatment with TGFβ resulted in a rapid increase in SMAD nuclear accumulation, which was maximal by 120 minutes (Figure 4A). Interestingly, we found that TGFβ concentrations substantially below the threshold required for induction of transcriptional responses (such as stimulation of COL1A2 and plasminogen activator inhibitor 1) were capable of inducing SMAD3/4 nuclear translocation (e.g., a concentration of TGFβ as low as 10 pM was sufficient to induce nuclear accumulation of endogenous SMAD3/4 in >60% of normal fibroblasts [24]).

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Figure 4. Increased nuclear SMAD accumulation in scleroderma fibroblasts. Control or scleroderma fibroblasts in parallel were incubated with transforming growth factor β1 (TGFβ1), fixed, and stained with fluorescein isothiocyanate (FITC)–conjugated SMAD3- or SMAD4-specific antibodies. A, Immunofluorescence confocal microscopic images from a representative experiment. Green color (FITC) indicates SMAD4; blue color (4′,6-diamidino-2-phenylindole) indicates the nucleus. a, Untreated control fibroblasts; b, fibroblasts treated with TGFβ; c, untreated scleroderma fibroblasts; d, scleroderma fibroblasts treated with TGFβ (original magnification × 100 in upper panels; × 630 in lower panels). B, The proportion of scleroderma or control fibroblasts unambiguously displaying a predominantly nuclear pattern of SMAD3 or SMAD4 immunofluorescence in the absence of added TGFβ was quantitated by scoring 100 individual cells in each culture in a blinded manner. Results shown represent the mean and SEM from 5 individual scleroderma (solid bars) and 3 individual control (open bars) fibroblast lines. ∗ = P < 0.05. C, Nuclear accumulation of SMAD2/3. Nuclear extracts were isolated from confluent scleroderma fibroblasts (S5, S12) or untreated or TGFβ-treated control fibroblasts (N1) and examined by immunoblot using antibodies to SMAD2/3 and histone H3.

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In contrast to control fibroblasts, 30–40% of unstimulated scleroderma fibroblasts showed predominantly nuclear localization of SMAD3 in the absence of exogenous TGFβ. The intensity of nuclear SMAD3 immunofluorescence showed some variability among individual fibroblasts. Elevated nuclear accumulation of SMAD3 persisted during serial in vitro passages of the scleroderma fibroblasts. Results were essentially identical when subcellular distribution of SMAD4 was compared between control and scleroderma fibroblasts. Incubation of fibroblasts with irrelevant IgG as negative control resulted in minimal fluorescence. Quantitation of results from multiple experiments with several fibroblast lines indicated that the proportion of fibroblasts displaying predominantly nuclear SMAD3 localization was 3.3-fold greater in scleroderma cultures (n = 6) than in control cultures (n = 3); for SMAD4, this proportion was 3.7-fold greater in scleroderma cultures (Figure 4B).

To confirm enhanced nuclear accumulation of SMAD3 in scleroderma fibroblasts, nuclear extracts were prepared from confluent cultures of control or scleroderma fibroblasts in parallel and examined by Western analysis. As shown in a representative immunoblot (Figure 4C), the accumulation of SMAD2/3 in the nucleus was ∼7-fold greater in scleroderma fibroblasts than in control fibroblasts. As expected, treatment of control fibroblasts with TGFβ rapidly induced an ∼10-fold increase in nuclear SMAD accumulation. We next compared the nuclear accumulation of phospho-SMAD2/3 in control and scleroderma fibroblasts. In the absence of TGFβ, only relatively low levels of phospho-SMAD2/3 could be detected in control fibroblasts, largely localized in the cytoplasm. In contrast, ∼30% of examined scleroderma fibroblasts showed strong expression and dominant nuclear localization of phospho-SMAD2/3 (Figures 5A and B).

