Characterization of SIS3, a Novel Specific Inhibitor of Smad3, and Its Effect on Transforming Growth Factor- 1-Induced Extracellular Matrix Expression

This is the first report that characterizes specific inhibitor of Smad3 (SIS3) as a potent and selective inhibitor of Smad3 function. In the reporter assay, the increased luciferase activity of p3TP-lux by the overexpression of constitutively active form of ALK-5 was abrogated by the treatment with SIS3 in a dosedependent manner. Immunoprecipitation revealed that SIS3 attenuated the transforming growth factor (TGF)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 TGF1-induced type I procollagen up-regulation in human dermal fibroblasts. We found that the addition of SIS3 attenuated the effects of TGF1 by reducing the transcriptional activity. SIS3 also inhibited the myofibroblast differentiation of fibroblasts by TGF1. 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. Transforming growth factor (TGF)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 TGF1 are triggered by the activation of serine/threonine kinase TGFreceptors. On activation by TGF1 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 TGFreceptor type I (Nakao et al., 1997). They are ligandspecific 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. TGF1 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 TGF1 (LeRoy et al., 1989), suggesting that activation of dermal fibroblast in scleroderma may be a result of stimulation by autocrine TGFsignaling. This notion is supported by our recent findings: 1) scleroderma fibroblasts express elevated This study was supported in part by a grant for scientific research from the Japanese Ministry of Education, by project research for progressive systemic sclerosis from the Japanese Ministry of Health and Welfare. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.017483. 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. 0026-895X/06/6902-597–607$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 2 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 17483/3082057 Mol Pharmacol 69:597–607, 2006 Printed in U.S.A. 597 at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from levels of TGFreceptors, and this correlates with the elevated levels of 2(I) collagen mRNA (Kawakami et al., 1998); 2) the blockade of TGFsignaling with anti-TGFantibodies or anti-TGF1 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 TGFsignaling 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 TGFsignaling can decrease collagen expression in vivo or in vitro (Yamamoto et al., 1999; Yamane et al., 2003a). Several investigators have reported possible inhibitors of TGFsignaling (Callahan et al., 2002; Kondo et al., 2004). A missense mutant of Smad2, Smad2D450E, that was not phosphorylated by TGFsignaling 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 TGFtype 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 TGF1-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 TGFsignaling, expressed its effects via the selective suppression of Smad3 phosphorylation. Furthermore, we also evaluated whether this reagent can abolish the ECM overexpression in the TGF1-treated normal dermal fibroblasts and scleroderma fibroblasts in vitro. Materials and Methods Reagents. Recombinant human TGF1 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) 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. Cell Cultures. Normal human dermal fibroblasts were obtained by skin biopsies from five healthy donors. Scleroderma fibroblasts were obtained by skin biopsies from the affected areas (dorsal forearm) of five patients with diffuse cutaneous systemic sclerosis and 2 years of skin thickening (Ihn et al., 2001b). 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-cm culture flasks in modified Eagle’s medium (MEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, and 50 g/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 se-