QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wendling, J. Articles by Verrecchia, F. Search for Related Content PubMed PubMed Citation Articles by Wendling, J. Articles by Verrecchia, F.

5-Fluorouracil Blocks Transforming Growth Factor-–Induced ₂ Type I Collagen Gene (COL1A2) Expression in Human Fibroblasts via c-Jun NH₂-Terminal Kinase/Activator Protein-1 Activation

Institut National de la Santé et de la Recherche Médicale U532, Skin Research Institute, Saint-Louis Hospital, Paris, France

Received February 28, 2003; accepted May 21, 2003

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
5-Fluorouracil (5-FU), a pyrimidine analog widely used in cancer chemotherapy and in glaucoma surgery, has recently shown some efficacy in the treatment of keloids, scars that overgrow the boundaries of original wounds. Given the physiopathological importance of transforming growth factor- (TGF-) in keloid and scar formation, we have examined whether the clinical benefits from 5-FU treatment may result from its capacity to interfere with TGF- signaling and resulting activation of type I collagen gene expression. Using various molecular approaches to study the mechanisms underlying 5-FU effects, we have demonstrated that 5-FU antagonizes TGF-–driven COL1A2 transcription and associated type I collagen production by dermal fibroblasts. In addition, 5-FU inhibits both SMAD3/4-specific transcription and formation of SMAD/DNA complexes induced by TGF-. 5-FU induces c-Jun phosphorylation and activates both AP-1-specific transcription and DNA binding. Overexpression of an antisense c-jun expression vector, or that of a dominant-negative form of MKK4 that interferes with c-Jun N-terminal kinase (JNK) activation, blocks the inhibitory activity of 5-FU on TGF-–induced COL1A2 transcription. Furthermore, in a cellular context devoid of JNK activity (i.e., JNK^–/– fibroblasts), 5-FU inhibits neither formation of SMAD/DNA complexes nor SMAD-driven COL1A2 transcription in response to TGF-. Together, these results identify 5-FU as a potent inhibitor of TGF-/SMAD signaling, capable of blocking TGF-–induced, SMAD-driven up-regulation of COL1A2 gene expression in a JNK-dependent manner. We thus provide a molecular explanation to the observed clinical benefits of 5-FU in the treatment of keloids and hypertrophic scars.

The role of the transforming growth factor- (TGF-) as the main factor inducing collagen gene expression leading to tissue fibrosis has been suggested by the observation that 1) TGF- expression often parallels increased type I collagen gene expression in fibrotic lesions (Nakatsukasa et al., 1990; Peltonen et al., 1990; Broekelmann et al., 1991) and 2) TGF- is a potent activator of extracellular matrix gene expression both in vitro and in vivo (reviewed in Verrecchia and Mauviel, 2002).

The TGF-s signal via specific serine/threonine kinase transmembrane receptors (TRI and TRII) that phosphorylate cytoplasmic mediators of the SMAD family (Massagué and Chen, 2000; Massagué and Wotton, 2000). The receptor-associated SMADs (R-SMADs), such as SMAD2 and SMAD3, interact directly with and are phosphorylated by activated TRI. Upon phosphorylation, they form heteromeric complexes with SMAD4, a common mediator for all SMAD pathways. These R-SMAD/SMAD4 complexes are translocated into the nucleus, where they may function as transcription factors, directly or in association with other DNA binding factors such as Fast and Sp1 (reviewed in Massagué and Chen 2000; Massagué and Wotton, 2000). Recently, using a combined cDNA microarray/promoter transactivation approach, we identified several skin fibrillar collagen genes, COL1A1, COL1A2, COL3A1, and COL5A2, to be direct SMAD3 targets downstream of TGF- (Verrecchia et al., 2001a).

5-FU was first used for its antimetabolite activity. In this molecule, the hydrogen atom in position 5' of uracil is replaced by the similarly sized atom of fluoride, and the molecule was designed to occupy the active sites of enzyme targets, thereby blocking metabolism. In the early 1980s, 5-FU was investigated as an adjunct to glaucoma filtering surgery, a procedure in which inhibition of wound healing is desirable to achieve surgical success (Gressel et al., 1984; Skuta et al., 1987). More recently, local 5-FU application has shown some success in the treatment and prevention of hypertrophic scars and keloids (Fitzpatrick 1999; Uppal et al., 2001).

In this work, we investigated the effects of 5FU on TGF-–induced type I collagen gene expression in human fibroblasts. We identify c-Jun N-terminal kinase (JNK) activation and subsequent c-Jun phosphorylation in response to 5-FU as critical for the antagonism exerted by 5FU against TGF-–induced SMAD signaling and resulting COL1A2 transactivation.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cell Cultures. Human dermal fibroblasts were established by explanting neonatal foreskins. Wild-type (wt) and JNK₁^–/–-JNK₂^–/– (JNK^–/–) immortalized fibroblasts were derived, respectively, from wild-type and JNK₁^–/–-JNK₂^–/– mouse embryos in which simultaneous targeted disruption of the jnk1 and jnk2 genes had been performed (Sabapathy et al., 1999). All fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and antibiotics (100 units/ml penicillin, 50 µg/ml streptomycin-G, and 25 µg/ml Fungizone). TGF-1 and 5-FU were purchased from R&D Systems Inc. (Minneapolis, MN) and Sigma Chemical (St. Louis, MO), respectively. In all experiments, TGF- was used at a concentration of 10 ng/ml, which maximally induces SMAD3/4 signaling in fibroblasts (F. Verrecchia and A. Mauviel, unpublished results). Unless noted otherwise, 5-FU was used at a concentration of 10 µM.

