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.
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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.