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Differential mRNA stability plays an important role in the regulation of gene expression. Several recent advances have helped to define the general pathways by which mRNA is degraded in prokaryotic cells, although many details remain to be elucidated. Much less is known about the pathways of degradation in eukaryotic cells, but recent studies on specific systems have highlighted both differences from and similarities to prokaryotic pathways.
The mechanisms by which fasting decreases liver insulin-like growth factor I (IGF-I) messenger RNA (mRNA) abundance have not been defined completely. In the present study, we have examined the effects of fasting in rats on hepatic IGF-I gene transcription, IGF-I pre-mRNA splicing, and cytoplasmic IGF-I mRNA stability. Using the in vitro nuclear run-on transcription technique, we observed that fasting did not change IGF-I gene transcription activity [76 +/- 32 densitometric units (DU) for fasted vs. 58 +/- 23 DU for control-fed rats; P = 0.1], whereas IGF-binding protein-1 (IGFBP-1) gene transcription, a positive control, was increased more than 2-fold (729 +/- 157 DU for fasted vs. 261 +/- 56 DU for control-fed rats; P < 0.05). This implies that fasting-induced reduction of liver IGF-I mRNA is due to events other than a decreased rate of IGF-I gene transcription. By measuring nonspliced (pre-mRNA) and spliced IGF-I transcripts in liver nuclear RNA using ribonuclease protection assays, we found that IGF-I pre-mRNA was increased in fasted rats (measured as the percentage of beta-actin: 34.0 +/- 5.5% for fasted vs. 8.1 +/- 3.8% for control-fed rats; P < 0.01), whereas spliced IGF-I transcript remained unchanged (measured as the percentage of beta-actin: 60.9 +/- 9.2% for fasted vs. 79.0 +/- 6.2% for control-fed rats; P = 0.75). We then compared this pattern of splicing to IGF-I pre-mRNA splicing in hypophysectomized rats subjected to GH stimulation and to IGFBP-1 pre-mRNA splicing in the same fasting experiment. One hour after GH injection, we observed a coordinate increase in both nonspliced and spliced IGF-I transcripts in liver nuclei of hypophysectomized rats. Fasting increased both IGFBP-1 pre-mRNA and spliced transcript. Taken together, these results indicate that the increase in IGF-I pre-mRNA in liver nuclei during fasting is caused by delayed pre-mRNA splicing, rather than increased IGF-I gene transcription. To examine the possible effect of fasting on hepatic IGF-I mRNA stability, we used an in vitro model of nutrient deprivation (fewer amino acids in culture medium) of rat hepatocyte primary culture. Each of the three major IGF-I mRNA species exhibited a shortened half-life in the amino acid-deprived media. The 7.5-kb IGF-I mRNA, however, was degraded faster than the two smaller IGF-I mRNA species. This may indicate that fasting decreases the stability of liver IGF-I mRNA in vivo. In summary, these results suggest that fasting regulates hepatic IGF-I gene expression mainly at the posttranscriptional level by delaying IGF-I pre-mRNA splicing, which attenuates mature IGF-I mRNA generation, and by accelerating the rate of degradation of IGF-I mRNA in cytoplasm.
Human alveolar macrophages have both lipopolysaccharide (LPS)-induced and constitutive phosphatidylinositol 3-kinase (PI3K) activity. We observed that blocking PI3K activity increased release of prostaglandin E2 after LPS exposure, and increasing PI3K activity (interleukin-13) decreased release of prostaglandin E2 after LPS exposure. This was not because of an effect of PI3K on phospholipase 2 activity. PI3K inhibition resulted in an increase in cyclooxygenase 2 (COX2) protein, mRNA, and mRNA stability. PI3K negatively regulated activation of the p38 pathway (p38, MKK3/6, and MAPKAP2), and an active p38 was necessary for COX2 production. The data suggest that PI3K inhibition of p38 modulates COX2 expression via destabilization of LPS-induced COX2 mRNA.
