Abrogation of Transforming Growth Factor-β Signaling by SMAD7 Inhibits Collagen Gel Contraction of Human Dermal Fibroblasts

Abstract

Human fibroproliferative disorders like hypertrophic scarring of the skin are characterized by increased contractility and excess extracellular matrix synthesis. A beneficial role of transforming growth factor (TGF)-β in wound healing was proposed; however, chronic stimulation by this cytokine leads to fibrosis. In the present report, the intracellular TGF-β signaling in fibroblasts derived from hypertrophic scars and normal skin was examined. In an attempt to intervene in profibrogenic TGF-β functions, ectopic expression of Smad7 or dominant negative Smads3/4 completely inhibited contractility of scar-derived and normal fibroblasts after suspension in collagen gels. Both cell types displayed constitutive Smad2/3 phosphorylation and (CAGA)9-MLP-Luc activity with expression and phosphorylation of Smad3 being predominant in hypertrophic scar-derived fibroblasts. Down-regulation of intrinsic signaling with various TGF-β antagonists, e.g. soluble TGF-β receptor, latency-associated peptide, and anti-TGF-β1 antibodies, confirms autocrine TGF-β stimulation of both cell populations. Further, Smad7 expression inhibited α1 (I) collagen and α-smooth muscle actin expression. In summary, our data indicate that autocrine TGF-β/Smad signaling is involved in contractility and matrix gene expression of fibroblasts from normal and hypertrophic scars. Smad7 inhibits these processes and may exert beneficial effects on excessive scar formation. Human fibroproliferative disorders like hypertrophic scarring of the skin are characterized by increased contractility and excess extracellular matrix synthesis. A beneficial role of transforming growth factor (TGF)-β in wound healing was proposed; however, chronic stimulation by this cytokine leads to fibrosis. In the present report, the intracellular TGF-β signaling in fibroblasts derived from hypertrophic scars and normal skin was examined. In an attempt to intervene in profibrogenic TGF-β functions, ectopic expression of Smad7 or dominant negative Smads3/4 completely inhibited contractility of scar-derived and normal fibroblasts after suspension in collagen gels. Both cell types displayed constitutive Smad2/3 phosphorylation and (CAGA)9-MLP-Luc activity with expression and phosphorylation of Smad3 being predominant in hypertrophic scar-derived fibroblasts. Down-regulation of intrinsic signaling with various TGF-β antagonists, e.g. soluble TGF-β receptor, latency-associated peptide, and anti-TGF-β1 antibodies, confirms autocrine TGF-β stimulation of both cell populations. Further, Smad7 expression inhibited α1 (I) collagen and α-smooth muscle actin expression. In summary, our data indicate that autocrine TGF-β/Smad signaling is involved in contractility and matrix gene expression of fibroblasts from normal and hypertrophic scars. Smad7 inhibits these processes and may exert beneficial effects on excessive scar formation. Hypertrophic scars are skin abnormalities secondary to traumatic or surgical injuries, characterized by excessive deposition and impaired degradation of collagen in dermis and subcutaneous tissues during the wound healing remodeling phase. Hypertrophic scarring can cause adverse cosmesis, loss of function, and strictures and adhesions following burns and abdominal surgery, as well as restriction of growth in children (1Brissett A.E. Sherris D.A. Facial Plast. Surg. 2001; 17: 263-272Crossref PubMed Scopus (167) Google Scholar). Transforming growth factor (TGF) 1The abbreviations used are: TGF, transforming growth factor; R-Smad, receptor-regulated Smad; HF, hypertrophic scar-derived fibroblasts; NF, normal fibroblasts; SMA, smooth muscle actin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; LAP, latency-associated peptide.1The abbreviations used are: TGF, transforming growth factor; R-Smad, receptor-regulated Smad; HF, hypertrophic scar-derived fibroblasts; NF, normal fibroblasts; SMA, smooth muscle actin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; LAP, latency-associated peptide.