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Figure 5. Increased nuclear phospho-SMAD accumulation in scleroderma fibroblasts. Control or scleroderma fibroblasts incubated with or without antibody (Ab) to TGFβ in parallel were fixed and stained with FITC-conjugated phospho-SMAD2/3 antibodies. A, Immunofluorescence confocal microscopic images from a representative experiment. Green color indicates phospho-SMAD2/3; blue indicates nuclei. a, Control fibroblasts; b, scleroderma fibroblasts; c, scleroderma fibroblasts incubated with anti-TGFβ antibody (original magnification × 100 in upper panels; × 400 in lower panels). B, The proportion of fibroblasts displaying predominantly nuclear localization of phospho-SMAD2/3 in the absence of added TGFβ was quantitated by scoring 100 individual cells in each culture in a blinded manner. Results shown represent the mean and SEM from 3 independent experiments with 4 individual scleroderma (solid bars) and 3 individual control (open bars) fibroblast lines. ∗ = P < 0.05. See Figure 4 for other definitions.

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SMAD7 acts as a component in negative feedback regulation of the fibroblast SMAD signaling pathway. By stably binding to the activated TGFβ receptor, SMAD7 competes with SMAD2/3 for receptor interaction (23). In previous studies, SMAD7 was shown to be located predominantly in the nucleus and to translocate into the cytoplasm upon stimulation by TGFβ. In those experiments, transformed cell lines and recombinant SMAD7 protein were used. We examined the subcellular localization of native SMAD7 in normal fibroblasts by confocal microscopy. The results showed that in contrast to transformed cells such as COS7, in normal fibroblasts SMAD7 was located predominantly in the cytoplasm, both in the absence and in the presence of exogenous TGFβ (results not shown). Therefore, SMAD7 subcellular localization appeared to be cell type dependent. The level of SMAD7 expression visualized by immunocytochemistry and its ligand-independent cytoplasmic localization were similar in control and scleroderma fibroblasts.

Effect of TGFβ blockade on SMAD subcellular distribution.

Because TGFβ is capable of inducing its own production in fibroblasts, autocrine signaling may contribute to sustaining of cellular responses to TGFβ. Autocrine stimulation by endogenous TGFβ has been implicated in the constitutively activated phenotype of scleroderma fibroblasts (12, 13). Because autocrine TGFβ stimulation could account for SMAD activation and nuclear accumulation observed in scleroderma fibroblasts, we examined the effect of blocking TGFβ-mediated signaling by two complementary approaches. For this purpose, first a neutralizing antibody recognizing all 3 isotypes of TGFβ was used, either alone or in combination with the naturally occurring TGFβ antagonist LAP. Confluent fibroblasts were washed extensively to remove secreted TGFβ, fresh media containing indicated concentrations of anti-TGFβ antibody (1–20 μg/ml) or nonimmune IgG were added, and the subcellular distribution of SMAD3/4 was then determined by confocal microscopy.

The results showed that exposure of scleroderma fibroblasts to the antibody for up to 48 hours, alone or in combination with the TGFβ antagonist LAP (5 μg/ml), failed to down-regulate the level of nuclear SMAD4 or SMAD3 (Figure 6A and data not shown). As an important control, ligand-induced SMAD nuclear import was examined. As expected, addition of anti-TGFβ antibody resulted in a dose-dependent inhibition of SMAD4 nuclear translocation induced by TGFβ in control fibroblasts. Anti-TGFβ antibody (10 μg/ml) was capable of reducing by >70% the SMAD nuclear translocation induced by TGFβ at concentrations of up to 100 pM (Figure 6A and data not shown). Therefore, failure to reverse enhanced SMAD nuclear accumulation in scleroderma fibroblasts by partial blockade of the autocrine TGFβ activation loop suggests that a mechanism independent of ligand-mediated TGFβ receptor stimulation may have been responsible.

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Figure 6. The effect of blockade of transforming growth factor β (TGFβ) signaling on SMAD nuclear accumulation. Confluent cultures of fibroblasts were incubated with neutralizing antibody (Ab) to TGFβ (α-TGF-β; 1–20 μg/ml) or with nonimmune IgG, followed by addition of TGFβ (10 pM) for 90 minutes. A, Cells were stained with SMAD4-specific antibodies and examined by confocal fluorescence microscopy. The proportion of control (open bars) or scleroderma (solid bars) fibroblasts displaying a predominantly nuclear pattern of immunofluorescence was quantitated by scoring 100 individual cells in each culture in a blinded manner. Results shown represent the mean and SEM of duplicate determinations from 3 independent experiments. B, Serum-starved normal (N1) or scleroderma (S5, S12) fibroblasts were incubated with anti-TGFβ antibody (10 μg/ml). Cells were harvested and whole cell lysates (200 μg) were immunoprecipitated with antibody to TGFβ receptor type I, followed by immunoblotting with antiphosphoserine antibody (p-Serine). Membranes were stripped and reprobed with antibody to IgG to confirm equal protein loading in each lane.