Pre-mRNA splicing is a widely used regulatory mechanism for controlling gene expression, and a family of conserved proteins, SR proteins, participate in both constitutive and alternative splicing. Here we describe a novel function for the SR protein ASF/SF2. We used an embryonic chicken cDNA library to screen for differential mRNA expression in the chicken B-cell line DT40-ASF, expressing or not expressing ASF/SF2. Remarkably, out of 3 x 10(6) clones screened, only one, isolated several times independently, showed ASF/SF2-related differential expression. The isolated cDNA, referred to here as PKCI-r (for PKCI-related), is closely related to the protein kinase C interacting protein (PKCI-1) gene. Transcript levels were increased approximately sixfold in ASF/SF2-depleted cells compared with cells expressing ASF/SF2, indicating a negative role for the SR protein. Strikingly, inhibition of ASF/SF2 expression had no significant effect on PKCI-r splicing, or transcription, but markedly increased the half-life of PKCI-r mRNA (6.6-fold). Similarly, increased mRNA stability was also observed upon expression of exogenous PKCI-r mRNA in cells depleted of ASF/SF2. ASF/SF2 bound to a discrete region containing a purine-rich sequence in the 3' UTR of the PKCI-r transcript, and deletion of this region eliminated ASF/SF2-mediated regulation of transcript stability. Together these data indicate a novel, direct effect of ASF/SF2 on PKCI-r mRNA stability. Therefore, ASF/SF2, and perhaps other SR proteins, affects gene expression in vertebrate cells through regulation of mRNA stability as well as splicing.
Using ROS17/2 rat osteosarcoma cells as a model system, we examined the possibility that endothelin (ET)-induced down-regulation of ETB receptor was accompanied by a decrease in levels of ETB receptor mRNA. Northern blot analysis showed that low doses of ET-1 and ET-3 caused a transient decrease in ETB receptor mRNA in the cells. The maximum decrease in the levels of ETB receptor mRNA (80%) occurred after 2-4 h of exposure of the cells to ETs and was followed by a gradual recovery to control levels by 24 h. The effects were dose-dependent (EC50-1 nM), and ET-1 and ET-3 were almost equipotent in eliciting the response. The addition of either ionomycin, a Ca2+ ionophore, or phorbol dibutyrate, a protein kinase C activator, mimicked the effect of ETs. These results suggested that ETs-induced down-regulation of ETB receptor mRNA was mediated by the activation of ETB receptor and that it may have involved ETB receptor coupled second messenger pathways. We also showed that ETB receptor mRNA had a long intracellular life span which suggested that ETs-induced down-regulation of ETB receptor mRNA may have been due to a decrease in the stability of mRNA, rather than inactivation of the transcription of mRNA.
We present evidence that pyrrolidine dithiocarbamate (PDTC) inhibits growth of p53-negative pancreatic adenocarcinoma cell lines via cell cycle arrest in the S-phase, while it has no effect on primary fibroblast proliferation. Growth inhibition of cancer cells is dependent on ROS and ERK1/2 induction as indicated by a significantly reduced PDTC-associated growth inhibition by the free radical scavenger N-acetyl-L-cysteine (NAC) or the MEK/ERK1/2 inhibitor (PD98059). Moreover, ERK1/2 induction is dependent on ROS production as demonstrated by a complete removal of PDTC-mediated ERK1/2 phosphorylation by NAC. p21(WAF1/CIP1) activation has a central role in growth inhibition by PDTC, as revealed by P21(WAF1/CIP1) silencing experiments with antisense oligonucleotide, and occurs via increased mRNA stability largely mediated by ROS/ERK induction. Conversely, PDTC does not affect P21(WAF1/CIP1) gene expression in primary fibroblasts, although it is able to activate p53 and the p53-regulated antioxidant SESN2. These results suggest that the resistance of fibroblasts to the cytotoxic action of PDTC may be related to the up-regulation of p53-dependent antioxidant genes. Finally, in vivo studies on PaCa44 cells subcutaneously xenografted in nude mice show that treatment with 100 or 200 mg/kg PDTC reduces of 30% or 60% the tumour volume, respectively, and does not cause any apparent form of toxicity.
