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1-Induced Extracellular Matrix Expression
Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan (M.J., K.T.); and Department of Dermatology and Plastic and Reconstructive Surgery, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan (H.I.)
Received July 30, 2005; accepted November 15, 2005
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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1-induced phosphorylation of Smad3 and interaction of Smad3 with Smad4. On the other hand, this reagent did not affect the phosphorylation of Smad2. Thereafter, we evaluated the ability of SIS3 in the suppression of the TGF-1-induced type I procollagen up-regulation in human dermal fibroblasts. We found that the addition of SIS3 attenuated the effects of TGF-1 by reducing the transcriptional activity. SIS3 also inhibited the myofibroblast differentiation of fibroblasts by TGF-1. Moreover, we demonstrated that SIS3 completely diminished the constitutive phosphorylation of Smad3 as well as the up-regulated type I collagen expression in scleroderma fibroblasts. Together, our study suggested that SIS3 is a useful tool to evaluate the TGF--regulated cellular mechanisms via selective inhibition of Smad3.
1 plays a critical role in a variety of biological processes, including proliferation, differentiation, extracellular matrix production, and apoptosis. The diverse cellular responses elicited by TGF-1 are triggered by the activation of serine/threonine kinase TGF- receptors. On activation by TGF-1 or related ligands, signaling from the receptors to the nucleus is mediated by phosphorylation of cytoplasmic mediators called Smads. The receptor-associated Smads, such as Smad2 and Smad3, interact directly with, and are phosphorylated by, activated TGF- receptor type I (Nakao et al., 1997
). They are ligand-specific and form, on phosphorylation, heteromeric complexes with Smad4. The latter functions as a common mediator for all Smad pathways. These complexes then are translocated into the nucleus, where they function as transcription factors, possibly in association with other proteins, such as Sp1. The third group of Smad proteins, the inhibitory Smads such as Smad6 or Smad7, prevents phosphorylation and/or nuclear translocation of receptor-associated Smads.
TGF-1 has been implicated in the development of fibrotic condition, including skin, lung, or liver. Systemic sclerosis or scleroderma is an acquired disorder that typically results in fibrosis of the skin and internal organs. Fibroblasts from affected scleroderma skin cultured in vitro produce excessive amounts of extracellular matrix (ECM), various collagens, mainly type I and III collagens, and display increased transcription of corresponding genes (Hitraya and Jimenez, 1996). Many of the characteristics of scleroderma fibroblasts resemble those of normal fibroblasts stimulated by TGF-1 (LeRoy et al., 1989), suggesting that activation of dermal fibroblast in scleroderma may be a result of stimulation by autocrine TGF- signaling. This notion is supported by our recent findings: 1) scleroderma fibroblasts express elevated levels of TGF- receptors, and this correlates with the elevated levels of 
2(I) collagen mRNA (Kawakami et al., 1998); 2) the blockade of TGF- signaling with anti-TGF- antibodies or anti-TGF-1 antisense oligonucleotides abolished the increased expression of human 2(I) collagen mRNA in scleroderma fibroblasts (Ihn et al., 2001b); and 3) Smad3 was constitutively phosphorylated in scleroderma fibroblasts (Asano et al., 2004). Thus, the blockade of autocrine TGF- signaling is thought to be one of the most reliable approaches in the treatment of scleroderma, and there have been several reports that actually show that the blockade of autocrine TGF- signaling can decrease collagen expression in vivo or in vitro (Yamamoto et al., 1999; Yamane et al., 2003a).
Several investigators have reported possible inhibitors of TGF- signaling (Callahan et al., 2002; Kondo et al., 2004). A missense mutant of Smad2, Smad2D450E, that was not phosphorylated by TGF- signaling suppressed the phosphorylation of Smad2, but it did not affect the phosphorylation of Smad3. Smad2D450E reduced hetero-oligomer formation of Smad2 with Smad4 but not of Smad3 with Smad4. Smad3D407E was not phosphorylated by the constitutively active form of the TGF- type I receptor and inhibited the phosphorylation of coexpressed wild-type Smad2 and Smad3. Furthermore, Smad3D407E reduced hetero-oligomer formation of both Smad2 and Smad3 with Smad4. On the other hand, SB-431542 has been characterized as a potent inhibitor of ALK-5 with greater selectivity against other kinases, including p38 mitogen-activated protein kinase (MAPK) and ALK-2, -3, -4, -6, or -7, which can prevent the TGF-1-induced elevation of fibronectin, plasminogen activator inhibitor-1, and 1(I)collagen mRNA (Laping et al., 2002).
