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Alterations in the Transforming Growth Factor (TGF)-β Pathway as a Potential Factor in the Pathogenesis of Peyronie’s Disease
european urology 51 (2007) 255–261
available at www.sciencedirect.com journal homepage: www.europeanurology.com
Sexual Medicine
Alterations in the Transforming Growth Factor (TGF)-b Pathway as a Potential Factor in the Pathogenesis of Peyronie’s Disease§ Simone M. Haag a, Ekkehard W. Hauck a,*, Carolin Szardening-Kirchner a, Thorsten Diemer a, Eun-Sook Cha b, Wolfgang Weidner a, Oliver Eickelberg b a b
Department of Urology and Pediatric Urology, Justus Liebig University Giessen, Rudolf-Buchheim-Str. 7, 35385 Giessen, Germany University of Giessen Lung Center, Department of Medicine II, Justus Liebig University Giessen, Aulweg 123, 35395 Giessen, Germany
Article info
Abstract
Article history: Accepted May 3, 2006 Published online ahead of print on May 19, 2006
Objectives: The development of fibrotic diseases is associated with alterations in the transforming growth factor b (TGF-b) pathway. We have investigated the expression and activity of Smad transcription factors of the TGF-b pathway in primary tunical fibroblasts derived from patients with Peyronie’s disease and from controls. Methods: Primary fibroblasts were established from biopsies obtained from plaques of 16 patients with Peyronie’s disease or the tunica albuginea of 8 control patients. The expression and activity of Smad transcription factors in control and TGF-b–stimulated primary fibroblasts were investigated at the RNA and protein level by reverse transcription-polymerase chain reaction, Western blotting, and immunofluorescence. Results: RNA expression levels of Smad3 and Smad4 were significantly increased in fibroblasts from patients with Peyronie’s disease. When stimulated with TGF-b1, fibroblasts showed rapid nuclear translocation of Smad2/3, as soon as 15 min after stimulation. This effect was more pronounced and exhibited an earlier onset in fibroblasts from patients with Peyronie’s disease, compared with controls. In addition, an increased nuclear retention time of Smad4 was observed in fibroblasts from patients with Peyronie’s disease. Conclusions: The expression and activity of Smad transcription factors of the TGF-b pathway is increased in fibroblasts of patients with Peyronie’s disease. Alterations in the TGF-b pathway seem to be a pathogenetic factor in the development of Peyronie’s disease.
Keywords: Peyronie’s disease Transforming growth factor Bone morphogenetic protein Smad Cell culture
# 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved.
§
The data of this study have been accepted for presentation at the 100th Annual Meeting of the American Association of Urology, San Antonio, Texas, USA, in 2005. * Corresponding author. Justus Liebig University Giessen, Department of Urology and Pediatric Urology, Rudolf-Buchheim-Str. 7, D-35385 Giessen, Germany. Tel. +49 641 99 44501; Fax: +49 641 99 44509. E-mail address: [email protected] (E.W. Hauck).
0302-2838/$ – see back matter # 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.eururo.2006.05.002
256
1.
european urology 51 (2007) 255–261
Introduction
Peyronie’s disease is characterized by a fibrotic and sometimes calcified plaque of the tunica albuginea and the adjacent tissue of the corpora cavernosa. The underlying molecular mechanism for the development of this hypertrophic scar tissue remains enigmatic [1]. Repetitive penile microtraumatization during sexual intercourse with subsequent induction of maladaptive wound healing and tissue fibrosis represents one of the main causes of plaque development [2]. In addition, initial fibrin deposition followed by inflammatory reactions has been shown to induce scar formation and plaque development in the tunica albuginea [2]. Recent studies have documented an increased expression of transforming growth factor b1 (TGF-b1) in plaques of patients with Peyronie’s disease [3]. This has provided evidence that TGF-b1 plays a crucial role in the pathogenesis of this disease [3]. In addition, genetic variations in the coding region of the TGF-b1 gene have been documented in Peyronie’s disease. An increased frequency of homozygosity in the single nucleotide polymorphism (SNP) G915C (G/G) has been detected in patients with Peyronie’s disease [4]. This SNP has been associated with elevated TGF-b1 serum levels and the occurrence of fibrotic disorders in other organ systems, such as pulmonary fibrosis [5]. Moreover, intratunical injection of cytomodulin, a synthetic heptapeptide with TGF-b-like activity, led to Peyronie’s disease-like conditions in an animal model, further suggesting a causal role for TGF-b in the pathogenesis of this disorder [6]. In recent years, novel aspects of the TGF-b pathway have been elucidated. TGF-b is a soluble and secreted growth factor of the TGF-b superfamily, which includes TGF-bs, activins and bone morphogenetic proteins (BMPs). TGF-b binds to specific serine/threonine kinase receptors at the cell surface, which triggers oligomerization of receptor isotypes, and subsequent phosphorylation and activation of intracellular signalling molecules, the Smad transcription factors. To date, eight different Smad molecules have been cloned and characterized, which have been categorized into three different subgroups: (1) receptor-regulated Smads (R-Smads), (2) common Smads (co-Smads), and (3) inhibitory Smads (I-Smads) [5]. Smad2 and 3, the two R-Smads, have been associated with TGF-b and activin signaling, whereas Smad1, 5 and 8 have been associated with BMP signaling. Phosphorylated R-Smads bind the co-Smad (Smad4), the complex of which then translocates into the nucleus to regulate cellular proliferation and/or differentiation via interaction with regulatory sequences in chromatin [7]. On the
basis of these observations, we sought to investigate the expression and function of the TGF-b pathway in primary fibroblasts derived from plaques of patients with Peyronie’s disease or from control patients. 2.
Patients and methods
2.1.
