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Smad3 pathway
Biochemical Pharmacology 85 (2013) 1594–1602
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Thalidomide inhibits fibronectin production in TGF-b1-treated normal and keloid fibroblasts via inhibition of the p38/Smad3 pathway Chan-Jung Liang a,1, Yu-Hsiu Yen a,b,1, Ling-Yi Hung a, Shu-Huei Wang a, Chi-Ming Pu a,c, Hsiung-Fei Chien d, Jaw-Shiun Tsai e, Chiang-Wen Lee f, Feng-Lin Yen g, Yuh-Lien Chen a,* a
Institute of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taiwan Department of Plastic Surgery, Hsinchu Cathay General Hospital, Taiwan Department of Plastic Surgery, Cathay General Hospital, Taiwan d Department of Surgery, National Taiwan University Hospital, Taiwan e Department of Family Medicine, National Taiwan University Hospital, Taiwan f Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Taiwan g Department of France and Cosmetic Science, Kaohsiung Medical University, Taiwan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 January 2013 Accepted 27 February 2013 Available online 13 March 2013
Keloids are characterized by the vigorously continuous production of extracellular matrix protein and aberrant cytokine activity in the dermis. There is a growing body of evidence that thalidomide, a-Nphthalimidoglutarimide, has anti-fibrotic properties. The aims were to examine possible therapeutic effects of thalidomide on fibronectin expression in transforming growth factor-b1 (TGF-b1)-treated normal fibroblasts (NFs) and keloid-derived fibroblasts (KFs) and the underlying mechanism of action, especially the involvement of mitogen-activated protein kinase (MAPKs) and Sma- and Mad-related family (Smads) pathways. In surgically removed human tissues, TGF-b1 and fibronectin immunoreactivity was high in keloid tissue, but barely detectable in normal tissue. TGF-b1 induced significant fibronectin expression in NFs and KFs and the effect was inhibited by pretreatment with thalidomide. TGF-b1 also induced phosphorylation of MAPKs (ERK1/2, p38, and JNK) and Smad2/3 and pretreatment with PD98059 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor) inhibited TGF-b1-induced fibronectin expression. Furthermore, pretreatment with thalidomide inhibited the TGF-b1-induced phosphorylation of p38 and Smad3, but not that of ERK1/2, JNK, and Smad2. In addition, thalidomide pretreatment inhibited the TGF-b-induced DNA binding activity of AP-1 and Smad3/4, caused fibronectin degradation by increasing the activity of matrix metalloproteinase 9, and decreased production of TGF-b1 and fibronectin and the number of fibroblasts in an in vivo keloid model. These results show that thalidomide has an antifibrotic effect on keloid fibroblasts that is caused by suppression of TGF-b1-induced p38 and Smad3 signaling. Our findings indicate that thalidomide may be a potential candidate drug for the treatment and prevention of keloids. ß 2013 Elsevier Inc. All rights reserved.
Keywords: Thalidomide Keloid Fibronectin Mitogen-activated protein kinases (MAPKs) Fibroblasts SMADS
1. Introduction Keloids are defined as benign fibroproliferative scars formed as an abnormal wound healing response to cutaneous injury [1]. Their pathology can be distinguished from that of normal skin by the vigorous continuous production of extracellular matrix proteins in the dermis and aberrant cytokine and growth factor activity [2].
* Corresponding author at: Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, No. 1, Section 1, Ren-Ai Rd, Taipei 100, Taiwan. Tel.: +886 2 23123456 88176; fax: +886 2 33931713. E-mail address: [email protected] (Y.-L. Chen). 1 The first two authors contributed equally to this study. 0006-2952/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2013.02.038
Keloid formation severely impairs quality of life by causing cosmetic and functional deformities, discomfort, and psychological stress [3,4]. Current treatments, such as surgical excision, corticosteroid injections, compression therapy, and laser therapy, are not successful for keloids [4,5] and there is an urgent need for a better understanding of keloid pathogenesis in order to develop better prevention and treatment approaches. Although the exact pathophysiology of keloid formation is unclear, previous studies have focused on investigating abnormal increases in growth factors and cytokines, such as transforming growth factor-b1 (TGF-b1), which plays an important role in wound healing and scar formation [6–8]. Deep injury of the skin by trauma or surgery stimulates TGF-b1 secretion by all major cell types participating in wound repair and tissue fibrosis, especially fibroblasts [9], and
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prolonged or impaired healing can lead to unbalanced TGF-b1 activity and excessive scar formation [8]. TGF-b1 induces the activation of intracellular effectors, such as mitogen-activated protein kinases (MAPKs) and Sma- and Mad-related proteins (Smads) [10,11]. Continuous activation or decreased inhibition of the signaling pathway leads to a persistent autocrine-positive feedback loop that could result in the overproduction of matrix proteins and subsequent fibrosis [4,8]. Thus, many research groups have suggested modulation of these signaling mediators as a means of treating keloids [12,13]. Thalidomide (a-N-phthalimidoglutarimide), a synthetic derivative of glutamic acid, was widely prescribed in Germany and Britain as a sedative-hypnotic, sleep-inducing, and antiemetic reagent in the early1960s, but was withdrawn from the market because it was found to be a potent teratogen [14,15]. Later, Sheskin [16] fortuitously found that it was extremely effective in the treatment of erythema nodosum leprosum and, subsequently, it was reported to have a variety of biological effects, including anti-angiogenic, anti-inflammatory, and immunomodulating properties [17,18]. It also has anti-hepatofibrotic effects, due to reduced TGF-b1 expression [19]. Because of its antifibrotic ability, we examined whether thalidomide could reduce the skin defects associated with keloids. We found that thalidomide treatment inhibited fibronectin production by normal fibroblasts (NFs) and keloid fibroblasts (KFs) during TGF-b1 stimulation in vitro. Furthermore, we demonstrated that phosphorylation of MAPKs and Smad2/3 was enhanced by TGF-b1 and that the p38 and Smad3 signaling pathways were inhibited by thalidomide. Using specific inhibitors and small interfering RNA (siRNA), we found that p38 and Smad3 are important signaling mediators in the inhibitory mechanism of thalidomide. Finally, treatment with thalidomide markedly inhibited fibronectin production in keloid nodules in an in vivo keloid mice model. Our findings suggest that thalidomide has potential as a therapeutic and preventive agent for keloids. 2. Materials and methods 2.1. NF and KF culture Keloid lesions were taken from the earlobe, foot, or chest of five patients (5 female, age range 28-55 years) who had not received previous treatment for keloids before surgical excision and control skin tissue was obtained from 4 volunteers (3 males and 1 female, aged 36-61 years). The study was approved by the Medical and Ethics Committee of the Cathay General Hospital. Primary fibroblast cultures were established as described previously [20]. The specimens were repeatedly washed in sterile Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, NY, USA) supplemented with 100 U/mL of penicillin, 0.1 g/mL of streptomycin, and 1.25 ng/ mL of Fungizone (Gibco) and cut into 5 mm 5 mm pieces, then the epidermis was scraped off and the dermis transplanted onto culture plates and incubated in the same medium as above supplemented with 10% fetal bovine serum (FBS, Biological Industries, Israel) at 37 8C in a humidified incubator in 5% CO2. The fibroblasts migrated over the plastic plates and, when they reached 90% confluence, were subcultured using 0.25% trypsin (Biological Industries) and 0.04% ethylenediaminetetraacetic acid (EDTA, Sigma, MO, USA). The cells were passaged and cells from passages 3 to 8 were used. 2.2. Cell viability assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay was used to assess cell viability in response to thalidomide treatment. NFs or KFs were plated at a
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density of 103 cells/well in 96-well plates, then, after overnight growth, were treated with different concentrations of thalidomide for 48 h, then cell viability was measured using the MTT assay. In brief, MTT (0.5 mg/mL) was applied to the cells for 4 h to allow the conversion of MTT into formazan crystals, then, after washing with phosphate-buffered saline (PBS), the cells were lysed with dimethyl sulfoxide and the absorbance at 550 nm read on a DIAS Microplate Reader (Dynex Technologies, VA, USA). The optical density obtained with the thalidomide-treated cells was normalized to that of cells incubated in control medium, which were considered 100% viable. 2.3. Analysis of protein expression in cell lysates and nodule tissues Western blot analyses were performed as described previously [21]. Briefly, for cultured cells, a total cell lysate was prepared by lysing the cells for 1 h at 4 8C in 20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM PMSF (Bionovas, Ontario, CA), pH 7.4, containing protease inhibitors (Cell Signaling, MA, USA), then centrifuging the lysate at 12,000 g for 1 h at 4 8C and taking the supernatant. Nodule tissues were homogenized in the same lysis buffer and the homogenate centrifuged at 12,000 g for 30 min at 4 8C and the supernatant used. The protein content of the supernatants was measured using the BioRad protein assay (Bio-Rad, Hercules, CA, USA), then an aliquot (25 mg/mL of protein) was subjected to 10% SDS-PAGE, the proteins were transferred onto PVDF membranes (Millipore, MA, USA), and the membranes were blocked by incubation for 1 h at room temperature (RT) with 10% non-fat milk in Tris-buffered saline containing 0.2% Tween 20 (TBST, Bionovas). The membranes were then incubated overnight at 4 8C with rabbit antibodies against human TGF b1 (1:2000 in PBS, GeneTex, TX, USA), human fibronectin (1:500 in PBS, GeneTex), human phospho-JNK, human phospho-ERK1/2, human phospho-p38, phospho-Smad2, or phospho-Smad3 (1:1000 in PBS, Cell Signaling), then for 1 h at RT with HRP-conjugated goat anti-rabbit IgG antibodies (1:5000 in PBS, Santa Cruz,CA, USA), bound antibodies being detected using Chemiluminescence Reagent Plus (Millipore). The intensity of each band was quantified using a densitometer. Antibodies against GAPDH (1:5000, Santa Cruz) were used as loading controls. 2.4. siRNA transfection To investigate the effects of silencing of p38 and Smad3, siRNA for p38 (CGAAUCAAUGAUGUGUAU) or Smad 3 (CAACAGCAAUGVAGCAGUG, Thermo, PA, USA) was used as concentrations of 10 nM and 50 nM. Cells were transfected with siRNA overnight using Oligofectamine (Invitrogen, CA, USA) in Opti-MEM (Invitrogen), then protein lysates were prepared as described above and Western blot analysis performed to validate the efficiency of silencing. 2.5. Nuclear extract preparation and electrophoretic mobility shift assay (EMSA) The preparation of nuclear protein extracts and the EMSA conditions have been described previously [21]. Nuclear proteins were extracted using NE-PER reagent (Pierce, IL, USA) according to the manufacturer’s protocol. The AP-1 and Smad3/4 binding activity of equal amounts (15 mg) of nuclear protein was analyzed using a LightShift Chemiluminescent EMSA kit (Pierce). The synthetic double-stranded oligonucleotides used as the probes in the gel shift assay were 50 -CGC TTG ATG AGT CAG CCG GAA-30 and 30 -GCG AAC TAC TCA GTC GGC CTT-50 for AP-1 and 50 -TCG AGA GCC AGA CAA AAA GCC-30 and 30 -AGC TCT CGG TCT GTT TTT CGG 50 for Smad3/4.