In this study, we showed that specific inhibitor of Smad3 (SIS3), a new inhibitor of TGF- signaling, expressed its effects via the selective suppression of Smad3 phosphorylation. Furthermore, we also evaluated whether this reagent can abolish the ECM overexpression in the TGF-1-treated normal dermal fibroblasts and scleroderma fibroblasts in vitro.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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1 and human platelet-derived growth factor-AA were obtained from R&D Systems (Minneapolis, MN). Antibodies for Smad2/3 (N-19), Smad3 (FL-425), phospho-Smad2/3, glutathione S-transferase (GST), c-Myc (9E10), Smad4, Smad7, phospho-extracellular signal-regulated kinase (ERK), ERK2, and p38 MAPK were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phosphoserine-specific antibody was from Zymed Laboratories (South San Francisco, CA). Anti-Smad2/3 antibody (S66220
[GenBank]
) was from BD Biosciences Transduction Laboratories (Lexington, KY). Anti-HA 3F10 antibody and FuGENE 6 were from Roche Diagnostics (Mannheim, Germany). FLAG M2 antibody was from Kodak IBI (New Haven, CT). The antibody for -actin or -smooth muscle actin (SMA) was from Sigma-Aldrich (St. Louis, MO). Antibody for phospho-Smad2, phosphoinositide 3-kinase p85, and phosphotyrosine (4G10) were from Upstate Biotechnology (Lake Placid, NY). The phospho-p38 MAPK (Thr180/Tyr182) rabbit polyclonal antibody was from New England Biolabs (Beverly, MA). Anti-type I collagen-UNLB was from Southern Biotechnology Associates (Birmingham, AL).
Synthesis of SIS3. Indole derivatives are regarded as structures that have high affinity to various receptors and then express important biological activities by binding with these receptors. SIS3 was composed of indole derivatives with 2'-phenyl as hydrophobic group to induce translocation into the nucleus. We synthesized SIS3, on the assumption that it acts as a ligand of nuclear receptors according to published methods of 2-(N-methylindolyl)acrylic acid, followed by condensation with the corresponding amine (Inhoffen et al., 1963; Yasufumi et al., 2003) (Fig. 1). SIS3 was stored as a solution in DMSO, and this solution was used after diluting it with medium for each assay.
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). Institutional review board approval and written informed consent were obtained according to the Declaration of Helsinki. Control donors were each matched with a scleroderma patient for age, sex, and biopsy site. Normal and patient samples were processed in parallel. Primary explant cultures were established in 75-cm2 culture flasks in modified Eagle's medium (MEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, and 50 µg/ml gentamicin. Fibroblast cultures independently isolated from different individuals were maintained as monolayers at 37°C in 95% air, 5% CO2 and studied between the third and sixth subpassages. Mouse dermal fibroblasts were also obtained from BALB/cA Jcl mice (CLEA Japan Inc., Tokyo, Japan). Mv1Lu cell line, COS-7 cell line, and NIH3T3 cell line were purchased from American Type Culture Collection (Manassas, VA). These cells were also maintained as described above.
Plasmid Construction. Generation of a series of 5'-deletion constructs consisting of the human collagen 2(I) gene fragments linked to the chloramphenicol acetyltransferase (CAT) reporter gene (COL1A2/CAT) was done as described previously (Ihn et al., 1996). –353m COL1A2/CAT construct with point mutations introduced into the potential Smad3 recognition site (located between nucleotides –263 and –258) of the –353
+58 base pair (bp) COL1A2/CAT deletion construct using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was as described previously (Asano et al., 2004). Mutation and deletion constructs were verified by sequencing.
Expression vectors for HA-tagged constitutively active form of TGF- type I receptor, ALK-5 (ALK-5TD); constitutively active form of activin type I receptor, ALK-4 (ALK-4QD); and FLAG-tagged-Smad3 or 6Myc-tagged-Smad4 containing six tandem copies of the Myc-epitope tag were kindly provided by Dr. Kohei Miyazono (University of Tokyo, Tokyo, Japan) (Nakao et al., 1997; Yagi et al., 1999, 2002). The p3TP-lux reporter plasmids and the pAR3-lux reporter plasmids were provided by Dr. Jeffrey Wrana (University of Toronto, Toronto, ON, Canada) (Carcamo et al., 1995; Hayashi et al., 1997). Xenopus laevis forkhead activin signal transducer 1 cDNA were kindly provided by Dr. Malcolm Whitman (Harvard Medical School, Boston, MA) (Yagi et al., 1999). Plasmids used in transient transfection assays were purified twice on CsCl gradients. At least two different plasmid preparations were used for each experiment.