Patients
We obtained biopsies from 16 patients with Peyronie’s disease undergoing surgery and 8 control patients. All patients enrolled in this study gave written consent, and the procedures were approved by the internal ethical review board of the University of Giessen School of Medicine. For patients with Peyronie’s disease, specimens were obtained directly out of the plaque during surgery, after degloving of the penis using the sleeve technique. In case of a plication procedure (n = 9), biopsies were obtained directly out of the plaque that could be easily identified by palpation with the help of a punch biopsy gun (Manan Pro-Mag 1.2, Medical Device Technologies Inc., Gainesville, FL, USA) with a 14-gauge biopsy needle (Manan SACN 14 ga 8 cm, Medical Device Technologies Inc.). The specimen was directly cut out of the incision line, when an incision and grafting procedure (n = 7) was performed. In all patients investigated, an additional specimen was obtained to confirm the clinical diagnosis of Peyronie’s disease by histology. In the control group, tunica albuginea was obtained from the incision line during insertion of a penile implant (n = 6) or from the ellipsoids excised during a Nesbit procedure (n = 2). The diagnosis of a normal tunica albuginea was also confirmed histologically with the use of an additional specimen. Biopsy samples were transferred into sterile vials containing 0.9% NaCl and transferred directly from the operation theatre into the laboratory for the initiation of cell cultures. A cell culture was initiated in all specimens. Fibroblasts grew out in 10 of 16 cases of Peyronie’s disease and in 6 of 8 cases of the control group.
2.2.
Cell culture
The tissues obtained were cleaned, cut and distributed onto cell culture dishes. Tissue pieces were then submerged in cell culture medium (Cell Growth Medium 2; C-39210; Promocell, Heidelberg, Germany), and fibroblasts were allowed to grow out from the tissues. After fibroblasts had grown out, the tissues were removed and the cells were trypsinized. Cells were further incubated at 37 8C in a humidified 5% CO2 atmosphere. ISCOVE-Medium basal (Cat# F0465; Biochrom AG, Berlin, Germany) supplemented with 10% fetal calf serum, 1% L-glutamine, and 50 mg/ml gentamicin was used to expand fibroblast cultures. The medium was changed every 2–3 days. Only cells between the 2nd and 12th passages were used throughout this study.
2.3. RNA isolation and reverse transcription-polymerase chain reaction Total RNA was isolated from fibroblasts with the use of the Rneasy Midi Kit (Quiagen, Hilden, Germany) according to the
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Table 1 – Primer sequences for Smads and HSC-70 Gene
Smad1 Smad2 Smad3 Smad4 Smad5 Smad7 HSC-70
Sequence Forward primer
Reverse primer
50 -GGAGACAGCTTTATTTCACCATATC-30 50 -GGGAGGTTCGATACAAGAGGCT-30 50 -AGCCATGTCGTCCATCCTG-30 50 -TTCACTGTTTCCAAAGGATCAAAA-30 50 -CTGTTCTTTCGGTAGCCACTGAC-30 50 -GACTTCTTCATGGTGTGCGG-30 50 -GCCCTTTATGGTGGTGAATGA-30
50 -CATCTGCCTCTGGAAAACTATTG-30 50 -TGTCATAGCATTGTGTGTGGTCC-30 50 -CCCATTGTTGTCAAGGAAGAAG-30 50 -CTTAACGTTATCAGGATGGTGGACTAC-30 50 -CCCCATATCTTCTGTTTCATAATGC-30 50 -GAGCAGGCCACACTTCAAACTA-30 50 -CTTGTTCTCTTTTCCCGTACTCTT-30
manufacturer’s instructions. Complementary DNA synthesis and reverse transcription-polymerase chain reaction (RT-PCR) were performed with the Omniscript-Kit (Quiagen, Hilden, Germany). Primers for Smad1, Smad2, Smad3, Smad4, Smad5, Smad7, and the housekeeping gene HSC-70 are listed in Table 1. PCRs were performed with 34 cycles at an annealing temperature of 57 8C and 60 8C for Smads and HSC-70, respectively. PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide.
2.4.
Cell fractionation and Western blotting
Fibroblast cultures were stimulated with TGF-b1 at a final concentration of 1 ng/ml, BMP-2 at 10 ng/ml and IFN-g at 50 ng/ml. Cytosolic and nuclear protein were extracted by differential salt lysis, as previously described [8]. Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and harvested in 1 ml of PBS with a rubber policeman. Samples were centrifuged for 1 min at 4000 g (4 8C), and cell pellets were resuspended in 100 ml low-salt buffer (20 mmol/l HEPES at pH 7.9, 10 mmol/l KCl, 0.1 mmol/l Na3VO4, 1 mmol/l EDTA, 1 mmol/l EGTA, 0.2% NP-40, 10% glycerol and a set of proteinase inhibitors, Complete). After 10 min of incubation on ice, the samples were centrifuged at 10,000 g for 1 min (4 8C) and the supernatants (cytosolic extracts) immediately frozen in a dry ice/ethanol bath. Pelleted nuclei were resuspended in 60 ml of high-salt buffer (20 mmol/l HEPES at pH 7.9, 420 mmol/l NaCl, 10 mmol/l KCl, 0.1 mmol/l Na3VO4, 1 mmol/l EDTA, 1 mmol/l EGTA, 20% glycerol, supplemented with Complete); nuclear proteins were extracted by shaking on ice for 30 min. Samples were centrifuged at 13,000 g for 10 min (4 8C) and the supernatants taken as nuclear extracts. Equal amounts of protein extracts were separated by SDSPAGE and immunoblotting. In brief, separated proteins were transferred to nitrocellulose membranes and blocked with 5% nonfat milk for 30 min. Membranes were washed three times with washing buffer (TBST: 1 mol/l Tris at pH 7.5, 4 mol/l NaCl, 0.1% Tween) and incubated 60 min with primary antibodies at room temperature (pSmad2/3Ser465/467, pSmad1/5/8Ser463/465 or pSTAT1Tyr701 (Cell Signaling Technology, Beverly, MA, USA), Smad2/3, STAT1 (BD Transduction Laboratories, Franklin Lakes, NJ, USA), Smad1/2/3, C23 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), Smad1 (Upstate, Waltham, MA, USA)). Washed membranes were incubated 45 min with HRPconjugated secondary antibodies, and signals were visualized with Super Signal West Pico Chemiluminescent Substrate
(Pierce Company, Rockford, IL, USA) and autoradiography according to the manufacturer’s instructions.
2.5.
