Recombinant Human Decorin Inhibits Cell
Proliferation and Downregulates TGF-β1
Production in Keloid Fibroblasts
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Abstract: Keloids remain a major problem for patients who have suffered deep injuries. The pathophysiology underlying keloid formation may be driven by the biological activity of transforming growth factor beta1 (TGF-b1). Decorin is a human proteoglycan that inactivates the effect of TGF-b1 and therefore displays a beneficial effect of antifibrosis in various tissues, such as kidney, muscle, and lung. Keloids are fibroproliferative disorders of the dermis that occur following wounding. This study investigated the effects of decorin on cell proliferation, TGF-b1 production, and collagen synthesis in keloid fibroblasts. Fibroblasts were extracted from explants of operative specimens obtained from keloid tissue and cultured in vitro with serial, diluted decorin concentrations (10, 50, 100, 200 nM) for 48 hours. The cell proliferation rates were measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide] colorimetric determination, and cell cycle distributions were analyzed with flow cytometry. Low-molecular-weight apoptotic DNA and TGF-b1 levels in supernatants were measured by enzyme-linked immunosorbent assay. Radioimmunoassay was used to detect the contents of type I and type III collagen amino-terminal propeptide (PINP, PIIINP) in supernatants. Fibroblast proliferation was significantly (P < 0.05) inhibited by decorin at all concentrations (12, 24, and 48 hours) and was dose-dependent. Decorin inhibited fibroblast proliferation by inducing cell growth arrest but not apoptosis. TGF-b1 and PINP levels were significantly (P < 0.05) lower in fibroblasts treated with 10, 50, 100, and 200 nM of decorin for 48 hours compared with fibroblasts not treated with decorin. There was no significant difference in PIIINP concentration between the decorin-treated group and the control group. The PINP/PIIINP ratios were significantly lower in fibroblasts treated with decorin (P < 0.05) compared to the control group. These results suggest that decorin has a down-regulatory effect on cell proliferation, TGF-b1 production, and collagen synthesis in keloid fibroblasts. Improved understanding of such regulatory mechanisms may eventually be of therapeutic significance in the control of keloid.
Aberrant wound healing is a significant problem that affects millions of patients yearly. Keloids, for example, are characterized by the formation of exuberant, firm scar tissue that does not flatten over time. The bulky and inelastic qualities of the scar, as well as the frequent occurrence of contractures, can severely restrict the mobility of joints and extremities, constrict orifices, immobilize structures, such as eyelids, and drastically compromise cosmetic appearance.1 Treatment for keloids is problematic, with no single modality producing uniformly satisfactory results.
Keloids are associated with an abnormal proliferation of fibroblasts and an overproduction of collagen and extracellular matrix (ECM).2 The pathological repair process underlying keloid formation may be mediated in part by the biological activity of transforming growth factor beta (TGF-β) during wound healing. Three isoforms of TGF-β (1, 2, and 3) have been identified in mammals. Only TGF-b3 is thought to possess an antiscarring effect.3 TGF-β1 exerts protean effects during wound healing, including increased production of collagen, increased expression of integrins, decreased expression of metalloproteinases, and increased expression of metalloproteinase inhibitors.4 Collectively, these effects of TGF-β1 occurring in excess could result in the abnormal accumulation of ECM, along with fibrosis and scar formation.
Decorin is a secreted 45 kDa proteoglycan with a core protein comprised primarily of leucine-rich repeats5 and is found in the ECM of several tissues, such as skin,6 cartilage,7 and bone.8 In-vitro binding studies have shown that some of them interact with several types of collagen and act as important regulators of collagen fibrillogenesis.9 In support of this hypothesis, a decorin-deficient mouse was found to have fragile skin with an abnormal organization of collagen fibers.10 Due to the leucine-rich repeats, decorin is thought to interact with several other proteins and lipid molecules.
Decorin has been reported to bind TGF-β1 and neutralize some of its activities.11,12 Decorin, either injected or synthesized in vivo from an expression vector, has been shown to have a beneficial effect of antifibrosis in various tissues, such as kidney,13 muscle,14 and lung.15 Keloids are fibroproliferative disorders of the dermis that occur following wounding. Furthermore, decorin has been demonstrated to inhibit proliferation of tumor cells16,17 and bone marrow macrophage colony-forming cells.18 However, there are few reports regarding the effect of decorin on keloid fibroblasts.
Thus, the present study was designed to examine the in-vitro effect of decorin on cell proliferation, TGF-β1 production, and collagen synthesis in keloid fibroblasts.
Materials and Methods Fibroblast cultures. The keloid fibroblast was established as a primary cell line from keloid tissue obtained from 9 patients whose scars extended above and beyond the sites of original injury. Scarring resulted in functional impairment and aesthetic deformity, and an operation was needed. Exemption to use operative specimens that would otherwise be discarded was obtained from the Human Subjects committee of Jinan University, China.