Transient Transfection. Fibroblasts were grown to 50% confluence in 100-mm dishes in MEM with 10% FCS. The medium was replaced with serum-free medium, and fibroblasts were transfected with promoter constructs, expression vectors, or corresponding empty constructs, using FuGENE6 as described previously (Ihn et al., 2002). To correct minor variations in transfection efficiency, pSV--galactosidase vector (Promega, Madison, WI) was included in all transfections. After 48 h of incubation, cells were harvested in 0.25 M Tris-HCl, pH 8, and fractured by freeze-thawing for CAT assay or luciferase assay. Extracts were normalized for protein content as measured by protein assay (Bio-Rad, Hercules, CA). Each experiment was performed in duplicate.
Cell Lysis and Immunoblotting. Human dermal fibroblasts were cultured until they were confluent, and then the media were collected. Remaining cells were washed with ice-cold phosphate-buffered saline twice and lysed in lysis buffer (Ihn et al., 2002; Asano et al., 2004). Aliquots of conditioned media (normalized for cell numbers) or cell lysates (normalized for protein concentrations as measured by the Bio-Rad reagent) were subjected to immunoblotting with antibodies for type I collagen, -SMA, Smad4, Smad7, or -actin.
For detection of phosphorylated levels of Smad2, p38 MAPK, or ERK, membranes were incubated with antibodies against phospho-Smad2, phospho-p38 MAPK, or phospho-ERK overnight at 4°C, respectively. As a loading control, the same membrane was then stripped and reprobed with antibodies against total Smad2/3 (N-19), p38 MAPK, or ERK2, respectively.
Immunoprecipitation. Phosphorylated levels of Smad3 or p85 were examined by immunoprecipitation using anti-Smad3 and anti-phosphoserine-specific antibodies or anti-p85 and anti-phosphotyrosine antibodies, respectively (Ihn et al., 2001a; Yamane et al., 2003b). The same membrane was then stripped and reprobed with anti-Smad2/3 (N-19) or p85 antibody to show the total amount of Smad3 or p85, respectively.
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). Forty-eight hours after transfection, the cells were solubilized, and the cell lysates were incubated with the anti-FLAG M2 antibody, followed by incubation with protein G-Sepharose beads. The immunocomplexes were subjected to sodium dodecyl sulfate-polyacrylamide gels electrophoresis and transferred to nitrocellulose membranes, which were used for immunoblotting using antibody for phosphoserine or Myc 9E10.
GST Pull-Down Assay. A GST pull-down assay was performed according to the method of Fernandez-Sanchez et al. (2003) with minor modification. The full-length Smad3 was cloned into pDEST27 (Invitrogen, Carlsbad, CA) and in vitro transcribed/translated using NHDF Nucleofector Kit (amaxa GmbH, Cologne, Germany). Fibroblasts were then grown in MEM with 10% FCS for 24 h, and the medium was replaced with serum-free medium. After 24 h of incubation, cells were harvested in lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1 mM EGTA, 1% Triton X-100, 50 mM sodium fluoride, 25 mM -glycerophosphate hydrate, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin. Proteins were purified with glutathione-Sepharose beads 4B (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Phosphorylated levels of Smad3 were analyzed by immunoblotting using phospho-Smad2/3 antibody. The same membrane was then stripped and reprobed with anti-GST antibody to show the total amount of Smad3.