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2.6. Gelatin zymography for MMP enzymatic activity Conditioned medium from control, TGF-b1-treated, or thalidomide+ TGF-b1 treated cells was collected and concentrated and equal amounts loaded onto 8% SDS polyacrylamide gels containing 1 mg/mL of gelatin for assessment of MMP activity. After gel electrophoresis, the gels were washed for 2 x for 30 min at RT in 2.5% Triton X-100 to remove the SDS and allow partial renaturation of the protein. The gels were then incubated overnight at 37 8C in 10 mM CaCl2, 150 mM NaCl, and 50 mM Tris-HCl, pH 7.5, stained with 0.2% Coomassie blue (Sigma), and photographed on a light box. Proteolysis was detected as a white zone in a dark blue field. For quantitative analysis, bands were scanned and the digital image analyzed. 2.7. Implantation of cultured human fibroblasts into nude mice and thalidomide treatment All procedures involving experimental animals were performed in accordance with the guidelines for animal care of the National Taiwan University and complied with the ‘‘Guide for the Care and Use of Laboratory Animals’’ NIH publication No. 86-23, revised 1985. We tested the therapeutic potential of thalidomide in the treatment of keloids using an in vivo model. Briefly, NFs and KFs (1 107 cells per injection) were implanted into the back muscle of Nu/Nu nude mice (The Jackson Lab, ME, USA) weighing 20 g using a syringe with a 26-gauge needle. At 7 days post-implantation, the mice were randomly grouped and given an intra-nodule injection of 100 mg/kg of thalidomide or vehicle (DMSO), then, 7 days later, blood samples were collected for assessment of liver and renal function and the animals sacrificed. Some nodule tissues were excised and fixed in formaldehyde for immunohistochemical analysis, while others were immediately frozen in liquid nitrogen for protein extraction and Western blot. 2.8. Hematoxylin and eosin staining and immunohistochemistry Normal and keloid nodules after 4% paraformaldehyde fixation and paraffin embedding were cut into 5-6 mm thick sections, washed with xylene to remove paraffin and hematoxylin, and subjected to eosin staining. Immunohistochemical staining was also performed on serial sections to detect expression of TGF-b1 and fibronectin. The first section was incubated for 1 h at 37 8C with rabbit monoclonal anti-human TGF-b1 antibody (1:50 dilution, GeneTex), washed with PBS, and incubated for 1 h at RT with HRP-conjugated goat anti-rabbit IgG antibodies (1:200 dilution in PBS, Sigma) and bound antibody visualized using 3,30 diaminobenzidine (Sigma). The second section was incubated with rabbit antibodies against human fibronectin (1:100, Gene Tex) for 1 h at 4 8C, then processed as above. 2.9. Statistical analysis Where applicable, the results are presented as the mean SEM. Statistical analysis was performed by ANOVA, followed by Fisher’s multiple comparison post test. A p value <0.05 was considered statistically significant. 3. Results 3.1. Thalidomide inhibits fibronectin expression in TGF-b1-treated NFs and KFs Expression of TGF-b1 and fibronectin in human keloid tissues was evaluated by immunohistochemistry. In surgically removed normal tissue from controls and keloid tissue, TGF-b1 and
Fig. 1. Expression of TGF-b1 and fibronectin in human keloid (A) and normal (B) skin tissues. Expression of TGF-b1 (top row) or fibronectin (center row) is shown by arrows; NC is the negative control without primary antibody. In each row, the rectangle in the panel to the left is magnified in the next panel to the right. Each bar = 100 mm.
fibronectin immunoreactivity was high in keloid tissue (Fig. 1A; each panel shows a higher magnification of the rectangular area on the previous panel), but barely detectable in normal tissue (Fig. 1B). These results show that fibronectin is highly expressed in fibroblasts in human keloid tissue sections, making this molecule an ideal target for selection of fibronectin-targeted agents. When the cytotoxicity of thalidomide for cultured NFs and KFs was assessed by the MTT assay after 48 h of incubation, cell viability was not affected by the presence of 39, 195, or 390 mM of thalidomide (95 1%, 97 3%, 98 2% of control). But 1950 or 3900 mM of thalidomide treatment caused a significant decrease in cell viability (78 2% and 42 1% of control, respectively). As shown in Fig. 2A, TGF-b1 induced significant expression of fibronectin in NFs (left panel) and KFs (right panel) in a dose-dependent manner, which peaked at 10 ng/mL. To examine whether thalidomide inhibited fibronectin production, NFs or KFs were incubated with different concentrations of thalidomide for 24 h, then with 10 ng/mL of TGF-b1 in the continued presence of thalidomide for 24 h and Western blotting was performed. As shown in Fig. 2B, TGF-b1 induced fibronectin expression in NFs and KFs that was, respectively, 2.1 0.2- and 5.1 0.4-fold higher than control levels. Pretreatment for 24 h with 39, 195, or 390 mM of thalidomide resulted, respectively, in TGF-b1-induced fibronectin expression that was 2.3 0.2-, 1.5 0.1-, or 0.5 0.1-fold higher in NFs and 5.1 0.3-, 4.1 0.3-, or 1.6 0.1-fold higher in KFs than in controls, the effects being significant at 390 mM in NFs and 195 and 390 mM in KFs. In all subsequent experiments, unless otherwise specified, 10 ng/mL of
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Fig. 2. Thalidomide reduced TGF-b1-induced fibronectin expression in NFs and KFs by Western blotting and immunofluorescent staining. (A) Fibroblasts from normal (NF) or keloid (KF) tissues were incubated with different doses of TGF-b1 for 24 h, then fibronectin expression was measured by Western blotting. GAPDH was used as a loading control. (B) NFs or KFs were incubated without or with 39, 195, or 390 mM of thalidomide for 24 h, then with or without 10 ng/mL of TGF-b1 in the continued presence of thalidomide for 24 h, when Western blotting for fibronectin was performed. The results are shown as the fold increase in fibronectin expression relative to that in untreated NFs. The data are the mean SEM (n = 4). *p < 0.05 compared to the value for untreated NFs or untreated KFs, respectively. yp < 0.05 vs TGF-b1-treated NFs or TGF-b1-treated KFs, respectively. (C) NFs or KFs were incubated with 100 mg/mL of thalidomide for 24 h, then with 10 ng/mL of TGF-b1 in the continued presence of thalidomide for 24 h, when immunofluorescent staining for fibronectin was performed. Experiments were repeated at least three times with similar results.
TGF-b1 and 390 mM of thalidomide were used. Interestingly, as shown in Fig. 2B, KFs expressed significantly more fibronectin than NFs in the absence of TGF-b1 treatment and thalidomide treatment inhibited fibronectin expression in KFs. Moreover, immunofluorescent staining (Fig. 2C) showed that fibronectin expression correlated with levels on Western blots and that thalidomide inhibited fibronectin expression in TGF-b1-treated NFs and KFs.
3.2. The thalidomide-induced reduction in TGF-b1-induced fibronectin expression is dependent on inhibition of p38 phosphorylation Since TGF-b1-activates the MAPK pathway [10], we next investigated whether the TGF-b1-induced fibronectin expression in NFs and KFs was mediated by activation of MAPKs. As
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Fig. 3. The thalidomide-mediated reduction in TGF-b1-induced fibronectin expression is dependent on inhibition of p38 phosphorylation. (A) NFs or KFs were incubated with 10 ng/mL of TGF-b1 for the indicated time, then the cell lysates were analyzed for MAPK phosphorylation by Western blotting using antibodies against p-ERK1/2 (top panels), p-p38 (center panels), or p-JNK (bottom panels). (B) The cells were preincubated for 1 h with the indicated concentration of PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), or SP600125 (JNK inhibitor), then with TGF-b1 in the continued presence of the inhibitor for 24 h and the cell lysate analyzed for fibronectin expression by Western blotting. (C) Western blot analysis showing the effect of thalidomide treatment on the phosphorylation of p-ERK1/2 (top panels), p-p38 (center panels), or p-JNK (bottom panels) in TGF-b1-treated NFs or KFs. Cells were incubated for 24 h with or without 390 mM of thalidomide, then with 10 ng/mL of TGF-b1 in the continued presence of the thalidomide for 1 h and aliquots of cell lysate containing equal amounts of protein were subjected to immunoblotting with the indicated antibodies. The data are expressed as a fold of the control value and are the mean SEM for 3 separate experiments. GAPDH was used as the loading control. *P < 0.05 vs untreated NFs or KFs. yP < 0.05 vs TGF-b1treated NFs or KFs. (D) NFs or KFs were left untreated or were transfected with p38 siRNA overnight, then incubated with 390 mM of thalidomide for 24 h, and with 10 ng/mL of TGFb1 in the continued presence of thalidomide for 24 h, when Western blotting was performed. The data are expressed as a fold of the control value and are the mean SEM for 3 separate experiments. GAPDH was used as the loading control. *P < 0.05 vs untreated NFs or KFs, respectively. yP < 0.05 vs TGF-b1-treated NFs or KFs, respectively. zP < 0.05 vs TGFb1-treated siSmad3-transfected KFs.