Transient Cell Transfections and Reporter Assays. Transient cell transfections were performed with Fugene-6 according to the manufacturer's protocol (Roche Diagnostics, Indianapolis, IN). pRSV–-galactosidase was cotransfected in every experiment to monitor transfection efficiencies. CAT activity was measured using [¹⁴C]chloramphenicol as substrate followed by thin-layer chromatography and quantitation with a PhosphorImager (Amersham Biosciences, Uppsala, Sweden). Luciferase activity was determined with a commercial assay kit (Promega, Madison, WI).

Plasmid Constructs. 3500COL1A2/CAT (gift from Francesco Ramirez, Mt. Sinai School of Medicine, New York, NY), and pRSV/AS-c-jun have been described previously (Boast et al., 1990; Chung et al., 1996). pAP1-TA-lux (Mercury pathway profiling vector; BD Biosciences Clontech, Palo Alto, CA) was used to evaluate AP-1–driven transcription. Dominant-negative MKK4 expression vector was used to modulate JNK activity (Verrecchia et al., 2003). To track JNK activity, we used a commercial reporter assay based on the modification of the mammalian one-hybrid system, consisting of a reporter plasmid, Gal4-Lux, bearing five Gal4 binding sites driving luciferase expression, and a transactivator plasmid encoding a chimeric protein, Gal4BD-c-Jun, consisting of the DNA binding domain of Gal4 (Gal4BD) fused to the transactivation domain of c-Jun, the latter requiring phosphorylation by JNK to fully transactivate Gal4-lux (Stratagene, La Jolla, CA).

Northern Blotting. Total RNA was obtained using RNeasy (Qiagen GmbH, Hilden Germany) and analyzed by Northern hybridization with ³²P-labeled cDNA probes for COL1A2 and GAPDH as described previously (Verrecchia et al., 2002). Quantitations were performed with a Storm 840 PhosphorImager (Amersham Biosciences).

Western Blot Analyses. Total protein cell extract (50 µg) in Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride) were denatured by heating at 95°C for 3 min before resolution by SDS-polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to Hybond enhanced chemiluminescence nitrocellulose filters (Amersham Biosciences), immunoblotted with either rabbit anti-type I collagen (Southern Biotech, Birmingham, AL), anti-phospho-c-Jun (Upstate Biotechnologies, Lake Placid, NY), anti-c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-actin (Sigma) antibodies, all at a dilution of 1:1000 in 1x phosphate-buffered saline/5% nonfat milk for 1 h. After incubation, filters were washed and incubated with a horseradish peroxidase-conjugated goat-anti-rabbit anti-mouse secondary antibody (Santa Cruz Biotechnology) for 1 h. Filters were then washed, developed according to chemiluminescence protocols (ECL, Amersham Biosciences), and revealed with a PhosphorImager.

Electrophoresis Mobility Shift Assays. A 3xCAGA SMAD3/4-specific oligonucleotide (Dennler et al., 1998) and a consensus AP-1 binding oligonucleotide (Promega Corp., Madison, WI) were used as probes. For supershift experiments, nuclear extracts (5–7 µg) were incubated overnight at 4°C with antisera before the binding reaction and separated electrophoretically on native 4% acrylamide gels.

	Results

Top Abstract Materials and Methods Results Discussion References

 
5-FU Does Not Affect Cell Viability at 24 and 48 h at a Concentration of 10 µM. Because high concentrations of 5-FU are cytotoxic, we first had to determine which concentrations of the drug would not induce significant cell death. This was particularly important because cellular toxicity may interfere significantly with cellular signaling outcomes and resulting gene expression. For this purpose, human dermal fibroblasts were incubated for 24 to 72 h with 5-FU concentrations ranging from 5 to 100 µM, at which point a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay was performed. As shown in Fig. 1, no significant cell mortality was observed at 24 and 48 h with concentrations of 5 and 10 µM. At 72 h, however, cytotoxicity was more than 30% for concentrations of 5-FU above 10 µM. We thus performed all subsequent experiments with 5-FU at a concentration of 10 µM, which exhibited no detectable cytotoxicity at either the 24- or 48-h time-point and induced less than 20% cell mortality at 72 h.

View larger version (19K): [in this window] [in a new window]   Fig. 1. 5-FU does not affect cell viability at 24 and 48 h with concentration of 10 µM. Succinate dehydrogenase activity was determined according to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay protocol (Sigma). The cells were incubated for 24 (), 48 (), and 72 h () with 5-FU concentrations ranging from 5 to 100 µM. Symbols indicate mean ± S.D. of three independent experiments performed, each with triplicate samples.