Fluorescence microscopy
Fibroblasts were grown on chamber slides and stimulated with TGF-b1 (1 ng/ml) or BMP-2 (10 ng/ml) for the indicated time points and fixed with methanol. After blocking with 5% fresh bovine serum, cells were incubated for 60 min at room temperature with primary antibodies at a dilution of 1:100. Cells were then washed three times with PBS and stained with fluorescein isothiocyanate–conjugated secondary antibodies (Zymax, Goat-Anti-Rabbit IgG and Goat-Anti-Mouse IgG, FITCConjugate, Zymed, San Francisco, CA, USA). Nuclei were then counterstained with DAPI (40 , 6-diamidino-2-phenylindole, dihydrochloride; Sigma, St. Louis, MO, USA) for 5 min; the slides were then covered and sealed with cover slips. Fluorescence images were obtained on the Leica AS-MDW workstation.
3.
Results
3.1.
Smad RNA expression in primary fibroblast cultures
TGF-b/BMP signaling is determined by the expression and activity of the Smad transcription factors. We initially sought to determine the expression levels of Smads by RT-PCR in primary fibroblasts derived from control patients (n = 4) and patients with Peyronie’s disease (n = 6). As depicted in Fig. 1, the expression levels of Smad1 and Smad5, and the loading control HSC-70 were similar in all samples investigated. Smad2 expression levels were similar in most samples, but very low in two samples obtained from patients with Peyronie’s disease. Smad3 and Smad4 RNA levels were higher in fibroblasts derived from plaques of patients with Peyronie’s disease, compared with controls (Fig. 1). Smad7 demonstrated a heterogeneous expression pattern, without a difference between control and Peyronie’s disease samples (Fig. 1; Table 2). 3.2.
Smad activation in response to TGF-b stimulation
TGF-b–induced effects are dictated by Smad expression levels and also by activation kinetics. We
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Fig. 1 – Smad RNA expression levels in primary fibroblasts derived from control patients (n = 4) and patients with Peyronie’s disease (n = 6). HSC-70 was used as a loading control; negative control (neg ctrl) was loaded in the last lane.
therefore characterized ligand-dependent Smad activation by phosphorylation and nuclear translocation of Smads. Fig. 2 depicts representative Western blot analyses of cytosolic and nuclear extracts obtained from fibroblasts cultures of control and Peyronie’s disease patients, and stimulated with TGF-b for 15, 30, 60 or 120 min. PhosphoSmad2/3 levels were detected in both cultures by Western blot analysis with activation state-specific antibodies. Smad2/3 phosphorylation characteristics differed between both cultures with respect to signal strength and onset. Fibroblasts from patients with Peyronie’s disease exhibited an earlier onset of phosphorylation, compared with control cells (15 min vs 30 min, respectively; Fig. 2). In addition, the phospho-Smad2/3 signal was stronger in fibroblasts from patients with Peyronie’s disease than in controls (Fig. 2). Smad2/3 is depicted for the
loading control, and the nuclear marker C23 as a control for the purity of the nuclear and cytosolic extraction. 3.3.
Smad translocation in response to TGF-b stimulation
We then analyzed whether Smad activation, as assessed by phosphorylation, coincided with nuclear translocation. We incubated fibroblast cultures for the indicated times (15, 30 or 60 min) with TGF-b, fixed the cells and performed immunofluorescence analysis of Smad3 and 4 localization. Translocation of Smad3 and 4 could be observed as early as 30 min after stimulation with TGF-b1 in control fibroblasts (Fig. 3). In contrast, nuclear translocation of Smad3 and 4 was observed as early as 15 min poststimulation in fibroblasts from Peyronie’s disease, corresponding to the earlier
Table 2 – Overview of the investigated Smads on RNA, protein and cellular levels Investigations
Smad1
Smad2
Smad3
Smad4
Smad5
Smad7
RNA: Smad expression levels
PD Control
+ +
(+) Partially very low +
++ +
++ +
+ +
Heterogeneous Heterogeneous
Protein: Smad activation in response to TGF-b stimulation
PD Control
Ø Ø
++ +
++ +
Ø Ø
Ø Ø
Ø Ø
Fibroblasts: Smad translocation in response to TGF-b stimulation
PD Control
Ø Ø
++ +
++ + Incomplete translocation
+ ++
Ø Ø
Ø Ø
TGF-b = transforming growth factor b.
european urology 51 (2007) 255–261
259
Fig. 2 – Representative Western blot analyses of cytosolic and nuclear extracts obtained from fibroblasts cultures of control and Peyronie’s disease stimulated with TGF-b for 15, 30, 60 or 120 min. TGF-b1 = transforming growth factor b1.
phosphorylation depicted in Fig. 2. After 60 min most of the Smad3/4 signal was still confined to the nuclear compartment, whereas it was predominantly cytosolic again for control fibroblasts (Fig. 3, last column).
4.
Discussion
In this study, we could demonstrate significant differences in the expression and activity of Smad
transcription factors in primary fibroblasts derived from control patients or patients with Peyronie’s disease. The Smads are key signal-transducing molecules of the TGF-b superfamily, a growth factor system essentially involved in the pathogenesis of fibrotic diseases. The TGF-b superfamily comprises over 30 distinct members, including TGF-bs, BMPs, activins, and growth and differentiation factors. These cytokines exhibit multifunctional and pleiotropic effects on cell proliferation, differentiation or migration [7].
Fig. 3 – Nuclear translocation of Smad3 and Smad4 in fibroblast cultures after stimulation with TGF-b for 15, 30 or 60 min. TGF-b1 = transforming growth factor b1.