shown in Fig. 2A, phosphorylation of ERK1/2, p38, and JNK in both NFs and KFs showed a significant increase at 10-30 min of TGF-b1 treatment and increased continuously up to at least 1 h. In addition, pretreatment for 1 h with the indicated concentrations of PD98059 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor), followed by and cotreatment with TGF-b1 for 24 h inhibited the increase in fibronectin expression (Fig. 3B). These results suggest that TGF-b1-induced fibronectin expression is mediated by activation of MAPKs in NFs and KFs. To examine whether thalidomide had an effect on the TGF-b1induced activation of MAPK signaling pathways in NFs and KFs, the cells were preincubated with thalidomide for 24 h and coincubated with thalidomide and TGF-b1 for 1 h. As shown in Fig. 3C, thalidomide treatment significantly inhibited TGF-b1induced p38 phosphorylation by 45% in NFs and 36% in KFs, but had no significant effect on ERK and JNK phosphorylation. As shown in Fig. 3D, transfection of NFs or KFs with 10 or 50 nM p38-specific siRNA inhibited the expression of p38 by 50% and that of fibronectin by 40%. Importantly, combined treatment with thalidomide and p38 siRNA reduced fibronectin expression in TGF-treated KFs by about 65%.
3.3. The thalidomide-induced reduction in TGF-b1-induced fibronectin expression in NFs and KFs is dependent on inhibition of phosphorylation of Smad3, but not Smad 2 Since Smad proteins act as intracellular signaling mediators of TGF-b [10], we examined levels of phosphorylated Smad2 and Smad3 in NFs and KFs treated with TGF-b1. As shown in Fig. 4A, treatment of NFs or KFs with different concentrations of TGF-b1 resulted in a significant increase in levels of phosphorylated Smad2 (top panels) and Smad3 (bottom panels). As the Smad signaling pathway plays a significant role in keloid formation [11,13], Western blotting was performed to examine whether thalidomide reduced TGF-b1-induced fibronectin expression in NFs and KFs through Smad phosphorylation. As shown in Fig. 4B, thalidomide significantly inhibited TGF-b1-induced Smad3 phosphorylation in NFs and KFs (bottom panels), but had no effect on Smad2 phosphorylation (top panels). To confirm the role of Smad3 in TGFb1-induced fibronectin production, fibroblasts were transfected with siRNAs targeting Smad3 and Western blotting performed. As shown in Fig. 4C, transfection of cells with 50 nM Smad3 siRNA resulted in a 70% reduction in Smad3 protein expression and markedly inhibited fibronectin expression in TGF-b1-treated NFs
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Fig. 4. The thalidomide-mediated reduction in TGF-b1-induced fibronectin expression in NFs and KFs is dependent on inhibition of phosphorylation of Smad3, but not Smad 2. (A) NFs or KFs were treated with 10 ng/mL of TGF-b1 for the indicated time, then the cell lysates were analyzed for phosphorylation of Smad2 and Smad3 by Western blotting. (B) Western blot analysis showing the effect of thalidomide treatment on the phosphorylation of Smad2 and Smad3 in TGF-b1treated NFs or KFs. Cells were incubated for 24 h with or without 390 mM of thalidomide, then were incubated with 10 ng/mL of TGF-b1 in the continued presence of thalidomide for 1 h and aliquots of cell lysates containing equal amounts of protein were subjected to immunoblotting with the indicated antibodies. (C) NFs or KFs were left untreated or were transfected with Smad siRNA overnight, then incubated with 100 mg/mL of thalidomide for 24 h, and with 10 ng/mL of TGF-b1 in the continued presence of thalidomide for 24 h, when Western blotting was performed. The data are expressed as a fold of the control value and are the mean SEM for 3 separate experiments. GAPDH was used as the loading control. *P < 0.05 vs untreated NFs or KFs, respectively. yP < 0.05 vs TGF-b1treated NFs or KFs, respectively. zP < 0.05 vs TGF-b1-treated siSmad3-transfected KFs.
and KFs by 60% and 45%, respectively. Taken together, these results demonstrate that Smad3, but not Smad2, is involved in thalidomide-mediated inhibition of TGF-b1-induced fibronectin production.
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Fig. 5. Thalidomide reduces the DNA binding activity of AP-1 and Smad3/4 in TGFb1-treated NFs and KFs by EMSA. (A, B) Nuclear extracts from untreated NFs or KFs with or without pretreatment with 390 mM of thalidomide, then incubated with 10 ng/mL of TGF-b1 for 1 h were tested for AP-1 (A) or Smad3/4 (B) DNA binding activity by EMSA. (C) NFs or KFs were preincubated for 1 h with the indicated concentration of tanshinone IIA (Tan IIA, an AP-1 inhibitor), then were treated with TGF-b1 in the continued presence of the inhibitor for 24 h and the cell lysates analyzed for fibronectin expression by Western blotting. *p < 0.05 compared to the value for untreated NFs or KFs, respectively. yp < 0.05 vs TGF-b1-treated NFs or KFs, respectively.
3.4. Thalidomide decreases AP-1 and Smad 3/4 binding activity in TGF-b1-treated NFs and KFs in an electrophoretic mobility shift assay Since the fibronectin gene promoter contains consensus binding sites for AP-1 and Smad3/4 [22], we examined whether thalidomide inhibited TGF-b1-induced fibronectin expression via an effect on these transcription factors in a gel-shift assay. As shown in Fig. 5, low basal levels of AP-1 (Fig. 5A) and Smad3/4 (Fig. 5B) binding activity were detected in untreated control cells, binding activity was significantly increased by 1 h treatment with TGF-b1, and this effect was blocked by 24 h pretreatment with thalidomide. Furthermore, the stimulatory effect of TGF-b1 on fibronectin levels was blocked by preincubation and co-incubation with 0-20 mM tanshinone IIA, an AP-1 inhibitor (Fig. 5C). These results show that thalidomide inhibits TGF-b1-induced fibronectin expression by inhibiting AP-1 and Smad3/4 activation. 3.5. Thalidomide induces fibronectin degradation through MMP-9 activity as shown by gelatin zymography Since MMP-9 activity is important in the degradation of extracellular matrix proteins [23], we next explored whether
thalidomide reduced the TGF-b1-induced increase in fibronectin expression by induction of MMP-9 activity using gelatin zymography. As shown in Fig. 6A, conditioned medium from cells treated with thalidomide (both with and without TGF) caused a significant increase in MMP-9 activity in NFs or KFs with or without TGF-b1 treatment and, as shown in Fig. 6B, co-incubation of the cultures with 10 mM GM6001 (an MMP inhibitor) markedly reduced the effects of thalidomide on fibronectin expression in TGF-b1-treated, but not control, NFs and KFs. 3.6. Thalidomide markedly inhibits nodule development and fibronectin production in an in vivo keloid model To evaluate the therapeutic potential of thalidomide in the treatment of keloids, we implanted cultured NFs and KFs into the backs of nude mice which were then randomly divided into three groups at day 7 after implantation, one of which was left untreated, while the other two were treated by intra-nodule injection with 100 mg/kg thalidomide or DMSO. No significant change in the weight and appearance of the mice or in liver and renal function
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Fig. 6. Thalidomide increases MMP-9 activity in TGF-b1-treated NFs or KFs by gelatin zymography. (A) NFs or KFs were incubated with or without 390 mM of thalidomide for 24 h, then with 10 ng/mL of TGF-b1 in the continued presence of thalidomide for 24 h and the cconditioned medium collected and tested by gelatin zymography. (B) NFs or KFs were incubated with or without 390 mM of thalidomide for 24 h, then 10 mM GM6001 (an inhibitor of MMP activity) was added to the mixture for 1 h, followed by 10 ng/mL of TGF-b1 for 24 h. The results are shown as the fold increase in expression relative to that in untreated controls. *P < 0.05 vs untreated NFs or KFs, respectively. yP < 0.05 vs TGF-b1-treated NFs or KFs, respectively. zP < 0.05 vs TGF-b1+ thalidomide-treated NFs or KFs, respectively.
was seen in any of the experimental groups. Histologically, the cellularity was higher in the KF nodules than in the NF nodules, as shown by hematoxylin and eosin staining, whereas blood vessels were equally well developed in NF and KF nodules (Fig. 7A). As shown in Fig. 7B, thalidomide treatment reduced cellularity, but not blood vessel formation, in the keloid nodules. We then examined TGF-b1 and fibronectin expression by immunohistochemistry. As shown in Fig. 7C, strong expression of TGF-b1 and fibronectin was seen in the nodules of KF-implanted mice, but not in those from NF-implanted mice and this effect was inhibited by about 90% by thalidomide treatment. In agreement with this, as shown in the Western blots in Fig. 7D, expression of TGF-b1 and fibronectin was high in keloid nodules from non-thalidomidetreated animals, but scarcely detectable in the thalidomide-treated mice. These observations show that thalidomide inhibits keloid development and TGF-b1 and fibronectin production in an in vivo keloid model. 4. Discussion Keloids occur as a result of a pathological wound-healing process, characterized by excessive production of extracellular matrix protein. In this study, we demonstrated that thalidomide treatment reduced fibronectin expression in vitro in TGF-b1treated NFs and KFs and in an in vivo keloid mice model. The
Fig. 7. Thalidomide reduces cell number and the production of TGF-b1 and fibronectin in an in vivo model. (A) NFs and KFs (1 107 cells per injection) were implanted into the back muscle of Nu/Nu nude mice. At 7 days post implantation, mice were randomly grouped and were left untreated or were given intra-nodule injections of 100 mg/kg of thalidomide or the vehicle (DMSO). All animals were sacrificed 14 days after implantation and the nodules excised (NF, NF-derived nodules; KF, KF-derived nodules). Representative photographs of NF and KF nodules excised from the back of nude mice by hematoxylin and eosin staining. (B) Average cell number and capillary density per high power field (HPF) in the excised nodules. *P < 0.05 vs untreated NFs. yP < 0.05 vs untreated or DMSO-treated KFs. (C) Expression of fibronectin and TGF-b1 in each nodule examined by immunostaining. (D) Expression of fibronectin and TGF-b1 in each nodule examined by Western blotting. GAPDH was used as the control.