5-FU Prevents TGF-–Induced COL1A2 Gene Transactivation in Human Fibroblasts. We first wanted to determine whether 5-FU antagonized TGF-–driven COL1A2 promoter transactivation in human dermal fibroblasts. As shown in Fig. 2A, and as expected from the literature, TGF- enhanced –3500COL1A2/CAT activity 4- to 5-fold above control levels. Addition of 5-FU had a negligible effect on basal COL1A2 promoter but completely abolished TGF- response. To determine the physiological relevance of such findings obtained with promoter reporter constructs in transient cell transfection experiments, we next examined whether endogenous COL1A2 expression followed the same pattern of regulation. As a first approach to address this point, the effect of 5-FU on TGF-–induced type I collagen gene expression was examined, taking the COL1A2 mRNA steady-state levels as an endpoint. As shown in Fig. 2B, Northern analysis of total RNA with a specific COL1A2 probe revealed an expected potent up-regulation by TGF- (lane 2 versus lane 1, top), that was completely prevented by 5-FU (lane 4 versus lane 2), whereas 5-FU alone did not modify basal COL1A2 expression (lane 3 versus lane 1). GAPDH mRNA steady-state levels were unaffected by treatments (Fig. 2B, bottom), attesting to the specificity of COL1A2 regulation.

View larger version (44K): [in this window] [in a new window]   Fig. 2. 5-FU inhibits TGF-–induced COL1A2 gene transactivation. A, subconfluent fibroblasts, placed in medium supplemented with 1% FCS, were transfected with –3500COL1A2/CAT. Three hours later, 5-FU (10 µM) was added, followed by TGF- (10 ng/ml) 1 h later, and incubations continued for 24 h before CAT assays. Bars indicate mean ± S.D. of at least three independent experiments performed, each with duplicate samples. B, subconfluent fibroblast cultures were treated with TGF- (10 ng/ml) and 5-FU (10 µM) for 24 h in medium containing 1% serum. After incubations, COL1A2 mRNA levels was detected by Northern blot analysis (top). Specificity of the modulation was confirmed using a specific GAPDH probe (bottom). C, 24 h after treatment of fibroblasts with TGF- (10 ng/ml) and 5-FU (10 µM) in medium containing 1% serum. Type I collagen production was detected by Western blot analysis of whole cell lysates (top). Specificity of the modulation was confirmed with an anti-actin antibody (bottom).

The modulation of TGF-–induced elevation of COL1A2 mRNA steady-state levels by 5-FU translated into altered type I collagen production, as determined by Western analysis of whole-cell lysates with an anti-type I collagen antibody (Fig. 2C). Specifically, elevated type I collagen production in response to TGF- (top, lane 2 versus lane 1) was prevented by addition of 5-FU (lane 4 versus lane 2). Actin levels (bottom) remained unchanged throughout the experiments. Together, these results provide evidence that 5-FU antagonizes TGF- induced type I collagen production, and this effect of 5-FU occurs at the level of COL1A2 transcription.

5-FU Reduces SMAD-DNA Interactions. The SMAD pathway is critical for activation of the human COL1A2 promoter by TGF- (Chen et al., 1999; Zhang et al., 2000; Verrecchia et al., 2001a). Therefore, one possibility to explain the inhibitory activity of 5-FU against TGF- would be that it interferes with the SMAD pathway. To test this hypothesis, two approaches were chosen. Firstly, we tested the capacity of 5-FU to alter SMAD3/4-specific transcription in a transient cell transfection assay with the SMAD3/4-specific reporter construct (CAGA)₉-lux (Dennler et al., 1998). As shown in Fig. 3A, TGF- up-regulated (CAGA)₉-lux activity about 12-fold above control values. 5-FU added 1 h before TGF- completely abolished such transactivation. To determine whether the blockade of SMAD3/4-dependent gene transcription by 5-FU was caused by a capacity of the latter to interfere with SMAD/DNA complex formation, EMSA experiments were carried out using nuclear extracts from fibroblasts treated with TGF- for 30 min, a time point at which maximal amounts of SMAD/DNA complexes are formed in response to TGF- (Vindevoghel et al., 1998a,b), pretreated or not with 5-FU for 60 min. As shown in Fig. 3B, SMAD/DNA complex formation induced by TGF- (lane 2 versus lane 1) was efficiently prevented when 5-FU was added 60 min before TGF- addition (lane 4 versus lane 2). Therefore, a good correlation exists between the inhibitory effect of 5-FU on TGF-/SMAD-dependent COL1A2 gene transactivation, described in Fig. 2, and on the formation of SMAD/DNA complexes.

View larger version (28K): [in this window] [in a new window]   Fig. 3. 5-FU inhibits SMAD3/4-dependent gene transcription and reduces SMAD-DNA interactions. A, subconfluent fibroblasts, placed in medium supplemented with 1% FCS, were transfected with (CAGA)9-lux. Three hours later, 5-FU (10 µM) was added, followed by TGF- (10 ng/ml) 1 h later, and incubations continued for 24 h before luciferase assays. Bars indicate mean ± S.D. of at least three independent experiments performed, each with duplicate samples. B, EMSAs were performed using the SMAD3/4-specific 3x CAGA oligonucleotide as a probe, together with nuclear extracts from fibroblast cultures treated for 30 min with TGF- (10 ng/ml) pretreated or not with 5-FU (10 µM, 60 min).