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european urology 51 (2007) 255–261
TGF-b regulates cellular processes by binding to specific cell-surface receptors, the type I and type II TGF-b receptors (TbRs). After ligand binding to the high-affinity type II receptor (TbRII), a heteromeric receptor complex of TGF-b/TbRI/TbRII is formed. The constitutively active TbRII kinase then activates TbRI by phosphorylation of the juxtamembrane GSregion (for glycine-serine rich). This event induces phosphorylation of the receptor-regulated Smad2/3 by the TbRI kinase. Activated Smad2/3 then forms heterotrimers with the co-Smad Smad4 and translocates to the nucleus. Negative feedback regulation of this pathway is given through inhibitory Smads (Smad6 and Smad7), which, when expressed, antagonize the phosphorylation of Smad2 and 3 by TbRI [9]. In our study, we chose to culture primary fibroblasts derived from the tunica albuginea or plaques of control patients or patients with Peyronie’s disease, respectively. We obtained viable and pure fibroblast cultures from 10 of 16 (62.5%) samples in the case of Peyronie’s disease. In the control group, viable fibroblast cultures were obtained 6 of 8 (75%) cases. Plaques of Peyronie’s disease patients contain a higher content of collagens, and in some cases calcification might decrease vital tissue, thus possibly explaining the lower percentage of viable cultures. The difficulty in obtaining fibroblasts from Peyronie’s disease specimens is well documented in the literature. In studies of Mulhall et al. [10], fibroblast cell cultures could be initiated in 5 of 10 (50%) plaque biopsies. In the study by Somers et al. [11], fibroblast cell cultures could be initiated in 19 of 23 (82.6%) plaque biopsies. In a recent study, El-Sakka et al. [3] demonstrated upregulation of TGF-b1, TGF-b2 and TGF-b3 protein expression in plaques of Peyronie’s disease patients. Consistent with these findings, we could detect increased Smad levels in primary fibroblasts cultures, documenting an overall expression increase of the TGF-b system in this disease. To the best of our knowledge, this finding has not been reported previously in Peyronie’s disease. Magee et al. [12] recently reported that Smad7, an inhibitory factor of the TGF-b pathway, was downregulated in Peyronie’s disease. Similar to our findings, Smad7 RNA expression was upregulated in primary fibroblasts from scleroderma patients, compared with normal fibroblasts, whereas the expression of Smad2, 3 and 4 were similar in both cultures [13]. To our knowledge, the more rapid translocation of Smad3 and 4 in Peyronie’s disease-derived fibroblasts has not been described before. A potential explanation of this finding is represented by the observation that Smads are also more potently and
rapidly phosphorylated in Peyronie’s disease, possibly because of the higher expression of TGF-b receptors on these cells, compared with normal tissue. Consistently, our study yielded similar results at the levels of RNA expression, Smad phosphorylation and nuclear translocation. A characteristic feature of the TGF-b system is its pleiotropic action (i.e., the observation that the biologic effect of TGF-b is strictly cell-type dependent). Although TGF-b induces cell proliferation in fibroblasts, it leads to potent growth arrest in epithelial cells, most likely triggered by similar signal transduction pathways [14]. In fibroblasts derived from chronic wounds, the concentration of pSmad2/3 was decreased in response to TGF-b1 more than 3-fold. Kim et al. [15] found decreased expression of TGF-b type II receptor in ulcer fibroblasts. Although the number of Peyronie’s disease and control specimens was small in our study, the results were very reproducible. Our study has shown differences in the TGF-b pathway of plaque-derived fibroblasts, compared with fibroblasts from tunica albuginea of controls. More studies with higher numbers and investigations on the TGF-b receptor levels will now dramatically increase our knowledge of the TGF-b system in Peyronie’s disease, and possibly unravel novel treatment regimen in this disease, which interferes with the TGF-b pathway.
5.
Conclusion
The expression and activity of Smad transcription factors of the TGF-b pathway are increased in fibroblasts of patients with Peyronie’s disease. We suggest that alterations in the TGF-b pathway represent a potential factor in the pathophysiology of Peyronie’s disease. Conflicts of interest This study was supported by a grant (Anschubfinanzierung) to E. W. Hauck donated by the Justus Liebig University, and by the Sofja Kovalevskaja Award to O. Eickelberg by the Alexander von Humboldt Foundation.
References [1] Gholami SS, Gonzalez-Cadavid NF, Lin CS, Rajfer J, Lue TF. Peyronie’s disease: a review. J Urol 2003;169:1234–41. [2] Devine Jr CJ, Somers KD, Jordan GH, Schlossberg SM. Proposal: trauma as the cause of the Peyronie’s lesion. J Urol 1997;157:285–90.
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[3] El-Sakka AI, Hassoba HW, Pillarisetty RJ, Dahiva R, Lue TF. Peyronie’s disease is associated with an increase in transforming growth factor-beta protein expression. J Urol 1997;158:1391–4. [4] Hauck EW, Hauptmann A, Schmelz HU, Bein G, Weidner W, Hackstein H. Prospective analysis of single nucleotide polymorphisms of the transforming growth factor b-1 gene in Peyronie’s disease. J Urol 2003;169:369. [5] Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signaling. Nature 2003;425:577–84. [6] El-Sakka AI, Hassoba HM, Chui RM, Bhatnagar RS, Dahiya R, Lue TF. An animal model of Peyronie’s like condition associated with an increase of transforming growth factor beta mRNA and protein expression. J Urol 1997;158:2284–90. [7] Lutz M, Knaus P. Integration of the TGF-beta pathway into the cellular signaling network. Cell Signal 2002;14:977–88. [8] Eickelberg O, Pansky A, Mussmann R. Transforming growth-factor-beta1 induces interleukin-6 expression via activating protein-1 consisting of JunD homodimers in primary human lung fibroblasts. J Biol Chem 1999;274: 12933.
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[9] Massague´ J. How cells read TGF-beta signals. Nat Rev Mol Cell Biol 2000;1:169–78. [10] Mulhall JP, Thom J, Lubrano T, Shankey TV. Basic fibroblast growth factor expression in Peyronie’s disease. J Urol 2001;165:419–23. [11] Somers KD, Dawson DM. Fibrin deposition in Peyronie’s disease plaque. J Urol 1997;157:311–5. [12] Magee TR, Qian A, Rajfer J, Sander FC, Levine LA, Gonzalez-Cadavid NF. Gene expression profiles in the Peyronie’s disease plaque. Urology 2002;59:451–7. [13] Asano Y, Ihn H, Yamane K, Kubo M, Tamaki K. Impaired Smad7-Smurf-mediated negative regulation of TGF-beta signaling in scleroderma fibroblasts. J Clin Invest 2004; 113:253–64. [14] Kretschmer A, Moepert K, Dames S, Sternberger M, Kaufmann J, Klippel A. Differential regulation of TGF-beta signaling through Smad2, Smad3 and Smad4. Oncogene 2003;22:6748–63. [15] Kim BC, Kim HT, Park SH, et al. Fibroblasts from chronic wounds show altered TGF-beta signaling and decreased TGF-beta Type II Receptor expression. J Cell Physiol 2003;195:331–6.