-β isoforms play an important role in the fibrogenic tissue response to injury in many organs, including skin (2Cowin A.J. Holmes T.M. Brosnan P. Ferguson M.W. Eur. J. Dermatol. 2001; 11: 424-431PubMed Google Scholar). Particularly, TGF-β1 is up-regulated in wound healing-related processes (3Roberts A.B. Sporn M.B. Assoian R.K. Smith M.J. Roche N.S. Wakefield L.M. Heine U.I. Liotta L.A. Falanga V. Kehrl J.H. Fauci A.S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4167-4171Crossref PubMed Scopus (2386) Google Scholar) and contributes to fibroblast activation and transdifferentiation to myofibroblasts. Myofibroblasts show enhanced production of extracellular matrix and up-regulation of cytokine and cytoskeletal protein expression and are the major cell type contributing to excessive wound contraction in hypertrophic scars (4Berry D.P. Harding K.G. Stanton M.R. Jasani B. Ehrlich H.P. Plast. Reconstr. Surg. 1998; 102: 124-134Crossref PubMed Scopus (148) Google Scholar). Smad proteins function as intracellular signaling mediators of TGF-β family members. Generally, they can be divided into receptor-regulated Smads (R-Smads), common mediator Smads, and inhibitory Smads (5ten Dijke P. Hill C.S. Trends Biochem. Sci. 2004; 29: 265-273Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar). After ligand binding to a type II receptor, type II receptor kinases phosphorylate the GS domain of type I receptors. The activated type I receptor kinases phosphorylate R-Smads (Smad1, -2, -3, -5, and -8) differentially at two serine residues in the SSXS motif at their extreme C termini. Phosphorylated R-Smads form oligomeric complexes with Smad4. These complexes translocate into the nucleus and regulate transcription of target genes. Among the R-Smads, Smad3 is especially implicated in specific pathways of tissue repair (6Flanders K.C. Int. J. Exp. Pathol. 2004; 85: 47-64Crossref PubMed Scopus (507) Google Scholar). Inhibitory Smad7 acts to oppose signal transducing R- and common mediator Smads by forming stable associations with activated type I receptors, thus preventing phosphorylation of R-Smads (7Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata N. Heldin N.E. Heldin C.H. Tendijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1534) Google Scholar). As a result of physiologic signal transduction by TGF-β, Smad7 expression is rapidly and transiently up-regulated in many cell types, thus acting as a negative feedback regulator (8Stopa M. Anhuf D. Terstegen L. Gatsios P. Gressner A.M. Dooley S. J. Biol. Chem. 2000; 275: 29308-29317Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Presently, there are only a few more or less successfully established therapies to treat hypertrophic scarring, including surgical excision, compression therapy, and local injection of corticosteroids. Experimental approaches to eliminate TGF-β effects were performed by applying cytokine-specific neutralizing antibodies during the early phase of wound healing and resulted in reduced scarring (9Shah M. Foreman D.M. Ferguson M.W. J. Cell Sci. 1995; 108: 985-1002Crossref PubMed Google Scholar). Additionally, experimental results referring to other fibroproliferative disorders indicate that gene therapeutic strategies targeting TGF-β might have potential to effectively prevent disease progression (10George J. Roulot D. Koteliansky V.E. Bissell D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12719-12724Crossref PubMed Scopus (321) Google Scholar, 11Isaka Y. Akagi Y. Ando Y. Tsujie M. Sudo T. Ohno N. Border W.A. Noble N.A. Kaneda Y. Hori M. Imai E. Kidney Int. 1999; 55: 465-475Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 12Ueno H. Sakamoto T. Nakamura T. Qi Z. Astuchi N. Takeshita A. Shimizu K. Ohashi H. Hum. Gene Ther. 2000; 11: 33-42Crossref PubMed Scopus (137) Google Scholar). Some of these approaches have successfully used Smad7 to inhibit TGF-β family signaling, e.g. during bleomycin-induced lung fibrosis (13Nakao A. Fujii M. Matsumura R. Kumano K. Saito Y. Miyazono K. Iwamoto T. J. Clin. Investig. 