5-FU Stimulates the cJun/AP-1 Signaling Pathway. We previously reported the ability of c-Jun to block TGF--induced COL1A2 promoter transactivation and its critical role in mediating the antagonistic effects of TNF- on SMAD-dependent gene transactivation induced by TGF- (Chung et al., 1996; Verrecchia et al., 2000, 2003). Furthermore, it has been shown that 5-FU stimulates JNK activity and subsequent c-Jun transcriptional activity (Eichhorst et al., 2000). We therefore investigated whether activation of the cJun/AP-1 signaling pathway by 5-FU may account for its antagonistic activity against TGF- signaling and resulting collagen gene modulation. First, the AP-1–specific reporter construct pAP1-TA-lux was used in transient cell transfection experiments to determine the effects of 5-FU on AP-1–dependent transcription. As shown in Fig. 4A, 5-FU (10 µM) enhanced pAP-1-TA-lux activity approximately 3.8-fold above control levels. Second, EMSAs were carried out with a radiolabeled consensus AP-1 oligonucleotide as a probe to determine whether 5-FU has the ability to induce AP-1/DNA complexes. As shown in Fig. 4B, a strong retarded band was detected (lane 2 versus lane 1) with nuclear extracts from fibroblasts treated with 5-FU for 1 h, which was supershifted with an anti-c-Jun antibody (lane 4). Together, these results demonstrate that 5-FU stimulates the cJun/AP-1 signaling pathway. To determine whether c-Jun is directly responsible for the antagonistic activities exerted by 5-FU against TGF-/SMAD signaling, we examined the effect of 5-FU in the presence of an antisense c-jun expression vector shown previously to efficiently antagonize TNF-–induced c-jun expression (Mauviel et al., 1996). As shown in Fig. 4C, overexpression of antisense c-jun prevented the inhibitory effect of 5-FU on TGF-–driven COL1A2 transactivation in human fibroblasts. These experiments provide evidence that c-Jun plays a critical role in mediating the inhibitory effect of 5-FU on TGF-/SMAD signaling and subsequent up-regulation of COL1A2 gene expression by TGF-.

View larger version (41K): [in this window] [in a new window]   Fig. 4. 5-FU stimulates the cJun/AP-1 signaling pathway. A, cells were transfected with pAP-1-Lux in presence or absence of 5-FU (10 µM, 24 h). B, EMSA was performed using the AP-1–specific oligonucleotide as a probe, together with nuclear extracts from control (lane 1, 3) and 5-FU–treated (10 µM, 1 h, lanes 2 and 4) fibroblast cultures. Supershift assay was carried out with an anti c-Jun antibody (lanes 3 and 4). C, subconfluent human fibroblasts were transfected with –3500COL1A2/CAT construct in presence or absence of an antisense c-Jun expression vector. Three hours after transfections, 5-FU (10 µM) was added, followed by TGF- (10 ng/ml) 1 h later. CAT assays were determined 24 h later. Bars indicate mean ± S.D. of at least three independent experiments performed, each with duplicate samples.

A Role for JNK in 5-FU–Driven Inhibition of SMAD-Dependent COL1A2 Gene Transcription Downstream of TGF-. We recently demonstrated that JNK-mediated phosphorylation of c-Jun is critical for c-Jun to antagonize TGF-/SMAD signaling and subsequent collagen gene transcription (Verrecchia et al., 2002, 2003). We therefore examined the capacity of 5-FU to induce c-Jun phosphorylation using a reporter assay based on the mammalian one-hybrid system (see Materials and Methods). As shown in Fig. 5A, 5-FU efficiently transactivated the Gal4-lux construct in the presence of Gal4BD-c-Jun, suggesting that 5-FU activates JNK and subsequent c-Jun phosphorylation. The ability of 5FU to activate c-Jun phosphorylation was further confirmed by Western blot analysis of phospho-c-Jun content in response to 5-FU. As shown in Fig. 5B, 5-FU stimulates c-Jun phosphorylation (lanes 2 versus lanes 1) to levels comparable with those achieved by TNF- (lane 3).

View larger version (26K): [in this window] [in a new window]   Fig. 5. 5-FU stimulates the AP-1 signaling pathway. A, cells were cotransfected with Gal4-Lux and a transactivator plasmid encoding a chimeric transactivator protein, Gal4BD-c-Jun, in the presence or absence of 5-FU (10 µM). Luciferase activity was determined 24 h later. Bars indicate mean ± S.D. of at least three independent experiments performed, each with duplicate samples. B, Western blot analysis of total protein extracts from fibroblasts treated with 5-FU (10 µM, lane 2) or TNF- (10 ng/ml, lane 3) for 1 h, using anti phospho-c-Jun and anti-c-Jun antibodies.

Second, transient transfection experiments using human dermal fibroblasts showed that expression of a dominant-negative mutant form of MKK4, blocking JNK activation, prevented in part the antagonistic effect of 5-FU against TGF-–driven COL1A2 transactivation (Fig. 6A).

View larger version (56K): [in this window] [in a new window]   Fig. 6. Critical role for JNK/cJun in 5-FU-driven inhibition of SMAD-dependent COL1A2 gene transcription downstream of TGF-. A, subconfluent human dermal fibroblasts were transfected with –3500COL1A2/CAT construct in presence or absence of a dominant-negative mutant form of mitogen-activated protein kinase kinase 4 (D/N MKK4). Three hours after transfections, 5-FU (10 µM) was added followed by TGF- (10 ng/ml) 1 h later. CAT assays was determined 24 h later. Top, autoradiogram of a representative experiment. Bottom, bars indicate mean ± S.D. of at least three independent experiments performed, each with duplicate samples. B, subconfluent wild-type and JNK–/– fibroblast cultures were transfected with –3500COL1A2/CAT construct. Three hours after transfections, 5-FU (10 µM) was added followed by TGF- (10 ng/ml) 1 h later. CAT assays were determined 24 h later. Top, autoradiogram of a representative experiment. Bottom, bars indicate mean ± S.D. of at least three independent experiments, each performed with duplicate samples.