anti-fibrotic effect of thalidomide was partly mediated by inhibition of both phosphorylation of p38 and Smad3 and activation of transcription factors AP-1 and Smad3/4. In addition, thalidomide treatment in the presence or absence of TGF-b1 increased MMP-9 activity, resulting in fibronectin degradation. More than 50 years have passed since thalidomide was found to be a teratogen in humans and laboratory animals. However, it is now known to be of great value because of its anti-inflammatory, immunosuppressive, anti-oncogenic, and anti-fibrotic properties in many experimental and clinical trials [17,24–26]. In vitro study has demonstrated that thalidomide inhibits TNF-a production by lipopolysaccharide-stimulated human blood monocytes [27]. Apart from its effect on TNF-a, thalidomide modulates adhesiveness in microvascular beds by modifying levels of surface cell adhesion molecules and inhibits nuclear transcription factor nuclear factor-kB (NF-kB) activity [28]. It has also been shown that thalidomide treatment before ischemic insult reduces earlyphase ischemia/reperfusion injury to the spinal cord in rabbits [29]. Moreover, thalidomide has been used to treat acute and chronic liver diseases, which are characterized by the deposition of extracellular matrix protein. Intraperitoneal injection of thalidomide inhibits lethal hepatic necroinflammation and accelerates recovery from rat liver cirrhosis induced by thioactamide, and the
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Fig. 8. A summary diagram showing that thalidomide reduced fibronectin expression in TGF-b1-treated NFs and KFs through the inhibition of both phosphorylation of p38 and Smad3 and activation of transcription factors AP-1 and Smad3/4. In addition, thalidomide treatment in the presence or absence of TGF-b1 increased MMP-9 activity, resulting in fibronectin degradation.
mechanism may involve decreased expression of TNF-a and TGF-b by Kupffer cells [26]. Expression of TGF-b mRNA and protein in rat liver cirrhosis is significantly inhibited by thalidomide [19]. Thalidomide also prevents bleomycin-induced pulmonary fibrosis in mice by inhibiting production of TGF-b1 and collagen [30]. Importantly, the safety of thalidomide has been confirmed by many research groups and it has been approved by the Food and Drug Administration for the treatment of erythema nodosum leprosum [25,31]. Thus, thalidomide has potential to treat keloids, which are also characterized by overproduction of extracellular matrix proteins. Here, we demonstrated that TGF-b1 induced significant fibronectin expression in NFs and KFs and that the effect was inhibited by thalidomide. To our knowledge, this is first report demonstrating that thalidomide reduces fibronectin expression in TGF-b1-treated NFs or KFs and in KFs without TGF-b1 treatment. Due to the close relationship between TGF-b1 signaling and fibronectin production [32], the blocking of TGF-b signaling has potential for decreasing fibronectin synthesis, thereby preventing the formation of keloids. The TGF-b1 signaling pathway in fibrogenesis is mainly mediated and modulated by MAPKs and Smads [10]. In this study, we analyzed the roles of various signaling pathways downstream of TGF-b1 and found that TGF-b1 activated the three MAPK cascades involving p38, ERK, and JNK and that TGFb1-induced fibronectin secretion was inhibited by ERK, JNK, and p38 inhibitors, showing that the increased synthesis of fibronectin induced by TGF-b1 is mediated by activation of ERK, JNK, and p38. Thalidomide treatment did not affect TGF-b1-induced ERK and JNK MAPK phosphorylation, but resulted in a significant reduction in phosphorylated p38 in both NFs and KFs. Furthermore, the present study demonstrated that TGF-b1 activated phosphorylation of Smad2 and Smad3 in NFs or KFs. Importantly, thalidomide blocked phosphorylation of Smad3, not that of Smad2, and Smad3 silencing decreased fibronectin production by NFs and KFs. These results are consistent with previous reports [33,34], which demonstrated that Smad3, but not Smad2, is required for the fibrotic response. In HK-2 cells, bone morphogenetic protein functions as an antifibrogenic factor that ameliorates the TGF-b1induced expression of fibronectin, a fibrosis marker, and this effect is mediated by inhibition of JNK and Smad2/3 activation and modulation of p38 activation [35]. The activated p38 pathway phosphorylates Smad3, thereby promoting Smad signaling and extracellular matrix deposition, leading to liver fibrosis [36]. Our data demonstrated that the major mechanism for the antifibrotic effect of thalidomide involved inhibition of the TGF-b1-induced activation of p38 and Smad3. Whether the reduced p38 activation induced by thalidomide regulates Smad signaling, thereby reducing fibronectin deposition and leading to keloids requires further investigation.
The TGF-b signal itself is predominantly transduced by a family of transcriptional factors, the Smad proteins [22]. Binding of TGF-b to its receptor activates receptor kinase, which then phosphorylates the receptor-regulated Smads Smad2 and Smad3, which then associate with Smad4, and the Smad complex translocates into the nucleus where it regulates the expression of target genes. In the nucleus, the Smad3/4 or Smad2/3/4 complex can activate transcription by binding directly to a certain sequence in DNA, the consensus sequence, which is present in the promoter region of several TGF-b target genes, including those important in fibrosis, e.g., those coding for fibronectin and collagen. Smad3 mediates TGF-b1-induced fibronectin expression in mesangial cells [37]. AP1 is another important signal transducer of TGF-b1 and plays an important role in its biological effects [38]. The AP-1 transcriptional complex is also a primary target of a number of MAPK pathways. Our results showed that TGF-b1 induced AP-1 and Smad 3/4 activity and that thalidomide reduced the activity of these transcription factors in an electrophoretic mobility shift assay. Previous reports and our present findings suggest that the Smad3/4 and AP-1 pathways participate in the activation of fibronectin synthesis. In addition, our study demonstrated that the thalidomide-induced suppression of fibronectin expression is mediated by inactivation of the AP-1 and Smad3/4 pathways in keloid development. Whether AP-1 components can cross-talk with Smad3 and that thalidomide affect the interaction need further investigation. Histologically, in an in vivo keloid model, the nodules from KFimplanted mice showed higher cellularity than those from NFimplanted mice, as demonstrated by hematoxylin and eosin staining. This result is consistent with the observation that KFs grow faster than NFs [39,40]. Thalidomide treatment significantly suppressed cellularity in KF nodules, but had no effect on the well developed blood vessels in keloid nodules in our study, whereas orally administered thalidomide was found to be an inhibitor of angiogenesis induced by basic fibroblast growth factor in a rabbit cornea micropocket assay [18]. These findings indicate that thalidomide may have different therapeutic potential in different diseases. TGF-b1 and fibronectin expression was high in the nodules from KF-implanted mice, but not in those from NFimplanted mice, and accumulation of TGF-b1 and fibronectin in keloid nodules was inhibited by thalidomide treatment. These observations suggest that thalidomide inhibits keloid development by inhibition of TGF-b1 and fibronectin production in an in vivo keloid model. The previous study reported that the E3 ligase protein cereblon (CRBN) was identified as a direct molecular target for the teratogenic effects of thalidomide [41]. CRBN levels were also shown to be critical for the antitumor activity of thalidomide in both in vitro model systems and in lenalidomide-resistant
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patients [42]. However, whether CRBN is involved in the antifibrotic effects of thalidomide on keloid need further study. In summary, we have demonstrated that thalidomide treatment resulted in less phosphorylation of p38 and Smad3 in NFs and KFs and that this was closely correlated with decreased TGF-b1induced expression of fibronectin (Fig. 8). In addition, thalidomide markedly reduced nodule development and TGF-b1 and fibronectin production in an in vivo keloid model. Together, these results suggest that thalidomide may have potential in the treatment and prevention of keloids and excessive scarring. Author contributions Conception and design, Chan-Jung Liang, Yu-Hsiu Yen, and Yuh-Lien Chen.; analysis and interpretation, Chan-Jung Liang, YuHsiu Yen, Ling-Yi Hung, and Yuh-Lien Chen.; drafting the manuscript for important intellectual content, Ling-Yi Hung, Shu-Huei Wang, Chi-Ming Pu, Hsiung-Fei Chien, Jaw-Shiun Tsai, Chiang-Wen Lee, and Feng-Lin Yen.; revising the manuscript for important intellectual content, Chan-Jung Liang, Yu-Hsiu Yen, and Yuh-Lien Chen.; and final approval of the manuscript, Chan-Jung Liang, Yu-Hsiu Yen, Ling-Yi Hung, Shu-Huei Wang, Chi-Ming Pu, Hsiung-Fei Chien, Jaw-Shiun Tsai, Chiang-Wen Lee, Feng-Lin Yen, and Yuh-Lien Chen.
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Conflict of interest The authors state there is no conflict of interest. Acknowledgments This work was supported by research grants from the National Science Council (NSC 99-2320-B-002-022-MY3) and Cathay General Hospital (CGH-MR-9927), Taiwan, Republic of China.