1999; 104: 5-11Crossref PubMed Scopus (380) Google Scholar) or by targeting renal fibrosis in post-obstructive kidney disease (14Terada Y. Hanada S. Nakao A. Kuwahara M. Sasaki S. Marumo F. Kidney Int. 2002; 61: 94-98Abstract Full Text Full Text PDF Scopus (96) Google Scholar). Furthermore, in an experimental model using bile duct ligation in rat, we were able to inhibit liver fibrogenesis by delivering Smad7 in a gene therapeutic approach (15Dooley S. Hamzavi J. Breitkopf K. Wiercinska E. Said H.M. Lorenzen J. ten Dijke P. Gressner A.M. Gastroenterology. 2003; 125: 178-191Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). In this study, we examined Smad-dependent TGF-β1 signaling in primary cultured fibroblasts isolated from surgically excised hypertrophic scars (HF). We identified Smad3/Smad4 signaling as the predominant mediator of autocrine stimulation in HF. Further, overexpression of Smad7 abrogated intrinsic contraction and α1 (I) procollagen mRNA and α-smooth muscle actin (SMA) expression, indicating the potential for this inhibitory Smad to blunt excessive scarring in patients. Materials—Taq polymerase and [32P]dCTP were purchased from Amersham Biosciences. All reagents for cell culture (Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), trypsin-EDTA, and phosphate-buffered saline) were from Invitrogen or BIOCHROM (Berlin, Germany). Monoclonal anti-FLAG and anti-β-actin antibodies were obtained from Sigma (M5, F-4042). Oligonucleotides were from MWG Biotech (Ebersberg, Germany) or Genset (Delaunay, France). The luciferase assay system was from Promega. Antisera for Smad2 and 3 were purchased from Zymed Laboratories Inc. (Berlin, Germany). The antibody for Smad7 was from Abcam, Cambridge, UK. Anti-phospho-Smad2 antiserum was from Cell Signaling Technology, New England Biolabs. Anti-phospho-Smad3 antiserum was kindly provided by P. ten Dijke, Netherlands Cancer Institute, Amsterdam. The anti-mouse horseradish peroxidase-conjugated secondary antibody was purchased from Zymed Laboratories Inc.. All other chemicals were obtained from commercial sources at the purest grade available. Cell Culture—Normal skin was harvested from patients undergoing plastic surgery procedures such as reduction mammaplasty or abdominoplasty. Hypertrophic scar tissue was obtained from plastic surgery scar corrections. Fibroblasts were isolated by using the explant culture technique as described elsewhere (16Weber L. Mauch C. Kirsch E. Muller P.K. Krieg T. J. Investig. Dermatol. 1986; 87: 217-220Crossref PubMed Scopus (13) Google Scholar) and grown in DMEM (glucose 1.0 g/liter, NaHCO3 3.7 g/liter, N-acetyl-l-alanyl-l-glutamine 1.0289 g/liter; BIOCHROM) supplemented with 10% FCS, 40 μg/ml gentamicin (Invitrogen) without additional growth factors in 75-cm2 culture flasks. All cultures were maintained at 37 °C, 5% CO2 in a humidified atmosphere. In case of 70–80% confluence, cells were subcultured or cryopreserved using 10% Me2SO. For adenoviral infection, fibroblasts were seeded in 58-cm2 Petri culture dishes (Greiner, Frickenhausen, Germany). Vectors and Gene Transfer—Adenoviral stocks for (CAGA)9-MLP-Luc were created using the Adeasy cloning and recombination procedure (Quantum, Appligene; www.quantum-appligene.com). Infections and coinfections of fibroblasts isolated from hypertrophic scars and normal skin were performed according to other reports (17Dooley S. Delvoux B. Streckert M. Bonzel L. Stopa M. ten Dijke P. Gressner A.M. FEBS Lett. 2001; 502: 4-10Crossref PubMed Scopus (179) Google Scholar). Routine infections were accomplished with 2 × 105 HF or normal fibroblasts (NF) at 50 multiplicity of infection with