In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.
Reduced arterial oxygen tension (i.e. hypoxia) is a powerful physiological stimulus that induces synthesis and release of dopamine from O2-sensitive (type I) cells in the mammalian carotid bodies. We reported recently that hypoxia stimulates gene expression for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis in type I cells of the carotid body. Efforts to identify the mechanisms regulating TH gene expression in O2-sensitive cells during hypoxia have been hampered by the lack of an appropriate model cell culture system. Here we report that TH gene expression in the rat pheochromocytoma cell line (PC12) is regulated during hypoxia in a manner similar to that measured in carotid body type I cells. PC12 cells might therefore be useful as an experimental model for identifying the molecular mechanisms that regulate TH gene expression during hypoxia. Nuclear runoff assays revealed that transcription of the wild type TH gene was enhanced during exposures to hypoxia lasting 12 h. Chloramphenicol acetyltransferase assays with constructs that contained different fragments of TH promoter revealed that the regulatory sequences that mediate the hypoxia-induced increase in transcription are located between bases -272 and +27 of the TH gene. Findings from experiments in which transcription was inhibited either with actinomycin D or 5,6-dichloro-1-D-ribofuranosylbenzimidazole, as well as pulse-chase experiments using 4-thiouridine showed that the half-life of TH mRNA was substantially increased during hypoxia. Thus, in the present paper we show that TH gene expression in PC12 cells during hypoxia is regulated by increases in both the rate of TH gene transcription and TH mRNA stability.
The MLA 144 gibbon T cell line is infected with a type C retrovirus and constitutively expresses interleukin-2 (IL-2) and granulocyte macrophage colony-stimulating factor (GM-CSF). IL-2 mRNA levels are 10-fold more abundant than GM-CSF in these cells. Comparable transcriptional rates for these lymphokines suggested the involvement of post-transcriptional mechanisms in selective IL-2 mRNA accumulation. IL-2 mRNA is exceptionally stable in MLA cells with a t1/2 of more than 8 h. The presence of reiterated AUUUA sequences in the 3'-untranslated region (UTR) has been shown to confer mRNA lability. The provirally altered MLA IL-2 allele encodes an mRNA in which three AUUUA motifs have been deleted. Six major cytoplasmic proteins bound in vitro transcribed RNA probes containing sequences from the 3'-UTR of normal human IL-2 (3'-IL-2), GM-CSF (delta 2R1), and the virally altered MLA IL-2 (3'-IL-2 PV) mRNA. Increased binding of these proteins to 3'-IL-2 PV was observed relative to 3'-IL-2 or delta 2R1. Northwestern blotting demonstrated similar differential ability of a 36- and 43-kDa protein to bind, as well as showed that these proteins colocalized by immunoblotting as hnRNP A1 and C, respectively. These findings suggest a direct correlation between differential binding of cytoplasmic proteins to AU-rich 3'-UTRs in vitro and lymphokine mRNA stability in vivo.
Modulation of mRNA stability provides a powerful means for controlling gene expression during the cell cycle, cell differentiation, the immune response, as well as many other physiological transitions. Through the years, many different methods have been developed for measuring mRNA stability. Frequently mRNA stability is studied indirectly by analyzing the steady-state level of mRNA. Therefore by inference, changes in mRNA abundance are thought to affect only the stability of the mRNA, an assumption that is not always correct. Alternatively, direct measurements of mRNA decay are performed in a number of ways, including kinetic labeling techniques and administration of transcriptional inhibitors. Due to the nature of these techniques, they either are technically demanding or introduce a significant change in cell physiology. In addition, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor-product relationships cannot be readily addressed by these methods. Here, we describe and discuss in detail two different transcriptional pulsing methods based on the c-fos serum-inducible promoter and the tetracycline-regulated promoter systems as an effort to better elucidate the mechanistic steps and regulation underlying differential and selective mRNA turnover in mammalian cells. Both systems allow unequivocal monitoring of deadenylation and decay kinetics as well as determination of precursor-product relationship. In addition, decay rate constants and half-lives are determined and used in both methods to quantitatively denote the mRNA stability. Thus, they provide a reliable way to determine subtle yet physiologically meaningful changes in mRNA stability. Application of one method or the other covers the study of mRNA turnover in most mammalian cell types under a wide range of physiological conditions.