available at www.sciencedirect.com journal homepage: www.europeanurology.com
Sexual Medicine
Alterations in the Transforming Growth Factor (TGF)-b Pathway as a Potential Factor in the Pathogenesis of Peyronie’s Disease§ Simone M. Haag a, Ekkehard W. Hauck a,*, Carolin Szardening-Kirchner a, Thorsten Diemer a, Eun-Sook Cha b, Wolfgang Weidner a, Oliver Eickelberg b a b
Department of Urology and Pediatric Urology, Justus Liebig University Giessen, Rudolf-Buchheim-Str. 7, 35385 Giessen, Germany University of Giessen Lung Center, Department of Medicine II, Justus Liebig University Giessen, Aulweg 123, 35395 Giessen, Germany
Article info
Abstract
Article history: Accepted May 3, 2006 Published online ahead of print on May 19, 2006
Objectives: The development of fibrotic diseases is associated with alterations in the transforming growth factor b (TGF-b) pathway. We have investigated the expression and activity of Smad transcription factors of the TGF-b pathway in primary tunical fibroblasts derived from patients with Peyronie’s disease and from controls. Methods: Primary fibroblasts were established from biopsies obtained from plaques of 16 patients with Peyronie’s disease or the tunica albuginea of 8 control patients. The expression and activity of Smad transcription factors in control and TGF-b–stimulated primary fibroblasts were investigated at the RNA and protein level by reverse transcription-polymerase chain reaction, Western blotting, and immunofluorescence. Results: RNA expression levels of Smad3 and Smad4 were significantly increased in fibroblasts from patients with Peyronie’s disease. When stimulated with TGF-b1, fibroblasts showed rapid nuclear translocation of Smad2/3, as soon as 15 min after stimulation. This effect was more pronounced and exhibited an earlier onset in fibroblasts from patients with Peyronie’s disease, compared with controls. In addition, an increased nuclear retention time of Smad4 was observed in fibroblasts from patients with Peyronie’s disease. Conclusions: The expression and activity of Smad transcription factors of the TGF-b pathway is increased in fibroblasts of patients with Peyronie’s disease. Alterations in the TGF-b pathway seem to be a pathogenetic factor in the development of Peyronie’s disease.
Keywords: Peyronie’s disease Transforming growth factor Bone morphogenetic protein Smad Cell culture
# 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved.
§
The data of this study have been accepted for presentation at the 100th Annual Meeting of the American Association of Urology, San Antonio, Texas, USA, in 2005. * Corresponding author. Justus Liebig University Giessen, Department of Urology and Pediatric Urology, Rudolf-Buchheim-Str. 7, D-35385 Giessen, Germany. Tel. +49 641 99 44501; Fax: +49 641 99 44509. E-mail address: [email protected] (E.W. Hauck).
0302-2838/$ – see back matter # 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.eururo.2006.05.002
256
1.
european urology 51 (2007) 255–261
Introduction
Peyronie’s disease is characterized by a fibrotic and sometimes calcified plaque of the tunica albuginea and the adjacent tissue of the corpora cavernosa. The underlying molecular mechanism for the development of this hypertrophic scar tissue remains enigmatic [1]. Repetitive penile microtraumatization during sexual intercourse with subsequent induction of maladaptive wound healing and tissue fibrosis represents one of the main causes of plaque development [2]. In addition, initial fibrin deposition followed by inflammatory reactions has been shown to induce scar formation and plaque development in the tunica albuginea [2]. Recent studies have documented an increased expression of transforming growth factor b1 (TGF-b1) in plaques of patients with Peyronie’s disease [3]. This has provided evidence that TGF-b1 plays a crucial role in the pathogenesis of this disease [3]. In addition, genetic variations in the coding region of the TGF-b1 gene have been documented in Peyronie’s disease. An increased frequency of homozygosity in the single nucleotide polymorphism (SNP) G915C (G/G) has been detected in patients with Peyronie’s disease [4]. This SNP has been associated with elevated TGF-b1 serum levels and the occurrence of fibrotic disorders in other organ systems, such as pulmonary fibrosis [5]. Moreover, intratunical injection of cytomodulin, a synthetic heptapeptide with TGF-b-like activity, led to Peyronie’s disease-like conditions in an animal model, further suggesting a causal role for TGF-b in the pathogenesis of this disorder [6]. In recent years, novel aspects of the TGF-b pathway have been elucidated. TGF-b is a soluble and secreted growth factor of the TGF-b superfamily, which includes TGF-bs, activins and bone morphogenetic proteins (BMPs). TGF-b binds to specific serine/threonine kinase receptors at the cell surface, which triggers oligomerization of receptor isotypes, and subsequent phosphorylation and activation of intracellular signalling molecules, the Smad transcription factors. To date, eight different Smad molecules have been cloned and characterized, which have been categorized into three different subgroups: (1) receptor-regulated Smads (R-Smads), (2) common Smads (co-Smads), and (3) inhibitory Smads (I-Smads) [5]. Smad2 and 3, the two R-Smads, have been associated with TGF-b and activin signaling, whereas Smad1, 5 and 8 have been associated with BMP signaling. Phosphorylated R-Smads bind the co-Smad (Smad4), the complex of which then translocates into the nucleus to regulate cellular proliferation and/or differentiation via interaction with regulatory sequences in chromatin [7]. On the
basis of these observations, we sought to investigate the expression and function of the TGF-b pathway in primary fibroblasts derived from plaques of patients with Peyronie’s disease or from control patients. 2.
Patients and methods
2.1.