single virus clones of the same virus stock preparation. Infection efficiency was proven by adenoviral constructs expressing β-galactosidase; each time, ∼90% of the cells were infected. Staining of infected cells was performed using an 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) staining kit (Roche Applied Science). After infection, cells were cultured for 1 day in DMEM containing 10% FCS and were then used for contraction testing in the collagen gel system. In parallel, part of the cells was used for molecular biology investigations. To perform luciferase assays, cells were cultured for 2 days in DMEM with 10% FCS and then infected with adenoviruses as indicated. Lysates for luciferase detection were prepared 24 h after adenoviral infections. Treatment with latency-associated peptide or anti-TGF-β1 antibody was performed 24 h after infection for 16 h. Adenoviral stocks for Smad7, Smad6, dominant negative Smad3 (ΔSmad3) and Smad4 (ΔSmad4), and LacZ were described previously (18Fujii M. Takeda K. Imamura T. Aoki H. Sampath T.K. Enomoto S. Kawabata M. Kato M. Ichijo H. Miyazono K. Mol. Biol. Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (366) Google Scholar) and kindly provided by A. Moustakas, Ludwig Institute for Cancer Research, Uppsala, Sweden. The adenovirus expressing a soluble Tβ RII (AdsTβ RII) was provided by R. Weiskirchen, Institute for Clinical Chemistry and Pathobiochemistry, University Hospital, RWTH-Aachen (19Herrmann J. B. E. S. Gressner A.M. R. 2004; PubMed Scopus Google Scholar). the contraction of normal and a modified in contraction model was used as previously described E. B. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In normal and hypertrophic scar-derived infected and were and of medium (Invitrogen) were to of collagen The was to with 2 To a collagen cell containing 3 of the prepared cell suspension was a of × 105 the cell collagen was provided in suspension culture (Greiner, 1 were for 1 h of collagen cell 2 of normal cell culture medium 10% were After and were from the and were obtained to the of gel contraction of each cell contraction was using the of was performed in of Cell and from and HF, infected and were prepared with of assay of were provided as (Roche Applied according to the To inhibit protein was a with a to were to a of a stock was by a on cell were by at × for at 40 of were by gel and to a for as previously described F. Delvoux B. C. J.H. Breitkopf K. Gressner A.M. Dooley S. 2002; Scopus Google Scholar). and of and was from and HF cell cultures with an kit according to the Germany). was performed as previously described K. K. M. FEBS Lett. PubMed Scopus Google Scholar) using of to transcription of α1 (I) of a from specific for α1 of collagen type 1 was on a described by F. 10: PubMed Scopus (380) Google containing a of results obtained from contraction was used by applying the with a for at at Smad7 in and hypertrophic the of fibroblasts to extracellular matrix is to and impaired of In fibroblasts are able to collagen into and the their wound We used this system to the of TGF-β signaling on the of HF and cells were infected with adenoviruses to suspension in a collagen in hypertrophic scar-derived fibroblasts was in normal fibroblasts As a HF and were infected with of fibroblasts displayed from of cells Smad7 overexpression in resulted in reduced contraction of the with to and to A of Smad7 was in HF, in of collagen in to HF or to from collagen indicate that Smad7 a reduction of in both cell of Smad7 may inhibit signal transduction of TGF-β family and Smad2/3 or To of the TGF-β in we dominant negative of Smads3/4 The of or was to blunt to Smad7 Gene by Smad7 in and TGF-β signal HF and were infected with an adenovirus containing a (CAGA)9-MLP-Luc including of a TGF-β response S. Itoh S. D. ten Dijke P. S. J. 1998; 17: PubMed Scopus Google Scholar). Infection of both cell were as examined by activity after infection Both cell show