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The effect of anti-TGFβ antibodies on subcellular distribution of phospho-SMAD2/3 was characterized. As shown in Figures 5A and B, ∼30% of scleroderma fibroblasts and <5% of control fibroblasts showed a predominantly nuclear localization of phospho-SMAD2/3, and anti-TGFβ antibody failed to significantly reduce the proportion of scleroderma fibroblasts showing nuclear phospho-SMAD2/3. Immunoblots of nuclear extracts demonstrated elevated levels of phospho-SMAD2/3 in scleroderma fibroblasts and further confirmed the failure of anti-TGFβ antibody to “normalize” phospho-SMAD2/3 distribution (results not shown).

In order to confirm that neutralizing anti-TGFβ antibody was capable of blocking autocrine TGFβ signaling, we examined the phosphorylation state of endogenous TGFβRI (which is primarily phosphorylated on serine residues in its GS domain upon activation by the ligand) on untreated control and scleroderma fibroblasts. For this purpose, TGFβRI was immunoprecipitated from whole cell lysates, followed by electrophoresis and immunoblotting with antiphosphoserine antibodies. The results showed that while TGFβRI was constitutively phosphorylated in both control and scleroderma fibroblasts due to autocrine signaling, scleroderma fibroblasts exhibited >2-fold higher TGFβRI phosphorylation than did control fibroblasts (Figure 6B). The levels of TGFβRI were similar in scleroderma and control fibroblasts (data not shown). Importantly, treatment with anti-TGFβ antibody caused a substantial reduction in TGFβRI phosphorylation in scleroderma fibroblasts, but not in control fibroblasts. These results indicate that under the conditions employed in these experiments, anti-TGFβ antibody effectively disrupted the autocrine TGFβ activation loop in scleroderma fibroblasts.

To further determine the potential contribution of autocrine TGFβ signaling in enhanced SMAD nuclear localization, we used a dominant-negative TGFβRII vector to disrupt TGFβ responses. The dominant-negative TGFβ receptor is unable to phosphorylate the type I receptor in response to any of the 3 TGFβ isoforms, thus interrupting intracellular TGFβ signaling. Normal and scleroderma fibroblasts in parallel were transduced with the dominant-negative TGFβRII or empty vector, followed 48 hours later by incubation with TGFβ for 90 minutes.

As shown in a representative experiment in Figure 7A, in normal fibroblasts transduced with dominant-negative TGFβRII, TGFβ-stimulated SMAD nuclear accumulation was markedly reduced (compare panels b and d). As expected, scleroderma fibroblasts showed a markedly higher level of basal nuclear SMAD accumulation in the absence of added TGFβ (panel e), and this was not reduced by the dominant-negative TGFβRII (panel f). A high level of dominant-negative TGFβRII expression in the same cells was confirmed by immunocytochemistry (lower panels). Quantitation of the results from several independent experiments confirmed the ability of adenovirus-mediated dominant-negative TGFβRII overexpression to reduce by >80% the TGFβ-induced nuclear migration of SMAD2/3 in normal fibroblasts, as well as its failure to “normalize” SMAD subcellular distribution in scleroderma fibroblasts (Figure 7B). Essentially identical results were noted with 3 separate scleroderma cell lines. Transduction with GFP vector had no effect on either TGFβ-induced SMAD nuclear migration in control fibroblasts or constitutive nuclear accumulation in scleroderma fibroblasts. Note the elevated nuclear accumulation of SMAD2/3 in scleroderma fibroblasts showing high levels of cellular dominant-negative TGFβRII expression 48 hours posttransduction in the representative images shown in Figure 7C.