Patients
We obtained biopsies from 16 patients with Peyronie’s disease undergoing surgery and 8 control patients. All patients enrolled in this study gave written consent, and the procedures were approved by the internal ethical review board of the University of Giessen School of Medicine. For patients with Peyronie’s disease, specimens were obtained directly out of the plaque during surgery, after degloving of the penis using the sleeve technique. In case of a plication procedure (n = 9), biopsies were obtained directly out of the plaque that could be easily identified by palpation with the help of a punch biopsy gun (Manan Pro-Mag 1.2, Medical Device Technologies Inc., Gainesville, FL, USA) with a 14-gauge biopsy needle (Manan SACN 14 ga 8 cm, Medical Device Technologies Inc.). The specimen was directly cut out of the incision line, when an incision and grafting procedure (n = 7) was performed. In all patients investigated, an additional specimen was obtained to confirm the clinical diagnosis of Peyronie’s disease by histology. In the control group, tunica albuginea was obtained from the incision line during insertion of a penile implant (n = 6) or from the ellipsoids excised during a Nesbit procedure (n = 2). The diagnosis of a normal tunica albuginea was also confirmed histologically with the use of an additional specimen. Biopsy samples were transferred into sterile vials containing 0.9% NaCl and transferred directly from the operation theatre into the laboratory for the initiation of cell cultures. A cell culture was initiated in all specimens. Fibroblasts grew out in 10 of 16 cases of Peyronie’s disease and in 6 of 8 cases of the control group.
2.2.
Cell culture
The tissues obtained were cleaned, cut and distributed onto cell culture dishes. Tissue pieces were then submerged in cell culture medium (Cell Growth Medium 2; C-39210; Promocell, Heidelberg, Germany), and fibroblasts were allowed to grow out from the tissues. After fibroblasts had grown out, the tissues were removed and the cells were trypsinized. Cells were further incubated at 37 8C in a humidified 5% CO2 atmosphere. ISCOVE-Medium basal (Cat# F0465; Biochrom AG, Berlin, Germany) supplemented with 10% fetal calf serum, 1% L-glutamine, and 50 mg/ml gentamicin was used to expand fibroblast cultures. The medium was changed every 2–3 days. Only cells between the 2nd and 12th passages were used throughout this study.
2.3. RNA isolation and reverse transcription-polymerase chain reaction Total RNA was isolated from fibroblasts with the use of the Rneasy Midi Kit (Quiagen, Hilden, Germany) according to the
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Table 1 – Primer sequences for Smads and HSC-70 Gene
Smad1 Smad2 Smad3 Smad4 Smad5 Smad7 HSC-70
Sequence Forward primer
Reverse primer
50 -GGAGACAGCTTTATTTCACCATATC-30 50 -GGGAGGTTCGATACAAGAGGCT-30 50 -AGCCATGTCGTCCATCCTG-30 50 -TTCACTGTTTCCAAAGGATCAAAA-30 50 -CTGTTCTTTCGGTAGCCACTGAC-30 50 -GACTTCTTCATGGTGTGCGG-30 50 -GCCCTTTATGGTGGTGAATGA-30
50 -CATCTGCCTCTGGAAAACTATTG-30 50 -TGTCATAGCATTGTGTGTGGTCC-30 50 -CCCATTGTTGTCAAGGAAGAAG-30 50 -CTTAACGTTATCAGGATGGTGGACTAC-30 50 -CCCCATATCTTCTGTTTCATAATGC-30 50 -GAGCAGGCCACACTTCAAACTA-30 50 -CTTGTTCTCTTTTCCCGTACTCTT-30
manufacturer’s instructions. Complementary DNA synthesis and reverse transcription-polymerase chain reaction (RT-PCR) were performed with the Omniscript-Kit (Quiagen, Hilden, Germany). Primers for Smad1, Smad2, Smad3, Smad4, Smad5, Smad7, and the housekeeping gene HSC-70 are listed in Table 1. PCRs were performed with 34 cycles at an annealing temperature of 57 8C and 60 8C for Smads and HSC-70, respectively. PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide.
2.4.
Cell fractionation and Western blotting
Fibroblast cultures were stimulated with TGF-b1 at a final concentration of 1 ng/ml, BMP-2 at 10 ng/ml and IFN-g at 50 ng/ml. Cytosolic and nuclear protein were extracted by differential salt lysis, as previously described [8]. Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and harvested in 1 ml of PBS with a rubber policeman. Samples were centrifuged for 1 min at 4000 g (4 8C), and cell pellets were resuspended in 100 ml low-salt buffer (20 mmol/l HEPES at pH 7.9, 10 mmol/l KCl, 0.1 mmol/l Na3VO4, 1 mmol/l EDTA, 1 mmol/l EGTA, 0.2% NP-40, 10% glycerol and a set of proteinase inhibitors, Complete). After 10 min of incubation on ice, the samples were centrifuged at 10,000 g for 1 min (4 8C) and the supernatants (cytosolic extracts) immediately frozen in a dry ice/ethanol bath. Pelleted nuclei were resuspended in 60 ml of high-salt buffer (20 mmol/l HEPES at pH 7.9, 420 mmol/l NaCl, 10 mmol/l KCl, 0.1 mmol/l Na3VO4, 1 mmol/l EDTA, 1 mmol/l EGTA, 20% glycerol, supplemented with Complete); nuclear proteins were extracted by shaking on ice for 30 min. Samples were centrifuged at 13,000 g for 10 min (4 8C) and the supernatants taken as nuclear extracts. Equal amounts of protein extracts were separated by SDSPAGE and immunoblotting. In brief, separated proteins were transferred to nitrocellulose membranes and blocked with 5% nonfat milk for 30 min. Membranes were washed three times with washing buffer (TBST: 1 mol/l Tris at pH 7.5, 4 mol/l NaCl, 0.1% Tween) and incubated 60 min with primary antibodies at room temperature (pSmad2/3Ser465/467, pSmad1/5/8Ser463/465 or pSTAT1Tyr701 (Cell Signaling Technology, Beverly, MA, USA), Smad2/3, STAT1 (BD Transduction Laboratories, Franklin Lakes, NJ, USA), Smad1/2/3, C23 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), Smad1 (Upstate, Waltham, MA, USA)). Washed membranes were incubated 45 min with HRPconjugated secondary antibodies, and signals were visualized with Super Signal West Pico Chemiluminescent Substrate
(Pierce Company, Rockford, IL, USA) and autoradiography according to the manufacturer’s instructions.
2.5.