intrinsic stimulation of gene with of luciferase activity being in HF with in overexpression of Smad7 reduced luciferase activity to in HF and of (CAGA)9-MLP-Luc by Smad7, LAP, and in and autocrine cells were with and Smad7, or an adenovirus a soluble TGF-β type II receptor (AdsTβ RII) as in of RII luciferase activity like Smad7, to autocrine stimulation of the cells and ligand of the To of we the cells with latency-associated peptide or with a anti-TGF-β1 antibody and obtained the in gene activity in HF and HF and of Smad3 with of is the of intracellular TGF-β signal to the of gene HF and phosphorylation of Smad3 and and phosphorylation of Smad3 were up-regulated in HF NF, activation of Smad2 was in both cell Further, Smad3 phosphorylation in HF is by overexpression of Smad7 indicating that Smad3 is involved in autocrine stimulation of contractility in HF and there is a Smad7 on Smad3 Smad3 expression is To the of expression of TGF-β signaling we expression of Smad7, and in HF using Smad4 and were in both cell types, Smad7 was at the protein in Smad7 α1 (I) in and of extracellular matrix proteins such as α1 (I) procollagen is for wound healing in scar a this protein is in excess J. Sci. PubMed Scopus Google Scholar). with this HF of α1 (I) procollagen mRNA with of Smad7 α1 (I) procollagen mRNA expression, indicating of TGF-β signaling in collagen expression Smad7, Smad6, Down-regulation of in muscle actin expression is for the of fibroblasts to and is in fibroblasts of hypertrophic scars (4Berry D.P. Harding K.G. Stanton M.R. Jasani B. Ehrlich H.P. Plast. Reconstr. Surg. 1998; 102: 124-134Crossref PubMed Scopus (148) Google the of wound Cell from and HF were for staining in indicating that HF increased expression with adenoviruses as described we Smad7 or in of and HF. Smad7, Smad6, up-regulated expression in HF, indicating that TGF-β, signaling in hypertrophic scar-derived protein expression The TGF-β of proteins involved in cell and tissue repair A. S. Heldin C.H. J. Cell Sci. 2001; PubMed Google Scholar). role in wound healing and scar TGF-β A.B. 2000; 11: PubMed Scopus Google Scholar) and may the expression of extracellular such as collagen and as well as is the major protein for P. P. C. D.A. J. Pathol. 1998; Google Scholar). injury of the skin by or surgery TGF-β expression in especially in fibroblasts. In case of of TGF-β may be in serum and to the of R. B. T. A. Plast. Reconstr. Surg. 1998; 102: PubMed Scopus Google Scholar). or impaired healing can to TGF-β activity and excessive have that strategies to TGF-β signaling are for of fibroproliferative disorders and kidney H. Sakamoto T. Nakamura T. Qi Z. Astuchi N. Takeshita A. Shimizu K. Ohashi H. Hum. Gene Ther. 2000; 11: 33-42Crossref PubMed Scopus (137) Google Scholar, A. Fujii M. Matsumura R. Kumano K. Saito Y. Miyazono K. Iwamoto T. J. Clin. Investig. 1999; 104: 5-11Crossref PubMed Scopus (380) Google Scholar, N. J.H. R. J. 2003; PubMed Scopus Google Scholar). more or less specific approaches dominant negative TGF-β proteins like such as growth or anti-TGF-β1 antibodies were A.M. R. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Inhibitory Smad7 was identified to TGF-β in and in by activated receptor complexes (7Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata N. Heldin N.E. Heldin C.H. Tendijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1534) Google Scholar, H. S. Y. J. J.L. D. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). We and have used overexpression of Smad7 to inhibit profibrogenic TGF-β effects in (13Nakao A. Fujii M. Matsumura R. Kumano K. Saito Y. Miyazono K. Iwamoto T. J. Clin. Investig. 1999; 104: 5-11Crossref PubMed Scopus (380) Google Scholar, Y. Hanada S. Nakao A. Kuwahara M. Sasaki S. Marumo F. Kidney Int. 2002; 61: 94-98Abstract Full Text Full Text PDF Scopus (96) Google Scholar, S. Hamzavi J. Breitkopf K. Wiercinska E. Said H.M. Lorenzen J. ten Dijke P. Gressner A.M. Gastroenterology. 