Third, we examined the effect of 5-FU on TGF-–driven COL1A2 transactivation in fibroblasts devoid of JNK activity, namely double JNK1-JNK2 knock-out fibroblasts (JNK^–/– fibroblasts). As shown in Fig. 6B, 5-FU inhibitory activity against TGF-–induced COL1A2 promoter activity was readily observed in wild-type fibroblasts but was completely absent in JNK^–/– fibroblasts providing definitive evidence for a crucial role of JNK in mediating 5-FU inhibitory effect against TGF-–induced type I collagen gene expression.

Next, EMSA experiments were carried out using nuclear extracts from wt and JNK^–/– fibroblasts treated with TGF- (30 min), preteated or not with 5-FU for 60 min. As shown in Fig. 7, SMAD/DNA complex formation was equally induced by TGF- in both wt and JNK^–/– fibroblasts (lanes 2 and 6). 5-FU efficiently reduced TGF-–induced SMAD/DNA complexes in wt (lane 4 versus lane 2); however, no effect of 5-FU on SMAD/DNA complex formation could be observed in JNK^–/– fibroblasts. Thus, it seems that a good correlation exists between the role of JNK on the inhibitory effect of 5-FU on SMAD-dependent COL1A2 gene transactivation, described in Fig. 6, and on the formation of SMAD/DNA complexes.

View larger version (74K): [in this window] [in a new window]   Fig. 7. Critical role for JNK in mediating 5-FU–driven inhibition of SMAD-DNA interactions. EMSAs were performed using the 3x CAGA oligonucleotide as a probe, together with nuclear extracts from wt and JNK–/– fibroblast cultures treated for 30 min with TGF-, pretreated or not with 5-FU (10 µM, 60 min).

Dose-Response Relationship of Effects of 5-FU. To ascertain the relationship between JNK activation and repression of TGF-–induced COL1A2 gene expression by 5-FU, we next performed a 5-FU dose-response experiment, in which collagen expression, JNK activation, and c-Jun phosphorylation were measured in parallel. As shown in Fig. 8, 5-FU at a concentration of 0.5 µM neither antagonized TGF-–induced COL1A2 gene expression (A, lane 3) nor activated JNK (as measured using a Gal4-c-Jun–based mammalian one-hybrid system) (B, lane 2) or c-Jun phosphorylation (C, lane 2). At 2.5 µM, 5-FU had little effect on COL1A2 expression, JNK activity. and c-Jun phosphorylation (Fig. 8, A, lane 4, and B and C, lanes 3.). Maximal JNK activity and c-Jun phosphorylation levels were observed at 5-FU concentrations of 5 and 10 µM, resulting in significant down-regulation of TGF-–induced COL1A2 gene expression (lanes 4 and 5).

View larger version (33K): [in this window] [in a new window]   Fig. 8. Dose-response relationship of effects of 5-FU on type I collagen gene expression, JNK activation, and c-Jun phosphorylation. A, subconfluent fibroblast cultures were treated with TGF- (10 ng/ml) and various concentrations of 5-FU (0.5, 2.5, 5, and 10 µM) for 24 h in medium containing 1% serum. After incubations, COL1A2 mRNA levels were determined by Northern blot analysis (top). Specificity of the modulation was confirmed using a specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (bottom). B, cells were cotransfected with Gal4-Lux, and a transactivator plasmid encoding a chimeric transactivator protein, Gal4BD-c-Jun, in the presence or absence of various concentration of 5-FU (0.5, 2.5, 5, and 10 µM). Luciferase activity, representative of JNK activity, was determined 24 h later. Bars indicate mean ± S.D. of at least three independent experiments performed, each with duplicate samples. C, Western blot analysis of total protein extracts from fibroblasts treated with 5-FU (lane 2, 0.5 µM; lane 3, 2.5 µM; lane 4, 5 µM; and lane 5, 10 µM) for 1 h, using anti phospho-c-Jun (top) and anti-c-Jun (bottom) antibodies.

Together, these results demonstrate a direct correlation between the capacity of 5-FU to activate JNK at concentrations above 2.5 µM and the antagonistic activity of 5-FU against TGF-–induced COL1A2 expression.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, in accordance with a previous work (Hendricks et al., 1993), human skin fibroblasts grown to confluence were exposed for 24 h to 5-FU at pharmacologically relevant drug concentrations. This treatment did not affect basal type I collagen synthesis, whereas TGF-–induced type I collagen synthesis was inhibited. We specifically demonstrated that 5-FU inhibits TGF--SMAD-driven COL1A2 transactivation, providing a molecular basis toward the understanding of the molecular mechanisms underlying the antagonistic activities of 5-FU with TGF-/SMAD.