Collagenase 3 degrades collagen fibrils and is necessary for bone resorption. Cortisol increases collagenase 3 mRNA in osteoblasts by stabilizing collagenase 3 transcripts. To understand mechanisms involved, we used RNA electrophoretic mobility shift assay and RNA turnover studies. Cortisol increased the binding of Ob cell cytosolic extracts to AU-rich sequences in the collagenase 3 3'-untranslated region (UTR). No cortisol-dependent protein complexes were formed with the coding region or the 5'-UTR. Functional assays, using transient transfections of CMV-driven c-fos collagenase 3'-UTR chimeric constructs, demonstrated that the 3'-UTR of collagenase 3 stabilizes c-fos mRNA in transcriptionally arrested Ob cells, cortisol prolongs the transcript half-life, and mutations of AU-rich sequences destabilize c-fos transcripts precluding the cortisol effect. Purification of osteoblast cytosolic extracts by ultracentrifugation, ion exchange, and RNA affinity chromatography, and polyacrylamide gel electrophoresis followed by mass spectroscopy identified specific proteins. RNA gel mobility supershift assays demonstrated that vinculin and far upstream element (FUSE)-binding protein 2 interacted with collagenase 3 3'-UTR sequences, and RNA interference demonstrated these proteins altered collagenase mRNA stability. In conclusion, AU-rich sequences of the 3'-UTR of collagenase 3 and vinculin and FUSE-binding protein 2 regulate collagenase mRNA stability in osteoblasts.
It is now well established that cAMP induces the transcription rate of the gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) and that this induction is dependent on a nucleotide domain located within the promoter-regulatory region of the gene (Short, J. M., Wynshaw-Boris, A., Short, H. P., and Hanson, R. W. (1986) J. Biol. Chem. 261, 9721-9726). We report here that cAMP also stabilizes phosphoenolpyruvate carboxykinase mRNA against degradation. Using two independent experimental approaches, we show that the half-life of the mRNA for phosphoenolpyruvate carboxykinase is extended when FTO-2B rat hepatoma cells are exposed to dibutyryl cyclic AMP (Bt2cAMP). In the first experiment, the rate of decay of phosphoenolpyruvate carboxykinase mRNA was determined in cells incubated in the presence of insulin, which has been shown to block the transcription rate of the gene for the enzyme. Under these conditions, the half-life of phosphoenolpyruvate carboxykinase mRNA was 30 min. However, in cells incubated in the presence of Bt2cAMP, the mRNA decayed with a half-life of 150 min. In the other experiment, mRNA stability was measured under steady state conditions, utilizing a "pulse-chase" approach. The apparent half-life of phosphoenolpyruvate carboxykinase mRNA increased from 40 min to over 250 min in Bt2cAMP-treated cells. No significant change in the stability of total cellular RNA was noted. Other experiments have shown that the transcription rate of the gene for phosphoenolpyruvate carboxykinase peaks within the first 20 min after exposing the cells to Bt2cAMP and then levels off, while the abundance of the mRNA reaches a maximum at about 90 min and remains at this level thereafter. Thus, the long term effect of cAMP on the expression of the gene coding for phosphoenolpyruvate carboxykinase occurs at least in part, through an alteration in the degradation rate of the mRNA for this enzyme.
Recent advances in the understanding of prokaryotic gene expression have led scientists to look beyond traditional promoter control for new methods of regulating gene expression. A promising, new technique centers on controlling the stability of messenger RNA. To exploit the potential of mRNA stability for gene expression control, it is important to understand the mechanisms of prokaryotic mRNA decay as well as the cellular factors that can be used to enhance bacterial gene expression through mRNA stabilization. Factors involved in controlling prokaryotic mRNA stability such as nucleases, secondary structures, translation influences, and transcription effects are discussed and analyzed within the context of three prevailing mRNA decay theories. Several strategies for manipulating mRNA stability in genetically-engineered cells are developed from these discussions and presented as a future direction in gene expression control. In the near future, it should be possible to use these strategies to control mRNA stability in such applications as pharmaceutical protein production and metabolic pathway design.