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Thalidomide inhibits fibronectin production in TGF-b1-treated normal and keloid fibroblasts via inhibition of the p38/Smad3 pathway Chan-Jung Liang a,1, Yu-Hsiu Yen a,b,1, Ling-Yi Hung a, Shu-Huei Wang a, Chi-Ming Pu a,c, Hsiung-Fei Chien d, Jaw-Shiun Tsai e, Chiang-Wen Lee f, Feng-Lin Yen g, Yuh-Lien Chen a,* a
Institute of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taiwan Department of Plastic Surgery, Hsinchu Cathay General Hospital, Taiwan Department of Plastic Surgery, Cathay General Hospital, Taiwan d Department of Surgery, National Taiwan University Hospital, Taiwan e Department of Family Medicine, National Taiwan University Hospital, Taiwan f Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Taiwan g Department of France and Cosmetic Science, Kaohsiung Medical University, Taiwan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 January 2013 Accepted 27 February 2013 Available online 13 March 2013
Keloids are characterized by the vigorously continuous production of extracellular matrix protein and aberrant cytokine activity in the dermis. There is a growing body of evidence that thalidomide, a-Nphthalimidoglutarimide, has anti-fibrotic properties. The aims were to examine possible therapeutic effects of thalidomide on fibronectin expression in transforming growth factor-b1 (TGF-b1)-treated normal fibroblasts (NFs) and keloid-derived fibroblasts (KFs) and the underlying mechanism of action, especially the involvement of mitogen-activated protein kinase (MAPKs) and Sma- and Mad-related family (Smads) pathways. In surgically removed human tissues, TGF-b1 and fibronectin immunoreactivity was high in keloid tissue, but barely detectable in normal tissue. TGF-b1 induced significant fibronectin expression in NFs and KFs and the effect was inhibited by pretreatment with thalidomide. TGF-b1 also induced phosphorylation of MAPKs (ERK1/2, p38, and JNK) and Smad2/3 and pretreatment with PD98059 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor) inhibited TGF-b1-induced fibronectin expression. Furthermore, pretreatment with thalidomide inhibited the TGF-b1-induced phosphorylation of p38 and Smad3, but not that of ERK1/2, JNK, and Smad2. In addition, thalidomide pretreatment inhibited the TGF-b-induced DNA binding activity of AP-1 and Smad3/4, caused fibronectin degradation by increasing the activity of matrix metalloproteinase 9, and decreased production of TGF-b1 and fibronectin and the number of fibroblasts in an in vivo keloid model. These results show that thalidomide has an antifibrotic effect on keloid fibroblasts that is caused by suppression of TGF-b1-induced p38 and Smad3 signaling. Our findings indicate that thalidomide may be a potential candidate drug for the treatment and prevention of keloids. ß 2013 Elsevier Inc. All rights reserved.
Keywords: Thalidomide Keloid Fibronectin Mitogen-activated protein kinases (MAPKs) Fibroblasts SMADS
1. Introduction Keloids are defined as benign fibroproliferative scars formed as an abnormal wound healing response to cutaneous injury [1]. Their pathology can be distinguished from that of normal skin by the vigorous continuous production of extracellular matrix proteins in the dermis and aberrant cytokine and growth factor activity [2].
* Corresponding author at: Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, No. 1, Section 1, Ren-Ai Rd, Taipei 100, Taiwan. Tel.: +886 2 23123456 88176; fax: +886 2 33931713. E-mail address: [email protected] (Y.-L. Chen). 1 The first two authors contributed equally to this study. 0006-2952/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2013.02.038
Keloid formation severely impairs quality of life by causing cosmetic and functional deformities, discomfort, and psychological stress [3,4]. Current treatments, such as surgical excision, corticosteroid injections, compression therapy, and laser therapy, are not successful for keloids [4,5] and there is an urgent need for a better understanding of keloid pathogenesis in order to develop better prevention and treatment approaches. Although the exact pathophysiology of keloid formation is unclear, previous studies have focused on investigating abnormal increases in growth factors and cytokines, such as transforming growth factor-b1 (TGF-b1), which plays an important role in wound healing and scar formation [6–8]. Deep injury of the skin by trauma or surgery stimulates TGF-b1 secretion by all major cell types participating in wound repair and tissue fibrosis, especially fibroblasts [9], and
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prolonged or impaired healing can lead to unbalanced TGF-b1 activity and excessive scar formation [8]. TGF-b1 induces the activation of intracellular effectors, such as mitogen-activated protein kinases (MAPKs) and Sma- and Mad-related proteins (Smads) [10,11]. Continuous activation or decreased inhibition of the signaling pathway leads to a persistent autocrine-positive feedback loop that could result in the overproduction of matrix proteins and subsequent fibrosis [4,8]. Thus, many research groups have suggested modulation of these signaling mediators as a means of treating keloids [12,13]. Thalidomide (a-N-phthalimidoglutarimide), a synthetic derivative of glutamic acid, was widely prescribed in Germany and Britain as a sedative-hypnotic, sleep-inducing, and antiemetic reagent in the early1960s, but was withdrawn from the market because it was found to be a potent teratogen [14,15]. Later, Sheskin [16] fortuitously found that it was extremely effective in the treatment of erythema nodosum leprosum and, subsequently, it was reported to have a variety of biological effects, including anti-angiogenic, anti-inflammatory, and immunomodulating properties [17,18]. It also has anti-hepatofibrotic effects, due to reduced TGF-b1 expression [19]. Because of its antifibrotic ability, we examined whether thalidomide could reduce the skin defects associated with keloids. We found that thalidomide treatment inhibited fibronectin production by normal fibroblasts (NFs) and keloid fibroblasts (KFs) during TGF-b1 stimulation in vitro. Furthermore, we demonstrated that phosphorylation of MAPKs and Smad2/3 was enhanced by TGF-b1 and that the p38 and Smad3 signaling pathways were inhibited by thalidomide. Using specific inhibitors and small interfering RNA (siRNA), we found that p38 and Smad3 are important signaling mediators in the inhibitory mechanism of thalidomide. Finally, treatment with thalidomide markedly inhibited fibronectin production in keloid nodules in an in vivo keloid mice model. Our findings suggest that thalidomide has potential as a therapeutic and preventive agent for keloids. 2. Materials and methods 2.1. NF and KF culture Keloid lesions were taken from the earlobe, foot, or chest of five patients (5 female, age range 28-55 years) who had not received previous treatment for keloids before surgical excision and control skin tissue was obtained from 4 volunteers (3 males and 1 female, aged 36-61 years). The study was approved by the Medical and Ethics Committee of the Cathay General Hospital. Primary fibroblast cultures were established as described previously [20]. The specimens were repeatedly washed in sterile Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, NY, USA) supplemented with 100 U/mL of penicillin, 0.1 g/mL of streptomycin, and 1.25 ng/ mL of Fungizone (Gibco) and cut into 5 mm 5 mm pieces, then the epidermis was scraped off and the dermis transplanted onto culture plates and incubated in the same medium as above supplemented with 10% fetal bovine serum (FBS, Biological Industries, Israel) at 37 8C in a humidified incubator in 5% CO2. The fibroblasts migrated over the plastic plates and, when they reached 90% confluence, were subcultured using 0.25% trypsin (Biological Industries) and 0.04% ethylenediaminetetraacetic acid (EDTA, Sigma, MO, USA). The cells were passaged and cells from passages 3 to 8 were used. 2.2. Cell viability assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay was used to assess cell viability in response to thalidomide treatment. NFs or KFs were plated at a
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density of 103 cells/well in 96-well plates, then, after overnight growth, were treated with different concentrations of thalidomide for 48 h, then cell viability was measured using the MTT assay. In brief, MTT (0.5 mg/mL) was applied to the cells for 4 h to allow the conversion of MTT into formazan crystals, then, after washing with phosphate-buffered saline (PBS), the cells were lysed with dimethyl sulfoxide and the absorbance at 550 nm read on a DIAS Microplate Reader (Dynex Technologies, VA, USA). The optical density obtained with the thalidomide-treated cells was normalized to that of cells incubated in control medium, which were considered 100% viable. 2.3. Analysis of protein expression in cell lysates and nodule tissues Western blot analyses were performed as described previously [21]. Briefly, for cultured cells, a total cell lysate was prepared by lysing the cells for 1 h at 4 8C in 20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM PMSF (Bionovas, Ontario, CA), pH 7.4, containing protease inhibitors (Cell Signaling, MA, USA), then centrifuging the lysate at 12,000 g for 1 h at 4 8C and taking the supernatant. Nodule tissues were homogenized in the same lysis buffer and the homogenate centrifuged at 12,000 g for 30 min at 4 8C and the supernatant used. The protein content of the supernatants was measured using the BioRad protein assay (Bio-Rad, Hercules, CA, USA), then an aliquot (25 mg/mL of protein) was subjected to 10% SDS-PAGE, the proteins were transferred onto PVDF membranes (Millipore, MA, USA), and the membranes were blocked by incubation for 1 h at room temperature (RT) with 10% non-fat milk in Tris-buffered saline containing 0.2% Tween 20 (TBST, Bionovas). The membranes were then incubated overnight at 4 8C with rabbit antibodies against human TGF b1 (1:2000 in PBS, GeneTex, TX, USA), human fibronectin (1:500 in PBS, GeneTex), human phospho-JNK, human phospho-ERK1/2, human phospho-p38, phospho-Smad2, or phospho-Smad3 (1:1000 in PBS, Cell Signaling), then for 1 h at RT with HRP-conjugated goat anti-rabbit IgG antibodies (1:5000 in PBS, Santa Cruz,CA, USA), bound antibodies being detected using Chemiluminescence Reagent Plus (Millipore). The intensity of each band was quantified using a densitometer. Antibodies against GAPDH (1:5000, Santa Cruz) were used as loading controls. 2.4. siRNA transfection To investigate the effects of silencing of p38 and Smad3, siRNA for p38 (CGAAUCAAUGAUGUGUAU) or Smad 3 (CAACAGCAAUGVAGCAGUG, Thermo, PA, USA) was used as concentrations of 10 nM and 50 nM. Cells were transfected with siRNA overnight using Oligofectamine (Invitrogen, CA, USA) in Opti-MEM (Invitrogen), then protein lysates were prepared as described above and Western blot analysis performed to validate the efficiency of silencing. 2.5. Nuclear extract preparation and electrophoretic mobility shift assay (EMSA) The preparation of nuclear protein extracts and the EMSA conditions have been described previously [21]. Nuclear proteins were extracted using NE-PER reagent (Pierce, IL, USA) according to the manufacturer’s protocol. The AP-1 and Smad3/4 binding activity of equal amounts (15 mg) of nuclear protein was analyzed using a LightShift Chemiluminescent EMSA kit (Pierce). The synthetic double-stranded oligonucleotides used as the probes in the gel shift assay were 50 -CGC TTG ATG AGT CAG CCG GAA-30 and 30 -GCG AAC TAC TCA GTC GGC CTT-50 for AP-1 and 50 -TCG AGA GCC AGA CAA AAA GCC-30 and 30 -AGC TCT CGG TCT GTT TTT CGG 50 for Smad3/4.