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Figure 7. The effect of adenovirus-mediated expression of dominant-negative transforming growth factor β receptor type II (TGFβRII) on SMAD nuclear accumulation in scleroderma fibroblasts. A, Confluent cultures of normal (a–d) or scleroderma (e and f) fibroblasts were transduced with empty vector (a, b, and e) or adenovirus expressing dominant-negative TGFβRII (c, d, and f), followed 48 hours later by addition of 10 pM TGFβ (b and d). Following 90 minutes of incubation, fibroblasts were stained with antibodies to SMAD2/3 (upper panels) or TGFβRII (lower panels) and examined by confocal fluorescence microscopy. B, The proportion of control (open bars) or scleroderma (solid bars) fibroblasts displaying a predominantly nuclear pattern of SMAD3 immunofluorescence was quantitated by scoring 100 individual cells in each culture in a blinded manner. Results shown represent the mean and SEM of duplicate determinations from several independent experiments with 3 separate scleroderma cell lines. C, Control (a) or scleroderma (b and c) fibroblasts were transduced with empty vector (a and b) or dominant-negative TGFβRII (c) and were stained 48 hours later with antibodies to SMAD2/3 or TGFβRII. Immunofluorescence confocal microscopic images from a representative experiment are shown. In c, note that immunostaining confirms substantial expression of dominant-negative TGFβRII (green) in scleroderma fibroblasts showing a high level of nuclear SMAD2/3 accumulation (pink). dnTβRII = dominant-negative TGFβRII. (Original magnification × 400.)

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SMAD-mediated transcriptional activity in scleroderma fibroblasts.

In order to investigate the potential functional significance of ligand-independent activation and nuclear accumulation of endogenous SMAD3 and SMAD4 in scleroderma fibroblasts, we examined SMAD-regulated luciferase reporter expression. The SMAD-responsive minimal pSBE4-luc construct was transiently transfected in control and scleroderma fibroblasts in parallel, and luciferase activity was determined after 48 hours. As shown in Figure 8, SMAD-inducible promoter activity was elevated in untreated lesional fibroblasts compared with controls. The activity of pGL3-luc was similar in lesional and control fibroblasts.

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Figure 8. SMAD-regulated transcriptional activity in fibroblasts. Subconfluent scleroderma (S) and control (N) fibroblasts in parallel were transiently transfected with pSBE4-luc and appropriate control plasmids, and luciferase (Luc) activities were determined after 48 hours of incubation. Results, normalized by renilla luciferase activity to correct for small variations in transfection efficiency between samples, represent the means of triplicate samples from 3 independent experiments. Bars show the group mean ± SEM. ∗ = P < 0.05.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The recent discovery and characterization of the SMAD protein family has provided novel opportunities for delineating the involvement of TGFβ in fibrosis (39). In normal fibroblasts, SMADs function as essential cytoplasmic effectors for TGFβ stimulation of collagen synthesis, as well as that of CTGF (40) and of TGFβ itself (41). Genetic targeting of the SMAD3 locus protected null mice from the development of radiation-induced fibrosis, indicating the fundamental physiologic role of SMAD3 in processes of tissue repair and fibrosis (42). Because the activated scleroderma fibroblast phenotype is thought to reflect enhanced stimulation by, or intrinsic responsiveness to, TGFβ, in the present study we examined the expression and regulation of the SMAD signaling pathway in these cells.

The results indicated that in a majority of scleroderma fibroblasts, SMAD3 protein and mRNA expression were elevated in comparison with control fibroblasts. Elevated levels of SMAD3 have also been observed in hepatic myofibroblasts (43) and in a murine model of renal fibrosis (44). Elevated SMAD3 expression could result in enhanced formation and nuclear import of transcriptionally active SMAD heterocomplexes, with increased transcription of TGFβ-regulated target genes. However, in light of the relatively modest and variable increase in SMAD3 mRNA levels in scleroderma versus control fibroblasts (∼60%), we consider it unlikely that elevated SMAD3 expression plays a major role in altered TGFβ signaling in scleroderma fibroblasts.