Fluorescence microscopy
Fibroblasts were grown on chamber slides and stimulated with TGF-b1 (1 ng/ml) or BMP-2 (10 ng/ml) for the indicated time points and fixed with methanol. After blocking with 5% fresh bovine serum, cells were incubated for 60 min at room temperature with primary antibodies at a dilution of 1:100. Cells were then washed three times with PBS and stained with fluorescein isothiocyanate–conjugated secondary antibodies (Zymax, Goat-Anti-Rabbit IgG and Goat-Anti-Mouse IgG, FITCConjugate, Zymed, San Francisco, CA, USA). Nuclei were then counterstained with DAPI (40 , 6-diamidino-2-phenylindole, dihydrochloride; Sigma, St. Louis, MO, USA) for 5 min; the slides were then covered and sealed with cover slips. Fluorescence images were obtained on the Leica AS-MDW workstation.
3.
Results
3.1.
Smad RNA expression in primary fibroblast cultures
TGF-b/BMP signaling is determined by the expression and activity of the Smad transcription factors. We initially sought to determine the expression levels of Smads by RT-PCR in primary fibroblasts derived from control patients (n = 4) and patients with Peyronie’s disease (n = 6). As depicted in Fig. 1, the expression levels of Smad1 and Smad5, and the loading control HSC-70 were similar in all samples investigated. Smad2 expression levels were similar in most samples, but very low in two samples obtained from patients with Peyronie’s disease. Smad3 and Smad4 RNA levels were higher in fibroblasts derived from plaques of patients with Peyronie’s disease, compared with controls (Fig. 1). Smad7 demonstrated a heterogeneous expression pattern, without a difference between control and Peyronie’s disease samples (Fig. 1; Table 2). 3.2.
Smad activation in response to TGF-b stimulation
TGF-b–induced effects are dictated by Smad expression levels and also by activation kinetics. We
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Fig. 1 – Smad RNA expression levels in primary fibroblasts derived from control patients (n = 4) and patients with Peyronie’s disease (n = 6). HSC-70 was used as a loading control; negative control (neg ctrl) was loaded in the last lane.
therefore characterized ligand-dependent Smad activation by phosphorylation and nuclear translocation of Smads. Fig. 2 depicts representative Western blot analyses of cytosolic and nuclear extracts obtained from fibroblasts cultures of control and Peyronie’s disease patients, and stimulated with TGF-b for 15, 30, 60 or 120 min. PhosphoSmad2/3 levels were detected in both cultures by Western blot analysis with activation state-specific antibodies. Smad2/3 phosphorylation characteristics differed between both cultures with respect to signal strength and onset. Fibroblasts from patients with Peyronie’s disease exhibited an earlier onset of phosphorylation, compared with control cells (15 min vs 30 min, respectively; Fig. 2). In addition, the phospho-Smad2/3 signal was stronger in fibroblasts from patients with Peyronie’s disease than in controls (Fig. 2). Smad2/3 is depicted for the
loading control, and the nuclear marker C23 as a control for the purity of the nuclear and cytosolic extraction. 3.3.
Smad translocation in response to TGF-b stimulation
We then analyzed whether Smad activation, as assessed by phosphorylation, coincided with nuclear translocation. We incubated fibroblast cultures for the indicated times (15, 30 or 60 min) with TGF-b, fixed the cells and performed immunofluorescence analysis of Smad3 and 4 localization. Translocation of Smad3 and 4 could be observed as early as 30 min after stimulation with TGF-b1 in control fibroblasts (Fig. 3). In contrast, nuclear translocation of Smad3 and 4 was observed as early as 15 min poststimulation in fibroblasts from Peyronie’s disease, corresponding to the earlier
Table 2 – Overview of the investigated Smads on RNA, protein and cellular levels Investigations
Smad1
Smad2
Smad3
Smad4
Smad5
Smad7
RNA: Smad expression levels
PD Control
+ +
(+) Partially very low +
++ +
++ +
+ +
Heterogeneous Heterogeneous
Protein: Smad activation in response to TGF-b stimulation
PD Control
Ø Ø
++ +
++ +
Ø Ø
Ø Ø
Ø Ø
Fibroblasts: Smad translocation in response to TGF-b stimulation
PD Control
Ø Ø
++ +
++ + Incomplete translocation
+ ++
Ø Ø
Ø Ø
TGF-b = transforming growth factor b.
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Fig. 2 – Representative Western blot analyses of cytosolic and nuclear extracts obtained from fibroblasts cultures of control and Peyronie’s disease stimulated with TGF-b for 15, 30, 60 or 120 min. TGF-b1 = transforming growth factor b1.
phosphorylation depicted in Fig. 2. After 60 min most of the Smad3/4 signal was still confined to the nuclear compartment, whereas it was predominantly cytosolic again for control fibroblasts (Fig. 3, last column).
4.
Discussion
In this study, we could demonstrate significant differences in the expression and activity of Smad
transcription factors in primary fibroblasts derived from control patients or patients with Peyronie’s disease. The Smads are key signal-transducing molecules of the TGF-b superfamily, a growth factor system essentially involved in the pathogenesis of fibrotic diseases. The TGF-b superfamily comprises over 30 distinct members, including TGF-bs, BMPs, activins, and growth and differentiation factors. These cytokines exhibit multifunctional and pleiotropic effects on cell proliferation, differentiation or migration [7].
Fig. 3 – Nuclear translocation of Smad3 and Smad4 in fibroblast cultures after stimulation with TGF-b for 15, 30 or 60 min. TGF-b1 = transforming growth factor b1.