SMAD signaling may be blocked by distinct mechanisms: 1) the inhibitory SMADs (SMAD6 or SMAD7) prevent R-SMAD phosphorylation and subsequent nuclear translocation of R-SMAD/SMAD4 heterocomplexes and 2) corepressors such as the viral oncoprotein E1A (Nishihara et al., 1999), the proto-oncogenes c-Ski and SnoN (Akiyoshi et al., 1999), TGIF (Wotton et al., 1999), Snip-1 (Kim et al., 2000), and SIP1 (Verschueren et al., 1999), which compete for R-SMAD/SMAD4 binding to CBP or p300. On the other hand, induction of Jun proteins by cytokines have been shown to directly interfere with the SMAD pathway either by preventing SMAD3 binding to cognate DNA sequences or by sequestering the transcriptional coactivator p300 (Dennler et al., 2000; Verrecchia et al., 2000, 2001b,c).

In accordance with a recent work showing that an AP-1–cis-element in the CD65L promoter are required for CD95L up-regulation by 5-FU (Eichhorst et al., 2000), we provide evidence that 5-FU stimulates c-Jun phosphorylation in dermal fibroblasts. In agreement with the original observation that c-Jun overexpression blocks TGF-–induced COL1A2 transactivation (Chung et al., 1996), we established that JNK/c-Jun play a central role in allowing the antagonistic activity of 5-FU against TGF-–induced type I collagen gene expression. The data establish a link between previous reports about the interference of c-Jun and JNK with SMAD signaling (Dennler et al., 2000) and the inhibition of TGF-–induced type I collagen gene expression by TNF- (Verrecchia et al., 2000, 2002). Specifically, one of the proposed mechanisms by which Jun proteins interfere with the SMAD pathway involves direct SMAD-Jun interaction, not compatible with SMAD/DNA complex formation (Verrecchia et al., 2001b,c). Also, overexpression of constituvely active forms of either MEKK1 or MKK4, kinases involved in JNK activation, enhance SMAD-Jun associations and repress SMAD-dependent transcription (Dennler et al., 2000).

Several different mechanisms could explain the excessive deposition of collagen in the fibrotic skin diseases. First, local expansion of synthetically active fibroblast populations, even with a normal rate of collagen production per cell, could lead to tissue accumulation of collagen. Second, the accumulation of collagen could result from accelerated production of collagen by fibroblasts, and such increase could reflect enhanced collagen gene expression at the transcriptional level (Uitto and Kouba, 2000). In this work, we established that 5-FU effects on type I collagen expression and subsequent collagen deposition could result from inhibition of TGF-–driven type I collagen transcription.

The basis of keloid formation is not fully understood. Some authors reported that keloid-derived fibroblasts produce increased amounts of collagen per cell compared with normal fibroblasts in culture (English and Shenefelt, 1999). They seem to function autonomously and demonstrate continued collagen synthesis in vitro. These fibroblasts and the collagen produced by them have been the target of antikeloid therapies (Low and Moy, 1992). Our work provides a molecular explanation for the observed clinical benefits of 5FU in the treatment of keloids.

	Acknowledgements

 
Drs. A. Atfi (Paris, France), S. Dennler, and J.-M. Gauthier (Glaxo-Wellcome, Les Ulis, France), provided us with cellular and molecular reagents essential for these studies. Their generosity is gratefully acknowledged.

	Footnotes

 
This work was supported by Institut National de la Santé et de la Recherche Médicale and by grants from the Association pour la Recherche contre le Cancer and Electricité de France (Service de Radioprotection) (to A.M.).

ABBREVIATIONS: TGF-, transforming growth factor-; R-SMAD, receptor-associated SMAD; 5-FU, 5-fluorouracil; JNK, c-Jun N-terminal kinase; wt, wild type; CAT, chloramphenicol acetyltransferase; TNF-, tumor necrosis factor-; EMSA, electrophoretic mobility shift assay.

Address correspondence to: Dr. Franck Verrecchia, INSERM U532, Institut de Recherche sur la Peau, Pavillon Bazin, Hôpital Saint-Louis, 1 avenue Claude Vellefaux, 75475 Paris cedex 10, France. E-mail: verrecchia{at}chustlouis.fr

	References

Top Abstract Materials and Methods Results Discussion References

 
Akiyoshi S, Inoue H, Hanai J, Kusanagi K, Nemoto N, Miyazono K, and Kawabata M (1999) c-Ski acts as a transcriptional co-repressor in transforming growth factor- signaling through interaction with SMADs. J Biol Chem 274: 35269–35277.[Abstract/Free Full Text]

Broekelmann TJ, Limper AH, Colby TV, and McDonald JA (1991) Transforming growth factor beta 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc Natl Acad Sci USA 88: 6642–6646.[Abstract/Free Full Text]

Boast S, Su MW, Ramirez F, Sanchez M, and Avvedimento EV (1990) Functional analysis of cis-acting DNA sequences controlling transcription of the human type I collagen genes. J Biol Chem 265: 13351–13356.[Abstract/Free Full Text]

Chen SJ, Yuan W, Mori Y, Levenson A, Trojanowska M and Varga J (1999) Stimulation of type I collagen transcription in human skin fibroblasts by TGF-beta: involvement of SMAD3. J Investig Dermatol 112: 49–57.[CrossRef][Medline]