Using a sterile technique under a laminar flow hood, the dermal specimen was minced into approximately 1-mm3 fragments with a sterile scalpel blade on a Petri dish. The specimens were washed in phosphate-buffered saline (PBS) solution with a combination of 1% penicillin, streptomycin sulfate, and amphotericin B (GIBCO, Invitrogen, Beijing, China). The specimens were then placed in 75-mm3 tissue culture flasks (T75; Falcon, Becton, Dickinson and Company, Franklin Lakes, NJ) with 10 mL of culture medium (10% fetal calf serum in Dulbecco-modified Eagle medium with 1% penicillin-streptomycin sulfate-amphotericin B [GIBCO]). The specimens were then maintained in a humidified incubator at 37°C with a 5% carbon dioxide atmosphere.
After 24 hours, the medium was changed to 7 mL of culture medium. The medium was then changed every 2 days until fibroblasts were seen growing outward from the explanted tissue under light microscopy. At that time, the tissue was removed. With sufficient outgrowth of fibroblasts, cells were subcultured into 75-mm2 culture flasks. Culture medium was changed every 3 to 4 days. Successive cultures were passed at confluence. Experiments were performed with early passage cells (passage 3 through 6). Cells at the same passage were used to examine the effect of decorin on fibroblast proliferation, apoptosis, TGF-β1 production, and collagen synthesis.
Cell proliferation assays. Cell proliferation assays were performed with minor modifications using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] uptake (Sigma Chemical Co., St. Louis, Mo) to monitor growth as described previously.17 Fibroblasts (1x104) were seeded into flat-bottomed 96-well plates in 100 µL of growth medium per well and allowed to attach and grow overnight. The medium was then replaced with 100 µL of growth medium containing 0, 10, 50, 100, and 200 nM of recombinant human decorin (R&D Systems, Minneapolis, Minn) and cultured for 12, 24, and 48 hours, respectively. At the end of the incubation, MTT (20 µL of a 5-mg/mL solution in PBS) was added and incubated at 37°C for 3 hours. The purple formazan (a product of MTT reduction via succinyl dehydrogenase in the mitochondria of living cells) was solubilized overnight at 37°C with 20% SDS and 50% dimethyl formamide. The MTT-containing culture fluid was removed from the wells, and the product was solubilized in 100 µL of DMSO for 5–10 minutes at room temperature. The plate was read at 570 nm in a plate reader (BioTech, Winooski, Vt). Eight replicate wells were used to obtain all data points, and all of the reported experiments were performed at least twice.
Flow cytometry analysis. Fibroblasts (1x106) were cultured in growth medium containing 0, 10, 50, 100, and 200 nM of recombinant human decorin. After a 48-hour incubation, cells were harvested and washed in ice-cold PBS. The cells were stained with propidium iodide (2 mg/mL, Oncogene Science, Cambridge, Mass) in 4 mM Na citrate buffer containing 3% Triton X-100 and RNase A (100 µg/mL). Twelve thousand cells were counted for each histogram, and cell cycle distributions were analyzed with the Multicycle program (Phoenix Flow Systems, San Diego, Calif). Four replicate wells were used to obtain all data points, and all samples were performed in triplicate.
Apoptosis assays. Low-molecular-weight apoptotic DNA created by internucleosomal cleavage was measured as previously described.19 Briefly, 1x106 fibroblasts were treated with 0, 10, 50, 100, and 200 nM of recombinant human decorin. After a 48-hour incubation, cells were harvested and washed in ice-cold PBS. The cells were lysed in 0.5 mL of lysis buffer (50 mmol/L Tris-HCl, 10 mmol/L EDTA, 1% SDS, pH 8.0) for 16 hours at 4°C, and the lysates were centrifuged (15,000 x g) to separate high-molecular-weight DNA (pellet) from cleaved low-molecular-weight DNA (supernatant). The DNA supernatants were phenol-extracted twice and precipitated. Low-molecular-weight apoptotic DNA was measured in supernatants using an enzyme-linked immunosorbent assay (ELISA) technique with Cell Death Detection ELISA Plus kits (Roche Diagnostics, Mannheim, Germany) that is directed against cytoplasmic histone-associated DNA fragments, according to the manufacturer’s instructions. Four replicate wells were used to obtain all data points, and all samples were performed in triplicate.