Growth arrest- and DNA damage-inducible protein alpha (gadd45alpha) is an important regulator for cell cycle, genomic stability, and cell apoptosis. In the present report, we demonstrated that NF-kappaB inhibition due to Ikkbeta deficiency enhanced the stability of gadd45alpha mNRA. Using embryo fibroblast cells derived from wild type (wt) or Ikkbeta gene knockout (Ikkbeta(-/-)) mice, reverse transcription-polymerase chain reaction revealed a three- to fourfold increase of gadd45alpha mRNA in Ikkbeta(-/-) cells compared with wt cells. The deficiency in Ikkbeta substantially decreased basal NF-kappaB activity and increased accumulation of reactive oxygen species (ROS). However, such deficiency had no effect on the basal expression or activity of Akt, FoxO3a, p53, and c-myc that regulate the transcription of gadd45alpha gene positively or negatively. Analysis of gadd45alpha mRNA stability showed a ROS-dependent increase in the half-life of gadd45alpha mRNA in Ikkbeta(-/-) cells. Immunoprecipitation experiments indicated an increased binding of a RNA stabilizing protein, nucleolin, to gadd45alpha mRNA in Ikkbeta(-/-) cells. The binding of nucleolin to gadd45alpha mRNA could be prevented by the antioxidant, N-acetyl-cysteine. Thus, these data are the first to suggest that inhibition of Ikkbeta-NF-kappaB signaling up-regulates the expression of gadd45alpha mNRA through a post-transcriptional, rather than a transcriptional, mechanism.
RhoB is a small GTP-binding protein that is involved in apoptotic signal transduction. We have cloned the mouse RhoB mRNA including a 1377 nucleotide 3'-untranslated region (UTR) that contains six AU-rich elements (AREs) as well as several uridine-rich stretches. There is 94% homology overall between the mouse and rat RhoB genes and 92% homology between the mouse and a putative human clone. Ultraviolet light (UVL) induces RhoB production through regulated changes in gene transcription and mRNA stabilization although the latter mechanism is unknown. We observed that UVL increased the half-life of RhoB mRNA from 63 min to 3.3 h in NIH/3T3 cells and from 87 min to 2.7 h in normal human keratinocyte cells. In vitro mobility shift assays demonstrated that HuR bound the 3'-UTR of RhoB at three distinct locations (nucleotides 1342-1696, 1765-1920 and 1897-1977) suggesting a regulatory role for this RNA-binding protein. HuR immunoprecipitations were positive for RhoB mRNA indicating an in vivo association, and Western blot analysis and immunofluorescence demonstrated that HuR rapidly partitions from the nucleus to the cytoplasm after UVL. Therefore, we propose a model in which UVL induces stress-activated signal transduction leading to nuclear/cytoplasmic shuttling of HuR and subsequent stabilization of RhoB mRNA.
Transforming growth factors (TGFs)-beta are multipotent in their biologic activity, regulating cell growth and differentiation as well as extracellular matrix deposition and degradation. Most of these activities involve modulation of gene transcription, but TGF-beta1 has been shown previously to substantially increase the expression of elastin by stabilization of tropoelastin mRNA through a signaling pathway that likely involves a phosphatidylcholine-specific phospholipase C, a protein kinase C, prenylated and acylated protein(s), and one or more tyrosine kinases. However, there is a 4- to 6-h lag period after the addition of TGF-beta1 before significant stimulation of elastin expression is observed and the question of whether the Smads are involved has not been addressed. In the present work, using cultured human fetal lung fibroblasts, we show through the use of specific inhibitors and transfection of a Smad 7 construct that in addition to de novo protein synthesis and active Smads, the extended activity of protein kinase C (PKC)-delta and the stress-activated protein kinase, p38, is required for TGF-beta1 to achieve elastin mRNA stabilization.