DNA Affinity Precipitation Assay. Two oligonucleotides containing biotin on the 5' nucleotide of the sense strand were used. The sequences of these oligonucleotides are as follows: 1) 3xCAGA oligo, 5'-TCGAGAGCCAGACAAGGAGCCAGACAAGGAGCCAGACACTCGAG, which is trimer of CAGA motif; and 2) 3xCAGA-M oligo, 5'-TCGAGAGCTACATAAAAAGCTACATATTTAGCTACATACTCGA, which is trimer of CAGA motif mutated (Asano et al., 2004; Jinnin et al., 2004). These oligonucleotides were annealed to their respective complementary oligonucleotides, and double-stranded oligonucleotides were gel-purified and used. Cell lysates were obtained using lysis buffer (Yagi et al., 2002). Poly(dI-dC) competitor was incubated with the cell lysates, followed by incubation with each double-stranded oligonucleotide. After the incubation, streptavidin-agarose (Sigma-Aldrich) was added to the reaction and incubated. The protein-DNA-streptavidin-agarose complex was washed and loaded onto a sodium dodecyl sulfate-polyacrylamide gel. Detection of Smad3 was performed with anti-Smad2/3 antibody (S66220
[GenBank]
).
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).
Measurement of [3H]Proline Incorporation. Cells (104/well) were plated into 96-well plates, and the medium was changed to serum-free medium supplemented with 50 µg/ml ascorbic acid for the duration of the experiment. Then, 0.05 µCi/µl [3H]proline {L-(2,3,4,5)-[3H]proline} (GE Healthcare) was added to the medium and incubated overnight. Medium was harvested from each well, and the incorporated radioactivity was counted in a liquid scintillation counter (Ziyadeh et al., 1994; Isono et al., 2000). Proline incorporation was corrected for the cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay in additional cells plated in parallel wells (Mosmann, 1983).
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction. Total RNA was extracted from the fibroblasts with Isogen (Nippongene, Tokyo, Japan). Complementary DNA was synthesized with ThermoScript reverse transcriptase (Invitrogen) (Li et al., 2003) and subjected to 25 cycles of PCR to amplify GAPDH, or 30 cycles of PCR to amplify human 2(I) collagen cDNA. The PCR cycle was 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min. The products were fractionated by agarose gel electrophoresis and stained with ethidium bromide. The nucleic acid sequences of the specific PCR primers were as follows: 2(I) collagen, 5'-ACT CAG CCA CCC AGA GTG GA-3' (sense) and 5'-TCT TGC AGT GGT AGG TGA TG-3' (antisense); and GAPDH, 5'-AAG AAG GTG GTG AAG CAG GC-3' (sense) and 5'-TCC ACC ACC CTG TTG CTG TA-3' (antisense).
Immunofluorescence. Fibroblasts were grown in four-well Lab-Tek chambers (Nalge Nunc, Naperville, IL) to subconfluence as described above. After 24 h of serum starvation, the cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100 in phosphate-buffered saline, and blocked with 10% FCS in 0.5% Triton X-100 in phosphate-buffered saline (Asano et al., 2004). The cells were stained with anti--SMA antibody as primary antibody, washed, and incubated with fluorescein isothiocyanate-conjugated secondary antibodies. To visualize the fluorescence, a Zeiss microscope (Carl Zeiss GmbH, Jena, Germany) was used.
Statistical Analysis. Statistical analysis was carried out with the Mann-Whitney U test for comparison of means. p values less than 0.05 were considered significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Type I Receptor. As an initial experiment, we compared the effects of SIS3 on the luciferase activity of p3TP-lux (which contains a part of the promoter region of the plasminogen activator inhibitor-1 gene and three tandem repeats of activator protein-1 binding sites of the collagenase 1 gene) (Carcamo et al., 1995; Hayashi et al., 1997) in Mv1Lu cells cotransfected with constitutively active form of TGF- type I receptor (ALK-5TD). As shown in Fig. 2, the overexpression of ALK-5TD increased the luciferase activity significantly, and its effect was abrogated by the treatment with SIS3 in a dose-dependent manner. Similar results were obtained using pAR3-lux (which contains the activin-responsive element of Mix.2 promoter in X. laevis) (Hayashi et al., 1997), constitutively active form of activin type I receptor (ALK-4QD), and an expression vector encoding the coactivator X. laevis forkhead activin signal transducer 1. Together, these results indicated that SIS3 inhibits the signaling activity of TGF-1 family.