TGF-β1 assays. Fibroblasts (5x105) were seeded into flat-bottomed 6-well plates, cultured in a commercially available serum-free medium (UltraCULTURE, BioWhittaker, Walkersville, Md) that was previously shown to sustain fibroblast cultures for durations similar to those used in this study20 and allowed to attach and grow overnight. The medium was then replaced with serum-free medium containing 0, 10, 50, 100, and 200 nM of decorin. Untreated cells (0 nM) were used for controls. After a 48-hour incubation, cell-free supernatant was collected, and cells were counted in duplicate using phase-contrast microscopy and a hemacytometer. The TGF-β1 levels were measured in supernatants using solid-phase ELISA with TGF-β1 ELISA kits for humans (R&D Systems), according to the manufacturer’s instructions. Samples with a concentration exceeding the limits of the standard curve were diluted and repeated. Eight replicate wells were used to obtain all data points, and all samples were performed in duplicate and averaged.
Determination of collagen propeptides. Cell-free supernatant was collected as previously mentioned. The concentration of PINP (amino-terminal propeptide of type I procollagen) and PIIINP (amino-terminal propeptide of type III procollagen) were measured by competitive radioimmunoassay methods21 with commercially available reagents (Orion Diagnostica, Espoo, Finland), according to the manufacturer’s instructions. The assays are based on the use of human antigens and polyclonal rabbit antibodies. A 50 mL aliquot (diluted with buffer 1:10 or 1:40) of the cell culture medium was used for the PINP assay and 200 mL of the dilution for the PIIINP analyses. Eight replicate wells were used to obtain all data points, and all samples were performed in duplicate and averaged.
Statistical analysis. Data were expressed as mean ± SEM. Statistical evaluation of the continuous data was performed by 1-way analysis of variance, followed by Dunnett’s t-test for between-group comparisons. The level of significance was considered to be P < 0.05.
Results Recombinant human decorin induces growth suppression in keloid fibroblasts. Keloid fibroblasts were cultured in the absence (0 nM) or presence of various concentrations of recombinant human decorin (10, 50, 100, 200 nM) for 12, 24, and 48 hours and analyzed using the MTT assay. Fibroblast proliferation was significantly (P < 0.05) inhibited by decorin at all concentrations and all time points, and this effect was dose-dependent. At decorin concentrations of 100 nM and 200 nM, fibroblast proliferation was completely inhibited (P < 0.001), and the expected temporal increase in absorbance units was completely abolished, indicating a static population (Figure 1).Figure 1
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Cell cycle distributions of decorin-treated keloid fibroblasts. The distribution of the DNA content of cells stained with propidium iodide showed that fibroblasts treated with 10, 50, 100, and 200 nM of decorin were blocked mainly at the G1 phase of the cell cycle (71.08 ± 3.21, 78.85 ± 4.57, 80.21 ± 5.43, 84.05 ± 5.63 percent, respectively), whereas fibroblasts growing in media without decorin (control group) showed a distribution corresponding to an active proliferating population (43.00 ± 4.40 percent of cells at G1 phase). The proportion of cells arrested in the G1 phase of the cell cycle was significantly increased (P < 0.01) in fibroblasts treated with 10, 50, 100, and 200 nM of decorin compared with fibroblasts not treated with decorin (Figure 2).
No apoptosis in decorin-treated keloid fibroblasts. Keloid fibroblasts treated with 10, 50, 100, and 200 nM of decorin for 48 hours and stained with propidium iodide revealed that there was no subdiploid peak corresponding to apoptotic cells (Figure 3A). In order to confirm this observation, apoptosis was further determined with the use of an ELISA kit that measures the presence of histone-associated DNA fragments, and there were no significant differences in the levels between the control group and the decorin-treated group (Figure 3B).Figure 3
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Figure 2
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Decorin downregulates TGF-β1 production in keloid fibroblasts. The TGF-β1 concentration in supernatants was divided by the number of viable cells to yield graphs of TGF-β1 concentration per cell (Figure 4). No TGF-β1 was detected in the serum-free media with or without the addition of decorin, as expected. Keloid fibroblasts without decorin addition exhibited the highest TGF-β1 concentration per cell. The TGF-β1 concentration per cell was significantly lower in fibroblasts treated with 10, 50, 100, and 200 nM of decorin (P < 0.05) compared with fibroblasts without decorin addition. TGF-β1 production per cell was not significantly different between fibroblasts treated with 10 nM of decorin and fibroblasts treated with 200 nM of decorin (Figure 4).