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2.6. Gelatin zymography for MMP enzymatic activity Conditioned medium from control, TGF-b1-treated, or thalidomide+ TGF-b1 treated cells was collected and concentrated and equal amounts loaded onto 8% SDS polyacrylamide gels containing 1 mg/mL of gelatin for assessment of MMP activity. After gel electrophoresis, the gels were washed for 2 x for 30 min at RT in 2.5% Triton X-100 to remove the SDS and allow partial renaturation of the protein. The gels were then incubated overnight at 37 8C in 10 mM CaCl2, 150 mM NaCl, and 50 mM Tris-HCl, pH 7.5, stained with 0.2% Coomassie blue (Sigma), and photographed on a light box. Proteolysis was detected as a white zone in a dark blue field. For quantitative analysis, bands were scanned and the digital image analyzed. 2.7. Implantation of cultured human fibroblasts into nude mice and thalidomide treatment All procedures involving experimental animals were performed in accordance with the guidelines for animal care of the National Taiwan University and complied with the ‘‘Guide for the Care and Use of Laboratory Animals’’ NIH publication No. 86-23, revised 1985. We tested the therapeutic potential of thalidomide in the treatment of keloids using an in vivo model. Briefly, NFs and KFs (1 107 cells per injection) were implanted into the back muscle of Nu/Nu nude mice (The Jackson Lab, ME, USA) weighing 20 g using a syringe with a 26-gauge needle. At 7 days post-implantation, the mice were randomly grouped and given an intra-nodule injection of 100 mg/kg of thalidomide or vehicle (DMSO), then, 7 days later, blood samples were collected for assessment of liver and renal function and the animals sacrificed. Some nodule tissues were excised and fixed in formaldehyde for immunohistochemical analysis, while others were immediately frozen in liquid nitrogen for protein extraction and Western blot. 2.8. Hematoxylin and eosin staining and immunohistochemistry Normal and keloid nodules after 4% paraformaldehyde fixation and paraffin embedding were cut into 5-6 mm thick sections, washed with xylene to remove paraffin and hematoxylin, and subjected to eosin staining. Immunohistochemical staining was also performed on serial sections to detect expression of TGF-b1 and fibronectin. The first section was incubated for 1 h at 37 8C with rabbit monoclonal anti-human TGF-b1 antibody (1:50 dilution, GeneTex), washed with PBS, and incubated for 1 h at RT with HRP-conjugated goat anti-rabbit IgG antibodies (1:200 dilution in PBS, Sigma) and bound antibody visualized using 3,30 diaminobenzidine (Sigma). The second section was incubated with rabbit antibodies against human fibronectin (1:100, Gene Tex) for 1 h at 4 8C, then processed as above. 2.9. Statistical analysis Where applicable, the results are presented as the mean SEM. Statistical analysis was performed by ANOVA, followed by Fisher’s multiple comparison post test. A p value <0.05 was considered statistically significant. 3. Results 3.1. Thalidomide inhibits fibronectin expression in TGF-b1-treated NFs and KFs Expression of TGF-b1 and fibronectin in human keloid tissues was evaluated by immunohistochemistry. In surgically removed normal tissue from controls and keloid tissue, TGF-b1 and
Fig. 1. Expression of TGF-b1 and fibronectin in human keloid (A) and normal (B) skin tissues. Expression of TGF-b1 (top row) or fibronectin (center row) is shown by arrows; NC is the negative control without primary antibody. In each row, the rectangle in the panel to the left is magnified in the next panel to the right. Each bar = 100 mm.
fibronectin immunoreactivity was high in keloid tissue (Fig. 1A; each panel shows a higher magnification of the rectangular area on the previous panel), but barely detectable in normal tissue (Fig. 1B). These results show that fibronectin is highly expressed in fibroblasts in human keloid tissue sections, making this molecule an ideal target for selection of fibronectin-targeted agents. When the cytotoxicity of thalidomide for cultured NFs and KFs was assessed by the MTT assay after 48 h of incubation, cell viability was not affected by the presence of 39, 195, or 390 mM of thalidomide (95 1%, 97 3%, 98 2% of control). But 1950 or 3900 mM of thalidomide treatment caused a significant decrease in cell viability (78 2% and 42 1% of control, respectively). As shown in Fig. 2A, TGF-b1 induced significant expression of fibronectin in NFs (left panel) and KFs (right panel) in a dose-dependent manner, which peaked at 10 ng/mL. To examine whether thalidomide inhibited fibronectin production, NFs or KFs were incubated with different concentrations of thalidomide for 24 h, then with 10 ng/mL of TGF-b1 in the continued presence of thalidomide for 24 h and Western blotting was performed. As shown in Fig. 2B, TGF-b1 induced fibronectin expression in NFs and KFs that was, respectively, 2.1 0.2- and 5.1 0.4-fold higher than control levels. Pretreatment for 24 h with 39, 195, or 390 mM of thalidomide resulted, respectively, in TGF-b1-induced fibronectin expression that was 2.3 0.2-, 1.5 0.1-, or 0.5 0.1-fold higher in NFs and 5.1 0.3-, 4.1 0.3-, or 1.6 0.1-fold higher in KFs than in controls, the effects being significant at 390 mM in NFs and 195 and 390 mM in KFs. In all subsequent experiments, unless otherwise specified, 10 ng/mL of
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Fig. 2. Thalidomide reduced TGF-b1-induced fibronectin expression in NFs and KFs by Western blotting and immunofluorescent staining. (A) Fibroblasts from normal (NF) or keloid (KF) tissues were incubated with different doses of TGF-b1 for 24 h, then fibronectin expression was measured by Western blotting. GAPDH was used as a loading control. (B) NFs or KFs were incubated without or with 39, 195, or 390 mM of thalidomide for 24 h, then with or without 10 ng/mL of TGF-b1 in the continued presence of thalidomide for 24 h, when Western blotting for fibronectin was performed. The results are shown as the fold increase in fibronectin expression relative to that in untreated NFs. The data are the mean SEM (n = 4). *p < 0.05 compared to the value for untreated NFs or untreated KFs, respectively. yp < 0.05 vs TGF-b1-treated NFs or TGF-b1-treated KFs, respectively. (C) NFs or KFs were incubated with 100 mg/mL of thalidomide for 24 h, then with 10 ng/mL of TGF-b1 in the continued presence of thalidomide for 24 h, when immunofluorescent staining for fibronectin was performed. Experiments were repeated at least three times with similar results.
TGF-b1 and 390 mM of thalidomide were used. Interestingly, as shown in Fig. 2B, KFs expressed significantly more fibronectin than NFs in the absence of TGF-b1 treatment and thalidomide treatment inhibited fibronectin expression in KFs. Moreover, immunofluorescent staining (Fig. 2C) showed that fibronectin expression correlated with levels on Western blots and that thalidomide inhibited fibronectin expression in TGF-b1-treated NFs and KFs.
3.2. The thalidomide-induced reduction in TGF-b1-induced fibronectin expression is dependent on inhibition of p38 phosphorylation Since TGF-b1-activates the MAPK pathway [10], we next investigated whether the TGF-b1-induced fibronectin expression in NFs and KFs was mediated by activation of MAPKs. As
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Fig. 3. The thalidomide-mediated reduction in TGF-b1-induced fibronectin expression is dependent on inhibition of p38 phosphorylation. (A) NFs or KFs were incubated with 10 ng/mL of TGF-b1 for the indicated time, then the cell lysates were analyzed for MAPK phosphorylation by Western blotting using antibodies against p-ERK1/2 (top panels), p-p38 (center panels), or p-JNK (bottom panels). (B) The cells were preincubated for 1 h with the indicated concentration of PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), or SP600125 (JNK inhibitor), then with TGF-b1 in the continued presence of the inhibitor for 24 h and the cell lysate analyzed for fibronectin expression by Western blotting. (C) Western blot analysis showing the effect of thalidomide treatment on the phosphorylation of p-ERK1/2 (top panels), p-p38 (center panels), or p-JNK (bottom panels) in TGF-b1-treated NFs or KFs. Cells were incubated for 24 h with or without 390 mM of thalidomide, then with 10 ng/mL of TGF-b1 in the continued presence of the thalidomide for 1 h and aliquots of cell lysate containing equal amounts of protein were subjected to immunoblotting with the indicated antibodies. The data are expressed as a fold of the control value and are the mean SEM for 3 separate experiments. GAPDH was used as the loading control. *P < 0.05 vs untreated NFs or KFs. yP < 0.05 vs TGF-b1treated NFs or KFs. (D) NFs or KFs were left untreated or were transfected with p38 siRNA overnight, then incubated with 390 mM of thalidomide for 24 h, and with 10 ng/mL of TGFb1 in the continued presence of thalidomide for 24 h, when Western blotting was performed. The data are expressed as a fold of the control value and are the mean SEM for 3 separate experiments. GAPDH was used as the loading control. *P < 0.05 vs untreated NFs or KFs, respectively. yP < 0.05 vs TGF-b1-treated NFs or KFs, respectively. zP < 0.05 vs TGFb1-treated siSmad3-transfected KFs.