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TGF-b regulates cellular processes by binding to specific cell-surface receptors, the type I and type II TGF-b receptors (TbRs). After ligand binding to the high-affinity type II receptor (TbRII), a heteromeric receptor complex of TGF-b/TbRI/TbRII is formed. The constitutively active TbRII kinase then activates TbRI by phosphorylation of the juxtamembrane GSregion (for glycine-serine rich). This event induces phosphorylation of the receptor-regulated Smad2/3 by the TbRI kinase. Activated Smad2/3 then forms heterotrimers with the co-Smad Smad4 and translocates to the nucleus. Negative feedback regulation of this pathway is given through inhibitory Smads (Smad6 and Smad7), which, when expressed, antagonize the phosphorylation of Smad2 and 3 by TbRI [9]. In our study, we chose to culture primary fibroblasts derived from the tunica albuginea or plaques of control patients or patients with Peyronie’s disease, respectively. We obtained viable and pure fibroblast cultures from 10 of 16 (62.5%) samples in the case of Peyronie’s disease. In the control group, viable fibroblast cultures were obtained 6 of 8 (75%) cases. Plaques of Peyronie’s disease patients contain a higher content of collagens, and in some cases calcification might decrease vital tissue, thus possibly explaining the lower percentage of viable cultures. The difficulty in obtaining fibroblasts from Peyronie’s disease specimens is well documented in the literature. In studies of Mulhall et al. [10], fibroblast cell cultures could be initiated in 5 of 10 (50%) plaque biopsies. In the study by Somers et al. [11], fibroblast cell cultures could be initiated in 19 of 23 (82.6%) plaque biopsies. In a recent study, El-Sakka et al. [3] demonstrated upregulation of TGF-b1, TGF-b2 and TGF-b3 protein expression in plaques of Peyronie’s disease patients. Consistent with these findings, we could detect increased Smad levels in primary fibroblasts cultures, documenting an overall expression increase of the TGF-b system in this disease. To the best of our knowledge, this finding has not been reported previously in Peyronie’s disease. Magee et al. [12] recently reported that Smad7, an inhibitory factor of the TGF-b pathway, was downregulated in Peyronie’s disease. Similar to our findings, Smad7 RNA expression was upregulated in primary fibroblasts from scleroderma patients, compared with normal fibroblasts, whereas the expression of Smad2, 3 and 4 were similar in both cultures [13]. To our knowledge, the more rapid translocation of Smad3 and 4 in Peyronie’s disease-derived fibroblasts has not been described before. A potential explanation of this finding is represented by the observation that Smads are also more potently and
rapidly phosphorylated in Peyronie’s disease, possibly because of the higher expression of TGF-b receptors on these cells, compared with normal tissue. Consistently, our study yielded similar results at the levels of RNA expression, Smad phosphorylation and nuclear translocation. A characteristic feature of the TGF-b system is its pleiotropic action (i.e., the observation that the biologic effect of TGF-b is strictly cell-type dependent). Although TGF-b induces cell proliferation in fibroblasts, it leads to potent growth arrest in epithelial cells, most likely triggered by similar signal transduction pathways [14]. In fibroblasts derived from chronic wounds, the concentration of pSmad2/3 was decreased in response to TGF-b1 more than 3-fold. Kim et al. [15] found decreased expression of TGF-b type II receptor in ulcer fibroblasts. Although the number of Peyronie’s disease and control specimens was small in our study, the results were very reproducible. Our study has shown differences in the TGF-b pathway of plaque-derived fibroblasts, compared with fibroblasts from tunica albuginea of controls. More studies with higher numbers and investigations on the TGF-b receptor levels will now dramatically increase our knowledge of the TGF-b system in Peyronie’s disease, and possibly unravel novel treatment regimen in this disease, which interferes with the TGF-b pathway.
5.
Conclusion
The expression and activity of Smad transcription factors of the TGF-b pathway are increased in fibroblasts of patients with Peyronie’s disease. We suggest that alterations in the TGF-b pathway represent a potential factor in the pathophysiology of Peyronie’s disease. Conflicts of interest This study was supported by a grant (Anschubfinanzierung) to E. W. Hauck donated by the Justus Liebig University, and by the Sofja Kovalevskaja Award to O. Eickelberg by the Alexander von Humboldt Foundation.
References [1] Gholami SS, Gonzalez-Cadavid NF, Lin CS, Rajfer J, Lue TF. Peyronie’s disease: a review. J Urol 2003;169:1234–41. [2] Devine Jr CJ, Somers KD, Jordan GH, Schlossberg SM. Proposal: trauma as the cause of the Peyronie’s lesion. J Urol 1997;157:285–90.
european urology 51 (2007) 255–261
[3] El-Sakka AI, Hassoba HW, Pillarisetty RJ, Dahiva R, Lue TF. Peyronie’s disease is associated with an increase in transforming growth factor-beta protein expression. J Urol 1997;158:1391–4. [4] Hauck EW, Hauptmann A, Schmelz HU, Bein G, Weidner W, Hackstein H. Prospective analysis of single nucleotide polymorphisms of the transforming growth factor b-1 gene in Peyronie’s disease. J Urol 2003;169:369. [5] Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signaling. Nature 2003;425:577–84. [6] El-Sakka AI, Hassoba HM, Chui RM, Bhatnagar RS, Dahiya R, Lue TF. An animal model of Peyronie’s like condition associated with an increase of transforming growth factor beta mRNA and protein expression. J Urol 1997;158:2284–90. [7] Lutz M, Knaus P. Integration of the TGF-beta pathway into the cellular signaling network. Cell Signal 2002;14:977–88. [8] Eickelberg O, Pansky A, Mussmann R. Transforming growth-factor-beta1 induces interleukin-6 expression via activating protein-1 consisting of JunD homodimers in primary human lung fibroblasts. J Biol Chem 1999;274: 12933.
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[9] Massague´ J. How cells read TGF-beta signals. Nat Rev Mol Cell Biol 2000;1:169–78. [10] Mulhall JP, Thom J, Lubrano T, Shankey TV. Basic fibroblast growth factor expression in Peyronie’s disease. J Urol 2001;165:419–23. [11] Somers KD, Dawson DM. Fibrin deposition in Peyronie’s disease plaque. J Urol 1997;157:311–5. [12] Magee TR, Qian A, Rajfer J, Sander FC, Levine LA, Gonzalez-Cadavid NF. Gene expression profiles in the Peyronie’s disease plaque. Urology 2002;59:451–7. [13] Asano Y, Ihn H, Yamane K, Kubo M, Tamaki K. Impaired Smad7-Smurf-mediated negative regulation of TGF-beta signaling in scleroderma fibroblasts. J Clin Invest 2004; 113:253–64. [14] Kretschmer A, Moepert K, Dames S, Sternberger M, Kaufmann J, Klippel A. Differential regulation of TGF-beta signaling through Smad2, Smad3 and Smad4. Oncogene 2003;22:6748–63. [15] Kim BC, Kim HT, Park SH, et al. Fibroblasts from chronic wounds show altered TGF-beta signaling and decreased TGF-beta Type II Receptor expression. J Cell Physiol 2003;195:331–6.