2003; 125: 178-191Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). In of latency-associated peptide, a of TGF-β, skin and fibrosis in a model for Y. J. Investig. Dermatol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). As a result of up-regulation of TGF-β1 and tissue growth factor mRNA and was Additionally, a TGF-β peptide was successfully used to contraction and scarring, wound in skin and skin wound J. 2002; PubMed Scopus Google Scholar). data were for an antibody that was able to scarring in M. Foreman D.M. Ferguson J. Cell Sci. PubMed Google Scholar). In our we Smad7 in fibroblasts from normal or hypertrophic scar tissue and on collagen Smad7 completely abrogated contraction of normal and scar-derived fibroblasts indicating of TGF-β signaling in this To the of autocrine TGF-β stimulation in and HF of were and the results were in with this of Smad7 and soluble TGF-β receptor type II as well as with latency-associated peptide or a anti-TGF-β1 antibody intrinsic (CAGA)9-MLP-Luc activity in both cell Furthermore, of anti-TGF-β1 antibody to like Smad7, completely inhibited collagen gel to TGF-β1 as mediator was by the of of TGF-β1 was in and HF, K. J. and S. and a more of role in skin fibroblasts to be A inhibitory of Smad7, and on contractility Smad of autocrine A of Smad expression and phosphorylation that Smad3 expression and intrinsic Smad3 phosphorylation were increased in HF NF, indicating a role of Smad3 in hypertrophic In with with Smad3 that displayed wound enhanced and reduced of and after with type K.C. A. Fujii M. A. A. A.B. J. Pathol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). is that Smad3 contributes to scarring and that of Smad3 signaling might wound Additionally, T. S. E. A.B. Cell 2003; PubMed Scopus Google Scholar) that is for contraction of type I collagen gels. In this study, fibroblasts from Smad2 and Smad3 type and were into type I collagen and with isoforms of A contraction of the was obtained with for Smad3 an role for Smad3 in wound healing processes like repair of A.B. M. M. T. A.B. Cell Biol. 1999; PubMed Scopus Google bleomycin-induced fibrosis in J. H. P. M.B. D. J. 2002; PubMed Scopus Google and activation of cells and liver fibrosis in B. D.A. 2001; PubMed Scopus Google Scholar) have In many cell types, TGF-β signaling Smad7 expression M. Moren A. S. Itoh S. Sampath K. B. Heldin C.H. Heldin N.E. ten Dijke P. Biochem. 1998; PubMed Scopus Google Scholar). the other this is in chronic that are by autocrine stimulation S. Delvoux B. B. K. Gressner A.M. 2000; PubMed Scopus Google Scholar). to Smad7 is in and HF. the of wound healing with hypertrophic scar wound and matrix the of fibroblasts to proteins such as type I collagen and is P. J. J. Full Text PDF PubMed Scopus Google Scholar). we have that expression and function of extracellular matrix proteins in and HF are at by In activated fibroblasts from hypertrophic α1 type I procollagen mRNA and protein expression are Smad7 expression autocrine TGF-β effects to expression of these target genes. expression of and was for fibroblasts with normal T. Nakao A. K. R. H. J. Dermatol. Sci. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In summary, our data that fibroproliferative of normal fibroblasts and fibroblasts during hypertrophic scar are and by signaling is thus important for contractility of and HF. pathways may to the of and collagen expression in these Smad7 is of TGF-β signaling, in expression of and extracellular fibrogenic proteins and we were able to a beneficial of Smad7 on progression in in a bile duct ligation model of liver fibrosis (15Dooley S. Hamzavi J. Breitkopf K. Wiercinska E. Said H.M. Lorenzen J. ten Dijke P. Gressner A.M. Gastroenterology. 2003; 125: 178-191Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). We that the in of Smad7 may be a approach to a gene therapeutic for the of hypertrophic scarring and other fibroproliferative disorders of the