shown in Fig. 2A, phosphorylation of ERK1/2, p38, and JNK in both NFs and KFs showed a significant increase at 10-30 min of TGF-b1 treatment and increased continuously up to at least 1 h. In addition, pretreatment for 1 h with the indicated concentrations of PD98059 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor), followed by and cotreatment with TGF-b1 for 24 h inhibited the increase in fibronectin expression (Fig. 3B). These results suggest that TGF-b1-induced fibronectin expression is mediated by activation of MAPKs in NFs and KFs. To examine whether thalidomide had an effect on the TGF-b1induced activation of MAPK signaling pathways in NFs and KFs, the cells were preincubated with thalidomide for 24 h and coincubated with thalidomide and TGF-b1 for 1 h. As shown in Fig. 3C, thalidomide treatment significantly inhibited TGF-b1induced p38 phosphorylation by 45% in NFs and 36% in KFs, but had no significant effect on ERK and JNK phosphorylation. As shown in Fig. 3D, transfection of NFs or KFs with 10 or 50 nM p38-specific siRNA inhibited the expression of p38 by 50% and that of fibronectin by 40%. Importantly, combined treatment with thalidomide and p38 siRNA reduced fibronectin expression in TGF-treated KFs by about 65%.
3.3. The thalidomide-induced reduction in TGF-b1-induced fibronectin expression in NFs and KFs is dependent on inhibition of phosphorylation of Smad3, but not Smad 2 Since Smad proteins act as intracellular signaling mediators of TGF-b [10], we examined levels of phosphorylated Smad2 and Smad3 in NFs and KFs treated with TGF-b1. As shown in Fig. 4A, treatment of NFs or KFs with different concentrations of TGF-b1 resulted in a significant increase in levels of phosphorylated Smad2 (top panels) and Smad3 (bottom panels). As the Smad signaling pathway plays a significant role in keloid formation [11,13], Western blotting was performed to examine whether thalidomide reduced TGF-b1-induced fibronectin expression in NFs and KFs through Smad phosphorylation. As shown in Fig. 4B, thalidomide significantly inhibited TGF-b1-induced Smad3 phosphorylation in NFs and KFs (bottom panels), but had no effect on Smad2 phosphorylation (top panels). To confirm the role of Smad3 in TGFb1-induced fibronectin production, fibroblasts were transfected with siRNAs targeting Smad3 and Western blotting performed. As shown in Fig. 4C, transfection of cells with 50 nM Smad3 siRNA resulted in a 70% reduction in Smad3 protein expression and markedly inhibited fibronectin expression in TGF-b1-treated NFs
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Fig. 4. The thalidomide-mediated reduction in TGF-b1-induced fibronectin expression in NFs and KFs is dependent on inhibition of phosphorylation of Smad3, but not Smad 2. (A) NFs or KFs were treated with 10 ng/mL of TGF-b1 for the indicated time, then the cell lysates were analyzed for phosphorylation of Smad2 and Smad3 by Western blotting. (B) Western blot analysis showing the effect of thalidomide treatment on the phosphorylation of Smad2 and Smad3 in TGF-b1treated NFs or KFs. Cells were incubated for 24 h with or without 390 mM of thalidomide, then were incubated with 10 ng/mL of TGF-b1 in the continued presence of thalidomide for 1 h and aliquots of cell lysates containing equal amounts of protein were subjected to immunoblotting with the indicated antibodies. (C) NFs or KFs were left untreated or were transfected with Smad siRNA overnight, then incubated with 100 mg/mL of thalidomide for 24 h, and with 10 ng/mL of TGF-b1 in the continued presence of thalidomide for 24 h, when Western blotting was performed. The data are expressed as a fold of the control value and are the mean SEM for 3 separate experiments. GAPDH was used as the loading control. *P < 0.05 vs untreated NFs or KFs, respectively. yP < 0.05 vs TGF-b1treated NFs or KFs, respectively. zP < 0.05 vs TGF-b1-treated siSmad3-transfected KFs.
and KFs by 60% and 45%, respectively. Taken together, these results demonstrate that Smad3, but not Smad2, is involved in thalidomide-mediated inhibition of TGF-b1-induced fibronectin production.
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Fig. 5. Thalidomide reduces the DNA binding activity of AP-1 and Smad3/4 in TGFb1-treated NFs and KFs by EMSA. (A, B) Nuclear extracts from untreated NFs or KFs with or without pretreatment with 390 mM of thalidomide, then incubated with 10 ng/mL of TGF-b1 for 1 h were tested for AP-1 (A) or Smad3/4 (B) DNA binding activity by EMSA. (C) NFs or KFs were preincubated for 1 h with the indicated concentration of tanshinone IIA (Tan IIA, an AP-1 inhibitor), then were treated with TGF-b1 in the continued presence of the inhibitor for 24 h and the cell lysates analyzed for fibronectin expression by Western blotting. *p < 0.05 compared to the value for untreated NFs or KFs, respectively. yp < 0.05 vs TGF-b1-treated NFs or KFs, respectively.
3.4. Thalidomide decreases AP-1 and Smad 3/4 binding activity in TGF-b1-treated NFs and KFs in an electrophoretic mobility shift assay Since the fibronectin gene promoter contains consensus binding sites for AP-1 and Smad3/4 [22], we examined whether thalidomide inhibited TGF-b1-induced fibronectin expression via an effect on these transcription factors in a gel-shift assay. As shown in Fig. 5, low basal levels of AP-1 (Fig. 5A) and Smad3/4 (Fig. 5B) binding activity were detected in untreated control cells, binding activity was significantly increased by 1 h treatment with TGF-b1, and this effect was blocked by 24 h pretreatment with thalidomide. Furthermore, the stimulatory effect of TGF-b1 on fibronectin levels was blocked by preincubation and co-incubation with 0-20 mM tanshinone IIA, an AP-1 inhibitor (Fig. 5C). These results show that thalidomide inhibits TGF-b1-induced fibronectin expression by inhibiting AP-1 and Smad3/4 activation. 3.5. Thalidomide induces fibronectin degradation through MMP-9 activity as shown by gelatin zymography Since MMP-9 activity is important in the degradation of extracellular matrix proteins [23], we next explored whether
thalidomide reduced the TGF-b1-induced increase in fibronectin expression by induction of MMP-9 activity using gelatin zymography. As shown in Fig. 6A, conditioned medium from cells treated with thalidomide (both with and without TGF) caused a significant increase in MMP-9 activity in NFs or KFs with or without TGF-b1 treatment and, as shown in Fig. 6B, co-incubation of the cultures with 10 mM GM6001 (an MMP inhibitor) markedly reduced the effects of thalidomide on fibronectin expression in TGF-b1-treated, but not control, NFs and KFs. 3.6. Thalidomide markedly inhibits nodule development and fibronectin production in an in vivo keloid model To evaluate the therapeutic potential of thalidomide in the treatment of keloids, we implanted cultured NFs and KFs into the backs of nude mice which were then randomly divided into three groups at day 7 after implantation, one of which was left untreated, while the other two were treated by intra-nodule injection with 100 mg/kg thalidomide or DMSO. No significant change in the weight and appearance of the mice or in liver and renal function
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Fig. 6. Thalidomide increases MMP-9 activity in TGF-b1-treated NFs or KFs by gelatin zymography. (A) NFs or KFs were incubated with or without 390 mM of thalidomide for 24 h, then with 10 ng/mL of TGF-b1 in the continued presence of thalidomide for 24 h and the cconditioned medium collected and tested by gelatin zymography. (B) NFs or KFs were incubated with or without 390 mM of thalidomide for 24 h, then 10 mM GM6001 (an inhibitor of MMP activity) was added to the mixture for 1 h, followed by 10 ng/mL of TGF-b1 for 24 h. The results are shown as the fold increase in expression relative to that in untreated controls. *P < 0.05 vs untreated NFs or KFs, respectively. yP < 0.05 vs TGF-b1-treated NFs or KFs, respectively. zP < 0.05 vs TGF-b1+ thalidomide-treated NFs or KFs, respectively.
was seen in any of the experimental groups. Histologically, the cellularity was higher in the KF nodules than in the NF nodules, as shown by hematoxylin and eosin staining, whereas blood vessels were equally well developed in NF and KF nodules (Fig. 7A). As shown in Fig. 7B, thalidomide treatment reduced cellularity, but not blood vessel formation, in the keloid nodules. We then examined TGF-b1 and fibronectin expression by immunohistochemistry. As shown in Fig. 7C, strong expression of TGF-b1 and fibronectin was seen in the nodules of KF-implanted mice, but not in those from NF-implanted mice and this effect was inhibited by about 90% by thalidomide treatment. In agreement with this, as shown in the Western blots in Fig. 7D, expression of TGF-b1 and fibronectin was high in keloid nodules from non-thalidomidetreated animals, but scarcely detectable in the thalidomide-treated mice. These observations show that thalidomide inhibits keloid development and TGF-b1 and fibronectin production in an in vivo keloid model. 4. Discussion Keloids occur as a result of a pathological wound-healing process, characterized by excessive production of extracellular matrix protein. In this study, we demonstrated that thalidomide treatment reduced fibronectin expression in vitro in TGF-b1treated NFs and KFs and in an in vivo keloid mice model. The
Fig. 7. Thalidomide reduces cell number and the production of TGF-b1 and fibronectin in an in vivo model. (A) NFs and KFs (1 107 cells per injection) were implanted into the back muscle of Nu/Nu nude mice. At 7 days post implantation, mice were randomly grouped and were left untreated or were given intra-nodule injections of 100 mg/kg of thalidomide or the vehicle (DMSO). All animals were sacrificed 14 days after implantation and the nodules excised (NF, NF-derived nodules; KF, KF-derived nodules). Representative photographs of NF and KF nodules excised from the back of nude mice by hematoxylin and eosin staining. (B) Average cell number and capillary density per high power field (HPF) in the excised nodules. *P < 0.05 vs untreated NFs. yP < 0.05 vs untreated or DMSO-treated KFs. (C) Expression of fibronectin and TGF-b1 in each nodule examined by immunostaining. (D) Expression of fibronectin and TGF-b1 in each nodule examined by Western blotting. GAPDH was used as the control.