SIS3 Reduces the TGF-1-Dependent Increase in Phosphorylation and DNA Binding of Smad3. Next, we investigated the TGF-1-induced phosphorylation state of Smad3 in the presence or absence of SIS3. As shown in Fig. 3, A and B, the stimulation of TGF-1 induced the marked phosphorylation of both endogenous and exogenous overexpressed Smad3 in human dermal fibroblasts. The pretreatment with 3 µM SIS3 reduced phosphorylation levels of Smad3 induced by TGF- by approximately 50%. To further confirm this finding, we investigated the DNA binding ability of Smad3 by DNA affinity precipitation in human dermal fibroblasts. As shown in Fig. 3C, TGF-1 also induced the strong binding of endogenous Smad3 with the 3xCAGA oligonucleotide, whereas the 3xCAGA-M oligonucleotide, which lacks the CAGA motif, did not bind with Smad3 even after treatment with TGF-1. Consistent with the results of immunoprecipitation, the pretreatment with 3 µM SIS3 decreased the levels of the DNA-Smad3 binding by approximately 50%.
When both Smad3 and Smad4 were simultaneously transfected into COS-7 cells and stimulated by the cotransfection with ALK-5TD, SIS3 inhibited the increased interaction between Smad3 and Smad4 as well as increased phosphorylated level of Smad3 (Fig. 3D). However, the phosphorylated levels of Smad2 (Fig. 3E) and the protein expression levels of Smad4 and Smad7, the inhibitory Smad (data not shown), were not affected by this reagent in the presence or absence of TGF-1. Furthermore, SIS3 did not affect the phosphorylation of other signaling pathways, such as MAPK/p38 induced by NaCl (Arbabi et al., 2000), ERK induced by fetal bovine serum (Thrane et al., 2001), or phosphoinositide 3-kinase by platelet-derived growth factor-AA (Wymann and Arcaro, 1994; data not shown). These results suggested that SIS3 attenuates the TGF-1-dependent increased promoter activity of p3TP-lux by selectively reducing the Smad3 phosphorylation, the DNA-Smad3 binding, and the interaction of Smad3 with Smad4.
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Effect of SIS3 on the ECM Expression Induced by TGF-1 in Normal Dermal Fibroblasts. We investigated whether SIS3 can also have an effect on the TGF-1-mediated ECM up-regulation. TGF- signaling contributes to the up-regulation of type I procollagen or -SMA expression via Smad3 in human dermal fibroblasts (Chen et al., 1999; Hu et al., 2003). Pretreatment of cells with SIS3 did not alter the protein levels of type I procollagen as well as -SMA in the absence of TGF-1, but the effect of TGF-1 was completely abrogated by 3 µM SIS3 (Fig. 4A). These results were confirmed by [3H]proline incorporation assay, which showed the effect of SIS3 on newly synthesized type I procollagen in human dermal fibroblasts as well as NIH3T3 cell line or mouse dermal fibroblasts (Fig. 4B). RT-PCR analysis revealed that the treatment of cells with 3 µM SIS3 reduced TGF-1-mediated up-regulation of 2(I) collagen mRNA (Fig. 4C). Note that the expression of GAPDH mRNA was not affected by SIS3, demonstrating that the indicated concentration of this reagent did not have generalized toxic effects. Thus, this inhibitory effect of SIS3 on the TGF-1-induced type I procollagen protein up-regulation was paralleled with the levels of 2(I) collagen mRNA.
To determine whether SIS3 affects the basal and the TGF-1-induced transcriptional activity of 2(I) collagen gene, we performed transient transfection assays using full-length COL1A2/CAT construct. As shown in Fig. 4D, SIS3 had no significant inhibitory effect on the basal 2(I) collagen promoter activity. In contrast, SIS3 reduced the TGF-1-induced 2(I) collagen promoter activity significantly in a dose-dependent manner. The pretreatment with 3 µM SIS3 reduced the promoter activity by approximately 50%.
To identify potential regulatory elements of the human 2(I) collagen gene by SIS3, we performed transient transfection assays using a series of 5' deletions of the COL1A2/CAT construct. As shown in Fig. 4E, the bp –353+58 construct responded at the highest level in the cells treated with TGF-1. The TGF-1-dependent increased promoter activity was decreased by the removal of a triple Sp1-binding site (bp –264+58 deletion construct) or a CAGA motif (bp –186+58 deletion construct) and was completely abrogated with the removal of another Sp1-binding site located at –125 bp (bp –108+58 deletion construct). The TGF-1-induced promoter activity of the bp –264+58 construct and the longer constructs was significantly reduced by treatment with SIS3. However, the inhibitory effect of SIS3 was completely diminished in the bp –186+58 construct and the subsequent deletion constructs. These data indicated that the responsive element of SIS3 in the 2(I) collagen promoter is located between bp –264 and –186. This region contains a CAGACA sequence (from bp –263 to –258) that was shown to be a functional Smad3-binding element (Chen et al., 1999). To further characterize the regulatory element of SIS3 in the 2(I) collagen promoter, we used the site-directed mutated construct –353m COL1A2/CAT, in which Smad3-binding site is mutated. Although mutating Smad3-binding sites resulted in the reduction of the response to TGF-1 by approximately 50%, the inhibitory effect of SIS3 was abolished in the mutated construct. Together, the experiments with deletion and substitution promoter mutants suggested that SIS3 inhibited the TGF-1 effect on type I procollagen expression at the transcriptional levels via the Smad3-binding site.