Steady-state levels of SMAD3 are determined in part by the rate of transcription of the SMAD3 gene and the intracellular degradation of SMAD3 protein. The factors determining the rate of SMAD3 transcription are currently unknown. Because we found in the present experiments that the increase in SMAD3 protein levels in scleroderma fibroblasts compared with control fibroblasts was of relatively greater magnitude than the increase in mRNA levels, it is possible that SMAD3 protein degradation was impaired; this is currently under investigation. Importantly, the expression of SMAD3 mRNA in both normal and scleroderma fibroblasts was selectively repressed by TGFβ. Inhibition of SMAD3 mRNA expression was a delayed response, with maximal effect after 48 hours of exposure. These results clearly indicate that TGFβ can regulate the steady-state levels of its own second messengers by pretranslational modulation, in addition to the well-documented proteasomal degradation pathways.

Repression of SMAD3 mRNA levels in response to TGFβ has been noted in epithelial and mesangial cells as well as in lung fibroblasts (45–47). In light of the dependence of TGFβ signaling intensity on the steady-state levels of its intracellular second messengers, inhibition of SMAD3 expression may thus be a negative feedback mechanism with autoregulatory function to prevent continuous SMAD signaling in response to TGFβ stimulation. The present results indicate that elevated SMAD3 steady-state levels seen in some scleroderma fibroblasts cannot be attributed to failure of TGFβ to repress SMAD3 gene expression in these cells.

In normal cells, TGFβ directly stimulates transcription of inhibitory SMAD7. Thus, SMAD7 induction serves as a critical intracellular “brake” on TGFβ-induced responses. The physiologic importance of this SMAD7-mediated autoinhibitory feedback loop is highlighted by recent observations with cultured cells in in vivo animal models. For example, tumor necrosis factor α–induced abrogation of TGFβ-dependent profibrotic responses was linked to the induction of endogenous SMAD7 (48). On the other hand, defective SMAD7 induction is implicated in the exaggerated TGFβ responsiveness characteristic of hepatic cells and myofibroblasts from chronically injured livers (49, 50). While the highest level of SMAD7 expression is normally found in the kidneys (23), in spontaneous renal fibrosis in TGFβ1-transgenic mice, and in renal fibrosis induced by anti–Thy-1 antibody, excessive matrix accumulation occurs in the setting of reduced SMAD7, resulting in exaggerated or sustained local TGFβ response (51, 52). Furthermore, a recent study demonstrated that SMAD7 basal expression levels and TGFβ-dependent inducibility were both markedly impaired in scleroderma fibroblasts in vivo and in vitro (53). These observations are intriguing, because SMAD7 deficiency could provide a mechanistic explanation for elevated TGFβ receptor expression and relative resistance to apoptosis that have been reported in scleroderma fibroblasts.

In the present studies, we failed to detect consistent intrinsic alterations in basal levels of SMAD7 expression or SMAD7 inducibility. Protein and mRNA expression of SMAD7, its regulation by TGFβ, and its subcellular localization in scleroderma fibroblasts were all comparable with those in normal fibroblasts. Therefore, our results do not support the hypothesis that in scleroderma, fibroblasts may be “sensitized” to the profibrotic effects of TGFβ because of inability to “apply the brakes” on TGFβ-induced cellular responses due to impaired SMAD7 regulation. It remains possible, however, that despite its apparently normal inducibility by TGFβ in vitro, SMAD7 in scleroderma fibroblasts somehow fails to inhibit receptor-mediated activation of SMAD2/3.

In the absence of ligand stimulation, receptor-activated SMADs and SMAD4 reside mostly in the cytoplasm, and their accumulation within the nucleus is a key event in the intracellular propagation of TGFβ signaling. Due to the presence of an N-terminal nuclear localization signal sequence, SMAD3 displays intrinsic nuclear import activity (54). In contrast, SMAD4 requires association with activated SMAD3 in order to accumulate in the nucleus (55). In normal fibroblasts, only a low level of nuclear SMAD accumulation could be detected in the absence of exogenous TGFβ. In contrast, the present studies revealed a substantial degree of endogenous SMAD phosphorylation and nuclear accumulation in scleroderma fibroblasts. These findings were highly consistent in each of the scleroderma fibroblast lines studied.