anti-fibrotic effect of thalidomide was partly mediated by inhibition of both phosphorylation of p38 and Smad3 and activation of transcription factors AP-1 and Smad3/4. In addition, thalidomide treatment in the presence or absence of TGF-b1 increased MMP-9 activity, resulting in fibronectin degradation. More than 50 years have passed since thalidomide was found to be a teratogen in humans and laboratory animals. However, it is now known to be of great value because of its anti-inflammatory, immunosuppressive, anti-oncogenic, and anti-fibrotic properties in many experimental and clinical trials [17,24–26]. In vitro study has demonstrated that thalidomide inhibits TNF-a production by lipopolysaccharide-stimulated human blood monocytes [27]. Apart from its effect on TNF-a, thalidomide modulates adhesiveness in microvascular beds by modifying levels of surface cell adhesion molecules and inhibits nuclear transcription factor nuclear factor-kB (NF-kB) activity [28]. It has also been shown that thalidomide treatment before ischemic insult reduces earlyphase ischemia/reperfusion injury to the spinal cord in rabbits [29]. Moreover, thalidomide has been used to treat acute and chronic liver diseases, which are characterized by the deposition of extracellular matrix protein. Intraperitoneal injection of thalidomide inhibits lethal hepatic necroinflammation and accelerates recovery from rat liver cirrhosis induced by thioactamide, and the
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Fig. 8. A summary diagram showing that thalidomide reduced fibronectin expression in TGF-b1-treated NFs and KFs through the inhibition of both phosphorylation of p38 and Smad3 and activation of transcription factors AP-1 and Smad3/4. In addition, thalidomide treatment in the presence or absence of TGF-b1 increased MMP-9 activity, resulting in fibronectin degradation.
mechanism may involve decreased expression of TNF-a and TGF-b by Kupffer cells [26]. Expression of TGF-b mRNA and protein in rat liver cirrhosis is significantly inhibited by thalidomide [19]. Thalidomide also prevents bleomycin-induced pulmonary fibrosis in mice by inhibiting production of TGF-b1 and collagen [30]. Importantly, the safety of thalidomide has been confirmed by many research groups and it has been approved by the Food and Drug Administration for the treatment of erythema nodosum leprosum [25,31]. Thus, thalidomide has potential to treat keloids, which are also characterized by overproduction of extracellular matrix proteins. Here, we demonstrated that TGF-b1 induced significant fibronectin expression in NFs and KFs and that the effect was inhibited by thalidomide. To our knowledge, this is first report demonstrating that thalidomide reduces fibronectin expression in TGF-b1-treated NFs or KFs and in KFs without TGF-b1 treatment. Due to the close relationship between TGF-b1 signaling and fibronectin production [32], the blocking of TGF-b signaling has potential for decreasing fibronectin synthesis, thereby preventing the formation of keloids. The TGF-b1 signaling pathway in fibrogenesis is mainly mediated and modulated by MAPKs and Smads [10]. In this study, we analyzed the roles of various signaling pathways downstream of TGF-b1 and found that TGF-b1 activated the three MAPK cascades involving p38, ERK, and JNK and that TGFb1-induced fibronectin secretion was inhibited by ERK, JNK, and p38 inhibitors, showing that the increased synthesis of fibronectin induced by TGF-b1 is mediated by activation of ERK, JNK, and p38. Thalidomide treatment did not affect TGF-b1-induced ERK and JNK MAPK phosphorylation, but resulted in a significant reduction in phosphorylated p38 in both NFs and KFs. Furthermore, the present study demonstrated that TGF-b1 activated phosphorylation of Smad2 and Smad3 in NFs or KFs. Importantly, thalidomide blocked phosphorylation of Smad3, not that of Smad2, and Smad3 silencing decreased fibronectin production by NFs and KFs. These results are consistent with previous reports [33,34], which demonstrated that Smad3, but not Smad2, is required for the fibrotic response. In HK-2 cells, bone morphogenetic protein functions as an antifibrogenic factor that ameliorates the TGF-b1induced expression of fibronectin, a fibrosis marker, and this effect is mediated by inhibition of JNK and Smad2/3 activation and modulation of p38 activation [35]. The activated p38 pathway phosphorylates Smad3, thereby promoting Smad signaling and extracellular matrix deposition, leading to liver fibrosis [36]. Our data demonstrated that the major mechanism for the antifibrotic effect of thalidomide involved inhibition of the TGF-b1-induced activation of p38 and Smad3. Whether the reduced p38 activation induced by thalidomide regulates Smad signaling, thereby reducing fibronectin deposition and leading to keloids requires further investigation.
The TGF-b signal itself is predominantly transduced by a family of transcriptional factors, the Smad proteins [22]. Binding of TGF-b to its receptor activates receptor kinase, which then phosphorylates the receptor-regulated Smads Smad2 and Smad3, which then associate with Smad4, and the Smad complex translocates into the nucleus where it regulates the expression of target genes. In the nucleus, the Smad3/4 or Smad2/3/4 complex can activate transcription by binding directly to a certain sequence in DNA, the consensus sequence, which is present in the promoter region of several TGF-b target genes, including those important in fibrosis, e.g., those coding for fibronectin and collagen. Smad3 mediates TGF-b1-induced fibronectin expression in mesangial cells [37]. AP1 is another important signal transducer of TGF-b1 and plays an important role in its biological effects [38]. The AP-1 transcriptional complex is also a primary target of a number of MAPK pathways. Our results showed that TGF-b1 induced AP-1 and Smad 3/4 activity and that thalidomide reduced the activity of these transcription factors in an electrophoretic mobility shift assay. Previous reports and our present findings suggest that the Smad3/4 and AP-1 pathways participate in the activation of fibronectin synthesis. In addition, our study demonstrated that the thalidomide-induced suppression of fibronectin expression is mediated by inactivation of the AP-1 and Smad3/4 pathways in keloid development. Whether AP-1 components can cross-talk with Smad3 and that thalidomide affect the interaction need further investigation. Histologically, in an in vivo keloid model, the nodules from KFimplanted mice showed higher cellularity than those from NFimplanted mice, as demonstrated by hematoxylin and eosin staining. This result is consistent with the observation that KFs grow faster than NFs [39,40]. Thalidomide treatment significantly suppressed cellularity in KF nodules, but had no effect on the well developed blood vessels in keloid nodules in our study, whereas orally administered thalidomide was found to be an inhibitor of angiogenesis induced by basic fibroblast growth factor in a rabbit cornea micropocket assay [18]. These findings indicate that thalidomide may have different therapeutic potential in different diseases. TGF-b1 and fibronectin expression was high in the nodules from KF-implanted mice, but not in those from NFimplanted mice, and accumulation of TGF-b1 and fibronectin in keloid nodules was inhibited by thalidomide treatment. These observations suggest that thalidomide inhibits keloid development by inhibition of TGF-b1 and fibronectin production in an in vivo keloid model. The previous study reported that the E3 ligase protein cereblon (CRBN) was identified as a direct molecular target for the teratogenic effects of thalidomide [41]. CRBN levels were also shown to be critical for the antitumor activity of thalidomide in both in vitro model systems and in lenalidomide-resistant
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patients [42]. However, whether CRBN is involved in the antifibrotic effects of thalidomide on keloid need further study. In summary, we have demonstrated that thalidomide treatment resulted in less phosphorylation of p38 and Smad3 in NFs and KFs and that this was closely correlated with decreased TGF-b1induced expression of fibronectin (Fig. 8). In addition, thalidomide markedly reduced nodule development and TGF-b1 and fibronectin production in an in vivo keloid model. Together, these results suggest that thalidomide may have potential in the treatment and prevention of keloids and excessive scarring. Author contributions Conception and design, Chan-Jung Liang, Yu-Hsiu Yen, and Yuh-Lien Chen.; analysis and interpretation, Chan-Jung Liang, YuHsiu Yen, Ling-Yi Hung, and Yuh-Lien Chen.; drafting the manuscript for important intellectual content, Ling-Yi Hung, Shu-Huei Wang, Chi-Ming Pu, Hsiung-Fei Chien, Jaw-Shiun Tsai, Chiang-Wen Lee, and Feng-Lin Yen.; revising the manuscript for important intellectual content, Chan-Jung Liang, Yu-Hsiu Yen, and Yuh-Lien Chen.; and final approval of the manuscript, Chan-Jung Liang, Yu-Hsiu Yen, Ling-Yi Hung, Shu-Huei Wang, Chi-Ming Pu, Hsiung-Fei Chien, Jaw-Shiun Tsai, Chiang-Wen Lee, Feng-Lin Yen, and Yuh-Lien Chen.
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Conflict of interest The authors state there is no conflict of interest. Acknowledgments This work was supported by research grants from the National Science Council (NSC 99-2320-B-002-022-MY3) and Cathay General Hospital (CGH-MR-9927), Taiwan, Republic of China.
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