Chung KY, Agarwal A, Uitto J, and Mauviel A (1996) An AP-1 binding sequence is essential for regulation of the human 2_I collagen (COL1A2) promoter activity by transforming growth factor-. J Biol Chem 271: 3272–3278.[Abstract/Free Full Text]

Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S and Gauthier JM (1998) Direct binding of SMAD3 and SMAD4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO (Eur Mol Biol Organ) J 17: 3091–3100.[CrossRef][Medline]

Dennler S, Prunier C, Ferrand N, Gauthier JM, and Atfi A (2000) c-Jun inhibits transforming growth factor -mediated transcription by repressing SMAD3 transcriptional activity. J Biol Chem 275: 28858–28865.[Abstract/Free Full Text]

Ehrlich HP, Desmouliere A, Diegelmann RF, Cohen IK, Compton CC, Garner WL, Kapanci Y, and Gabbiani G (1994) Morphological and immunochemical differences between keloid and hypertrophic scar. Am J Pathol 145: 105–113.[Abstract]

Eichhorst ST, Muller M, Li-Weber M, Schulze-Bergkamen H, Angel P, and Krammer PH (2000) A novel AP-1 element in the CD95 ligand promoter is required for induction of apoptosis in hepatocellular carcinoma cells upon treatment with anticancer drugs. Mol Cell Biol 20: 7826–7837.[Abstract/Free Full Text]

English RS and Shenefelt PD (1999) Keloids and hypertrophic scars. Dermatol Surg 25: 631–638.[CrossRef][Medline]

Fitzpatrick RE (1999) Treatment of inflamed hypertrophic scars using intralesional 5-FU. Dermatol Surg 25: 224–232.[CrossRef][Medline]

Gressel MG, Parrish RK, and Folberg R (1984) 5-fluorouracil and glaucoma filtering surgery: I. An animal model. Ophthalmology 91: 378–383.[Medline]

Hendricks T, Martens MF, Huyben CM, and Wobbes T (1993) Inhibition of basal and TGF beta-induced fibroblast collagen synthesis by antineoplastic agents. Implications for wound healing. Br J Cancer 67: 545–550.[Medline]

Jimenez SA and Saitta B (1999) Alterations in the regulation of expression of the alpha 1(I) collagen gene (COL1A1) in systemic sclerosis (scleroderma). Springer Semin Immunopathol 21: 397–414.[Medline]

Kim RH, Wang D, Tsang M, Martin J, Huff C, de Caestecker MP, Parks WT, Meng X, Lechleider RJ, Wang T, et al. (2000) A novel SMAD nuclear interacting protein, SNIP1, suppresses p300-dependent TGF-beta signal transduction. Genes Dev 14: 1605–1616.[Abstract/Free Full Text]

Low SQ and Moy RL (1992) Scar wars strategies. Target collagen. J Dermatol Surg Oncol 18: 981–986.[Medline]

Nakatsukasa H, Nagy P, Evarts RP, Hsia CC, Marsden E, and Thorgeirsson SS (1990) Cellular distribution of transforming growth factor-beta 1 and procollagen types I, III and IV transcripts in carbon tetrachloride-induced rat liver fibrosis. J Clin Investig 85: 1833–1843.

Nishihara A, Hanai J, Imamura T, Miyazono K, and Kawabata M (1999) E1A inhibits transforming growth factor- signaling through binding to SMAD proteins. J Biol Chem. 274: 28716–28723.[Abstract/Free Full Text]

Mauviel A, Chung KY, Agarwal A, Tamai K, and Uitto J (1996) Cell-specific induction of distinct oncogenes of the Jun family is responsible for differential regulation of collagenase gene expression by transforming growth factor- in fibroblasts and keratinocytes. J Biol Chem 271: 10917–10923.[Abstract/Free Full Text]

Massagué J and Chen YG (2000) Controlling TGF-beta signaling. Genes Dev 14: 627–644.[Free Full Text]

Massagué J and Wotton D (2000) Transcriptional control by the TGF-beta/SMAD signaling system. EMBO (Eur Mol Biol Organ) J 19: 1745–1754.[CrossRef][Medline]

Peltonen J, Kähäri L, Jaakkola S, Kähäri VM, Varga J, Uitto J, and Jimenez SA (1990) Evaluation of transforming growth factor beta and type I procollagen gene expression in fibrotic skin diseases by in situ hybridization. J Investig Dermatol 94: 365–371.[CrossRef][Medline]

Sabapathy K, Jochum W, Hochedlinger K, Chang L, Karin M, and Wagner EF (1999) Defective neural tube morphogenesis and altered apoptosis in the absence of both JNK1 and JNK2. Mech Dev 89: 115–124.[CrossRef][Medline]

Skuta GL, Assil K, Parrish RK, Folberg R, and Weinreb RN (1987) Filtering surgery in owl monkeys treated with the antimetabolite 5-fluorouridine 5'-monophosphate entrapped in multivesicular liposomes. Am J Ophthalmol 103: 714–716.[Medline]

Uitto J and Kouba D (2000) Cytokine modulation of extracellular matrix gene expression: relevance to fibrotic skin diseases. J Dermatol Sci 24: S60–S69.