Similar ligand-independent SMAD activation has been described in hepatic stellate cells derived from fibrotic livers (56) and in dermal fibroblasts derived from keloid lesions (57). Constitutive SMAD activation is therefore likely to contribute to the profibrotic phenotype of scleroderma fibroblasts. One of the significant profibrotic effects of TGFβ is its ability to inhibit production of the matrix-degrading enzyme collagenase 1. Repression of collagenase in normal fibroblasts by TGFβ is mediated through SMAD3 (58). Therefore, the present results demonstrating signal-independent activation of SMAD3 in scleroderma fibroblasts may provide a mechanistic explanation to account for the reduced collagenase expression previously described in scleroderma (59). Recent reports indicate that optimal induction of COL1A2 transcription by TGFβ involves an interaction between activated SMAD3 and the ubiquitous DNA binding transcription factor Sp1 (32, 33). In this regard, it is of interest that Sp1 has been shown to be constitutively phosphorylated and activated in scleroderma fibroblasts (60). These findings suggest that for maximal transactivation of COL1A2 in scleroderma fibroblasts, SMAD activation and Sp1 phosphorylation may both be required.

The mechanisms responsible for constitutive SMAD activation in scleroderma fibroblasts are currently unknown. Autocrine stimulation by endogenous TGFβ may provide a possible explanation. Indeed, a key role for autocrine stimulation by TGFβ in the activated phenotype of scleroderma fibroblasts has been suggested, and elevated collagen synthesis was reduced by disrupting endogenous TGFβ signaling (12, 13). It has been recently demonstrated that SMAD3/4 undergo continuous nucleocytoplasmic shuttling, and their relative levels within the nucleus are directly dictated by the level of TGFβ receptor activity (61). However, in the present studies, we found that neutralizing anti-TGFβ antibody and LAP failed to normalize SMAD subcellular distribution in scleroderma fibroblasts despite a decrease in TGFβRI phosphorylation. Furthermore, transduction of dominant-negative TGFβ receptor that prevented TGFβ-induced SMAD activation in normal fibroblasts failed to reduce SMAD nuclear accumulation in scleroderma fibroblasts, suggesting that constitutive SMAD nuclear accumulation was not due to autocrine signaling by endogenous TGFβ.

It is conceivable that in the present experiments, neither neutralizing antibody nor transduction of dominant-negative TGFβRII was able to completely abrogate fibroblast activation mediated through endogenous TGFβ. In that case, the “ligand-independent” SMAD activation we observed in scleroderma fibroblasts may in fact reflect autocrine TGFβ stimulation. This possibility cannot be ruled out, given that scleroderma fibroblasts in some studies (13, 14), although not in others (62), were shown to display elevated expression of TGFβ receptors and may thus have been “sensitized” to TGFβ (13, 14); SMAD nuclear migration in fibroblasts can be induced by very low concentrations of TGFβ. Alternatively, it is possible that non-TGFβ ligands trigger cellular SMAD activation and nuclear migration. For example, a recent report indicated that insulin-like growth factor binding protein 3 (IGFBP-3) could by itself induce SMAD2/3 phosphorylation and potentiate TGFβ-stimulated cellular responses (63). The ability of IGFBPs to induce TGFβ-independent SMAD phosphorylation/activation may be particularly relevant in the pathogenesis of scleroderma, since IGFBP gene expression is increased in scleroderma fibroblasts (64).

In summary, the present results provide evidence for consistent alterations in the activation states of intracellular TGFβ/SMAD signaling components in the absence of exogenous TGFβ in scleroderma fibroblasts. These alterations may contribute to sensitizing scleroderma fibroblasts to TGFβ or other stimuli that utilize the SMAD pathway, resulting in enhanced fibrotic responses elicited by extracellular signals in pathologic fibrosis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We are grateful to Dr. R. Derynck (University of California, San Francisco), Dr. P. ten Dijke (Ludwig Institute for Cancer Research, Uppsala, Sweden), Dr. J. Massague (Memorial Sloan-Kettering Cancer Center, New York, NY), and Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD) for providing us with reagents, and we thank members of our laboratory staff for helpful discussions.

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  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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