Effect of SIS3 on the TGF-1-Mediated Myofibroblast Differentiation of Dermal Fibroblasts. It is well known that -SMA expression is the established marker of myofibroblast differentiation. As shown in Fig. 4A, immunoblotting revealed that -SMA expressed little in normal cell lysates. Exogenous TGF- 1 induced the -SMA expression in cultured normal fibroblasts, and SIS3 decreased the -SMA expression in a dose-dependent manner. We further confirmed this result by immunocytochemistry. The stimulation of dermal fibroblasts with TGF- 1 increased the -SMA expression, which was abolished by the pretreatment with 3 µM SIS3 (Fig. 5). This result suggested that SIS3 can inhibit the myofibroblast differentiation induced by TGF-1.
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signaling. There is an expectation that SIS3 may reduce the up-regulated expression of 2(I) collagen gene through the inhibition of phosphorylated Smad3 in scleroderma fibroblasts. To confirm this, we investigated the effect of SIS3 on the up-regulated expression of 2(I) collagen gene in scleroderma fibroblasts. As shown in Fig. 6, A and B, 3 µM SIS3 reduced the up-regulated expression of type I procollagen protein as well as -SMA in scleroderma fibroblasts to the same extent as that in normal fibroblasts. We next investigated the effect of SIS3 on the phosphorylation and the DNA binding ability of Smad3 in scleroderma fibroblasts. The DNA-Smad3 binding as well as Smad3 phosphorylation is reported to be increased in scleroderma fibroblasts (Asano et al., 2004). The treatment with 3 µM SIS3 abolished the phosphorylation (Fig. 6C) and DNA binding ability of Smad3 (data not shown) in scleroderma fibroblasts. These results are consistent with the results of normal dermal fibroblasts stimulated by exogenous TGF-1. Furthermore, the treatment with 3 µM SIS3 also reduced bp –353+58 COL1A2/CAT promoter activity in scleroderma fibroblasts completely, but –353m COL1A2/CAT was not affected by this reagent (Fig. 6D). This result suggested that the inhibitory effect of SIS3 on the up-regulated type I collagen expression in scleroderma fibroblasts involves Smad3.
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-SMA (Jelaska and Korn, 2000), and Smad3 mediates the TGF--induced -SMA expression (Hu et al., 2003). Therefore, there is a good possibility that the autocrine TGF- signaling contributes to the maintenance of -SMA expression in scleroderma fibroblasts. Thus, we evaluated the effect of SIS3 on the expression of -SMA in scleroderma fibroblasts. As shown in Fig. 7, scleroderma fibroblasts, which looked like the TGF-1-treated normal fibroblasts (Fig. 5), expressed -SMA much more than normal fibroblasts. SIS3 (3 µM) decreased the expression of -SMA to the same extent as that in normal fibroblasts, which is consistent with the result of immunoblotting (Fig. 6A). These results indicated that SIS3 restores myofibroblast differentiation of scleroderma fibroblasts.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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1. Activin, a member of the TGF-1 family, also expresses its effect via Smad2, Smad3, and Smad4 (Nakao et al., 1997). Together, because both TGF-1 and activin expressed their effects via the common pathway, it is likely that SIS3 can inhibit the effects of TGF-1 and activin.
Further study revealed that the effects of SIS3 were mediated by the suppression of the Smad3 phosphorylation, the DNA-Smad3 binding, and the interaction of Smad3 with Smad4. On the other hand, this reagent affected neither the phosphorylation of Smad2, the expression of Smad4 or Smad7, nor the phosphorylation of other signaling pathways, such as p38, p85, or ERK, in the presence or absence of TGF-1. These results indicate that SIS3 can inhibit the function of Smad3 selectively in TGF- family signaling pathway.