Uppal RS, Khan U, Kakar S, Talas G, Chapman P, and McGrouther AD (2001) The effects of a single dose of 5-fluorouracil on keloid scars: a clinical trial of timed wound irrigation after extralesional excision. Plast Reconstr Surg 108: 1218–1224.[CrossRef][Medline]

Verrecchia F, Chu ML, and Mauviel A (2001a) Identification of novel TGF-/SMAD gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach. J Biol Chem 276: 17058–17062.[Abstract/Free Full Text]

Verrecchia F and Mauviel A (2002) Transforming growth factor-beta signaling through the Smad pathway: role in extracellular matrix gene expression and regulation. J Investig Dermatol 118: 211–215.[CrossRef][Medline]

Verrecchia F, Pessah M, Atfi A, and Mauviel A (2000) Tumor necrosis factor- inhibits transforming growth factor-/SMAD signaling in human dermal fibroblasts via AP-1 activation. J Biol Chem 275: 30226–30231.[Abstract/Free Full Text]

Verrecchia F, Tacheau C, Schorpp-Kistner M, Angel P, and Mauviel A (2001b) Induction of the AP-1 members c-Jun and JunB by TGF-beta/SMAD suppresses early SMAD-driven gene activation. Oncogene 20: 2205–2211.[CrossRef][Medline]

Verrecchia F, Tacheau C, Wagner EF and Mauviel A (2003) A central role for the Jun-N-terminal kinase pathway in mediating the antagonistic activity of pro-inflammatory cytokines against transforming growth factor--driven SMAD3/4 specific gene expression. J Biol Chem 278: 1585–1593.[Abstract/Free Full Text]

Verrecchia F, Vindevoghel L, Lechleider RJ, Uitto J, Roberts AB, and Mauviel A (2001c) SMAD3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner. Oncogene 20: 3332–3340.[CrossRef][Medline]

Verrecchia F, Wagner EF, and Mauviel A (2002) Distinct involvement of the Jun-N-terminal kinase and NFkB pathways in the repression of the human COL1A2 gene by TNF-alpha. EMBO (Eur Mol Biol Organ) Rep 3: 1069–1074.[CrossRef][Medline]

Verschueren K, Remacle JE, Collart C, Kraft H, Baker BS, Tylzanowski P, Nelles L, Wuytens G, Su MT, Bodmer R, et al. (1999) SIP1, a novel zinc finger/homeodomain repressor, interacts with SMAD proteins and binds to 5'-CACCT sequences in candidate target genes. J Biol Chem 274: 20489–20498.[Abstract/Free Full Text]

Vindevoghel L, Kon A, Lechleider RJ, Uitto J, Roberts AB, and Mauviel A (1998a) Smad-dependent transcriptional activation of human type VII collagen gene (COL7A1) promoter by transforming growth factor-. J Biol Chem 273: 13053–13057.[Abstract/Free Full Text]

Vindevoghel L, Lechleider RJ, Kon A, de Caestecker MP, Uitto J, Roberts AB, and Mauviel A (1998b). SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta. Proc Natl Acad Sci USA 95: 14769–14774.[Abstract/Free Full Text]

Wotton D, Lo RS, Lee S, and Massague J (1999) A SMAD transcriptional corepressor. Cell 97: 29–39.[CrossRef][Medline]

Zhang W, Ou J, Inagaki Y, Greenwel P, and Ramirez F (2000) Synergistic cooperation between Sp1 and SMAD3/SMAD4 mediates transforming growth factor beta1 stimulation of 2_I-collagen (COL1A2) transcription. J Biol Chem 275: 39237–39245.[Abstract/Free Full Text]

This article has been cited by other articles:

				X.-Q. Wang, Y.-K. Liu, Z.-Y. Wang, Wei Jun, Y.-z. Jiang, Qing Chun, and S.-L. Lu Antimitotic Drug Injections and Radiotherapy: A Review of the Effectiveness of Treatment for Hypertrophic Scars and Keloids International Journal of Lower Extremity Wounds, September 1, 2008; 7(3): 151 - 159. [Abstract] [PDF]

				B. Wang, A. Omar, T. Angelovska, V. Drobic, S. G. Rattan, S. C. Jones, and I. M. C. Dixon Regulation of collagen synthesis by inhibitory Smad7 in cardiac myofibroblasts Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H1282 - H1290. [Abstract] [Full Text] [PDF]

				N. Poulalhon, D. Farge, N. Roos, C. Tacheau, C. Neuzillet, L. Michel, A. Mauviel, and F. Verrecchia Modulation of Collagen and MMP-1 Gene Expression in Fibroblasts by the Immunosuppressive Drug Rapamycin: A DIRECT ROLE AS AN ANTIFIBROTIC AGENT? J. Biol. Chem., November 3, 2006; 281(44): 33045 - 33052. [Abstract] [Full Text] [PDF]

				C.-K. Oh, H.-S. Son, Y.-W. Kwon, H.-S. Jang, and K.-S. Kwon Intralesional Fluorouracil Injection in Infantile Digital Fibromatosis Arch Dermatol, May 1, 2005; 141(5): 549 - 550. [Full Text] [PDF]

				S. Wang and R. Hirschberg Bone Morphogenetic Protein-7 Signals Opposing Transforming Growth Factor {beta} in Mesangial Cells J. Biol. Chem., May 28, 2004; 279(22): 23200 - 23206. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wendling, J. Articles by Verrecchia, F. Search for Related Content PubMed PubMed Citation Articles by Wendling, J. Articles by Verrecchia, F.