Our previous results in mouse osteoclasts suggested that calcitonin (CT) alters CT receptor (CTR) mRNA stability. The CTR mRNA transcript contains several adenylate/uridylate (AU)-rich destabilizing elements in the 3' untranslated region (3'UTR). When the 3'UTR of mouse CTR mRNA was labeled by [alpha-(32)P]-uridine 5-triphosphate, interactions were observed between the transcript and several cell extracts, including those from the osteoclast progenitor monocyte/macrophage cell line, RAW 264.7. The molecular masses of the interacting proteins ranged from approximately 35 to 50 kDa, similar to AU-rich RNA-binding factor 1 (AUF1) and Hu antigen R (HuR). Radiolabeled 3'UTR transcripts bound with a 40-kDa protein, which could be extracted from cells transfected with AUF1 p40. To confirm the binding specificity, a pSG5 vector construct, containing the AUF1 p40 with an hemagglutinin tag, was transiently transfected into NIH3T3 cells. The extracts were incubated with poly(A)-added CTR3'UTR. The reaction mixture was immunoprecipitated using an antihemagglutinin antibody and precipitated mRNA species were extracted and reverse transcribed using oligo-dT primers. It was found that PCR primers specific for the 3'UTR of CTR mRNA sequence generated a PCR signal. No signal was observed when mutated AUF1 p40 was transfected. In a manner similar to the AUF1 binding, HuR was also found to bind to the 3'UTR. Specific binding of AUF1 p40 and HuR was also found with RNA extracted from mouse osteoclasts. Treatment of osteoclasts with CT did not significantly affect the expression of AUF1 but decreased the levels of HuR and its mRNA. The role of CTR3'UTR in mRNA stability was further tested by expressing luciferase reporter constructs that did, or did not, contain the CTR3'UTR, under the control of the tetracycline-regulatory system. The results showed that the addition of 3'UTR considerably shortened the mRNA half-life of the luciferase reporter gene. These results suggest that AUF1 p40, HuR, and the 3'UTR of the CTR mRNA transcript could be involved in posttranscriptional regulation of CTR mRNA expression.
TGF-beta is implicated in the pathogenesis of fibrotic disorders. It has been shown that Smad3 promotes the human alpha2(I) collagen (COL1A2) gene expression by TGF-beta1 in human dermal fibroblasts. Here, we investigated the role of phosphatidylinositol 3-kinase (PI3K) in the COL1A2 gene expression in normal and scleroderma fibroblasts. In normal fibroblasts, the PI3K inhibitor, LY294002, significantly decreased the basal and the TGF-beta1-induced increased stability of COL1A2 mRNA. The TGF-beta1-induced COL1A2 promoter activity, but not the basal activity, was significantly attenuated by LY294002 or the dominant negative mutant of p85 subunit of PI3K, while the constitutive active mutant of p110 subunit of PI3K did not affect the basal or the TGF-beta1-induced COL1A2 promoter activity. LY294002 significantly decreased the phosphorylation of Smad3 induced by TGF-beta1. Furthermore, the transient overexpression of 2xFYVE, which induces the mislocalization of FYVE domain proteins, decreased the TGF-beta1-induced Smad3 phosphorylation to a similar extent to LY294002. In scleroderma fibroblasts, the blockade of PI3K significantly decreased the mRNA stability and the promoter activity of the COL1A2 gene. Furthermore, LY294002 and the transient overexpression of 2xFYVE completely diminished the constitutive phosphorylation of Smad3. These results indicate that 1) the basal activity of PI3K is necessary for the COL1A2 mRNA stabilization in normal and scleroderma fibroblasts, 2) there is an unidentified FYVE domain protein specifically interacting with Smad3, and 3) the basal activity of PI3K and the FYVE domain protein are indispensable for the efficient TGF-beta/Smad3 signaling in normal fibroblasts and for the establishment of the constitutive activation of TGF-beta/Smad3 signaling in scleroderma fibroblasts.