Propagation of TGF- signals is mediated by the direct association of the receptor-associated Smads (Smad2 and Smad3) with the TGF- receptor complex. The receptor-associated Smads are then directly phosphorylated by the type I TGF- receptor kinase on the last two serines of a conserved specific phosphorylation motif, SSXS, located at the extreme carboxyl terminus of the MH2 domain (Attisano and Tuen Lee-Hoeflich, 2001). Thus, one of the mechanisms by which SIS3 specifically inhibited phosphorylation of Smad3 may involve this SSXS motif. However, SIS3 did not affect Smad2, which also contains SSXS motif. We assumed that SIS3 acts as ligand of nuclear receptors. Chou et al. (2003) reported that TGF--regulated Smads can have transcriptional crosstalk with nuclear receptor hepatocyte nuclear factor-4. Interaction of some orphan nuclear receptors with Smad3 through SIS3 may inhibit the phosphorylation of Smad3 specifically via SSXS motif.
Thereafter, we evaluated whether SIS3 suppresses the TGF-1-induced type I procollagen protein or 2(I) collagen gene expression in human dermal fibroblasts. We found that SIS3 did not decrease the basal expression of type I procollagen protein or 2(I) collagen gene but that the addition of SIS3 attenuated the TGF-1 effects on type I collagen expression by reducing its transcriptional activity.
Moreover, we demonstrated that SIS3 also decreased the excessive type I procollagen expression or the 2(I) collagen promoter activation, which may be because SIS3 abolished autocrine TGF- signaling pathway in scleroderma fibroblasts. Note that 3 µM SIS3 completely diminished the constitutive phosphorylation and the DNA binding of Smad3 as well as the excessive type I procollagen expression or the 2(I) collagen promoter activation in scleroderma fibroblasts.
We also examined the effects of SIS3 on -SMA expression in normal and scleroderma fibroblasts. SMA-positive fibroblasts, so-called myofibroblasts, are found in scleroderma and in a number of other fibrotic conditions (Sappino et al., 1990). Kirk et al. (1995) suggested that the myofibroblast phenotype might correspond to "activated fibroblast" phenotype found in systemic sclerosis. These activated fibroblasts have a high synthetic capacity for ECM proteins, growth factors/cytokines, growth factor receptors, integrins, and oxidants (Thannickal and Fanburg, 1995). The presence/activation of myofibroblasts seems to be a consistent finding in the pathology of human fibrotic diseases involving diverse organ systems such as the lung, liver, and kidney (Border and Noble, 1994). Thus, persistent myofibroblast proliferation and/or survival represents a pathological repair process that can result in aberrant architectural remodeling of tissues associated with end-stage fibrosis and organ failure. On the other hand, TGF- signaling contributes to the up-regulation of -SMA expression in cultured fibroblasts via Smad3 (Hu et al., 2003). Our results suggested that SIS3, which inhibits TGF- signaling, could be used as a therapeutic intervention for scleroderma by turning back scleroderma fibroblasts abnormally overexpressing -SMA via the inhibition of Smad3.
Together, our data suggest that SIS3 is a useful reagent to evaluate TGF--regulated cellular mechanisms by the selective inhibition of Smad3 and that SIS3 can block excessive ECM production from the TGF-1-treated normal fibroblasts and scleroderma fibroblasts, the model of cells with autocrine TGF- signaling in vitro. Our study also indicated that the increased phosphorylation and the DNA binding ability of Smad3 in scleroderma fibroblasts is one of the causes of excessive ECM deposition in this disease, although the pathogenesis of this disease is still unclear. Because TGF- is a potent stimulus for ECM synthesis, the inhibition of Smad3 may be beneficial in other fibrotic disorders. Therefore, further in vivo and in vitro studies are necessary.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: TGF, transforming growth factor; ECM, extracellular matrix; MAPK, mitogen-activated protein kinase; SIS3, specific inhibitor of Smad3; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; SMA, smooth muscle actin; DMSO, dimethyl sulfoxide; MEM, modified Eagle's medium; FCS, fetal calf serum; CAT, chloramphenicol acetyltransferase; bp, base pair(s); HA, hemagglutinin; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; PCR, polymerase chain reaction; AU, arbitrary units; SB-431542, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide.
Address correspondence to: Dr. Hironobu Ihn, Department of Dermatology and Plastic and Reconstructive Surgery, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan. E-mail: ihn-der{at}kaiju.medic.kumamoto-u.ac.jp
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