Type I and type III procollagen levels in supernatants. The results of type I and type III collagen amino-terminal propeptide determinations are shown in Figure 5. Keloid fibroblasts without decorin addition exhibited the highest PINP concentration in supernatants. The PINP concentration was significantly lower in fibroblasts treated with 10, 50, 100, and 200 nM of decorin (P < 0.05) compared with fibroblasts without decorin addition. There was no significant difference in PIIINP concentration between the decorin-treated group and the control group (Figure 5A). The PINP/PIIINP ratios were significantly lower in fibroblasts treated with 10, 50, 100, and 200 nM of decorin (P < 0.05) compared with the control group (Figure 5B).Figure 5
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Figure 4
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Discussion The abnormal biological behavior of fibroblasts played a central role in keloid formation and development.22 For instance, keloid fibroblasts were overproliferative and produced high levels of collagen, fibronectin, elastin, and proteoglycan compared with normal fibroblasts.23 Regulated cell proliferation and growth did not require the interaction of growth factors with their cellular receptors as a prerequisite for the completion of the cell cycle. Cell proliferation was also linked with changes in the interactions of cells with their macromolecular environment.24 Hence, the extracellular matrix surrounding the cells was of similar importance for growth control as were the interactions between soluble growth factors and their cellular receptors. Proteoglycans of the extracellular matrix have recently been recognized to not only play a part in providing shape and biomechanical strength to organs and tissues but also exhibit direct and indirect cell signaling properties.24 Muir et al25 showed that a citric acid buffer extract of human dermis inhibited growth of human diploid fibroblasts in monolayer culture, and the active principle was a proteoglycan.26
Decorin is the predominant proteoglycan in normal skin. However, scar tissue showed a marked reduction in the amount of decorin as compared to normal skin.27 Fibroblasts isolated from reticular dermis reportedly synthesized less decorin than cells from papillary dermis, while fibroblasts responsible for healing might come from deep dermal survival fibroblasts in scar-healing wounds.28 Decorin appeared to be expressed early and abundantly in normally healing wounds.29 These data suggest that reduction of decorin is possibly related to excessive scar formation.
In this article, the authors reported that recombinant human decorin inhibited proliferation of keloid fibroblasts and that this effect was dose-dependent. The inhibitory effect of decorin was confirmed by flow cytometry, because fibroblasts treated with decorin were blocked mainly at the G1 phase of the cell cycle. Moreover, the inhibition of proliferation was not due to lower cell viability because apoptosis was not detected.
Evidence suggested that several cytokines were important components in the process of wound healing and scar formation. Among these, TGF-β1 played a central role in wound healing,22 both as a potent regulator of cellular proliferation and as a modulator of the interaction of cells with extracellular matrix. In wound healing, TGF-β1 functioned by stimulating the synthesis and deposition of extracellular matrix proteins, along with decreasing metalloproteinases and increasing metalloproteinase inhibitors in the wound environment.23
TGF-β1 was a multifunctional growth factor that influences many important physiologic processes, such as cellular proliferation and differentiation.30 TGF-β1 stimulated normal human dermal fibroblasts to synthesize collagen.23 It was reported that TGF-β1 mRNA was greater in keloid tissue than in normal skin.31 The in-vitro cultured fibroblasts derived from the keloid also expressed TGF-β1 mRNA in a level significantly higher than that of the normal fibroblasts.32 TGF-β1 protein was at higher levels in keloid fibroblast cultures compared with normal human dermal fibroblast cultures.33 The scar formation in adult rodent wounds could be inhibited by neutralizing wound TGF-β1 with anti-TGF-β1 antibody.34,35 Thus, TGF-β1 might be the driving force behind the excessive scar formation seen in keloid.
In the present study, the use of a serum-free protocol allowed analysis of the effect of decorin on per-cell TGF-β1 production. As the fibroblasts replicated, they were bathed in only their own autocrine growth factors instead of in serum that contains exogenous growth factors. The method used to evaluate cell growth measured only viable cells. This allowed the authors to determine the average TGF-β1 concentration per viable cell.
This study demonstrated that decorin decreased the per-cell concentration of TGF-β1 in keloid fibroblasts grown in serum-free media, and higher concentrations of decorin trended toward progressive decrements in the TGF-β1 level. Al-Attar et al36 demonstrated that a persistent autocrine loop of TGF-β1 might exist and contribute to scar formation. It could be speculated, therefore, that the addition of decorin may lead to improved wound healing by reducing the level of autocrine TGF-β1 production.
The authors’ results showed that decorin inhibited the secretion of type I, not type III, collagen in keloid fibroblasts. It was reported that scar fibroblast selectively increased the biosynthesis of type I collagen; the abnormal metabolism resulted in the deposition of collagen in scar and altered the steady-state ratio of collagen type I/III in the process of wound healing.23,37,38 Accordingly, decorin actively regulated the ratio of collagen type I/III in the metabolism of keloid fibroblasts by inhibiting the secretion of type I collagen.
The authors proposed that a mechanism by which decorin decreased keloid fibroblast proliferation and collagen synthesis was by downregulating TGF-β1 production. Alteration of the concentration of TGF-β1 in keloid, by the addition of decorin, could possibly modify the wound environment and convert it from one of excessive scarring to one in which the normal processes of extracellular matrix accumulation during repair terminate appropriately. Furthermore, the biological activity of TGF-β1 in keloid could be diminished via gene therapy through the use of antisense oligonucleotides.
Fibroblasts isolated from reticular dermis reportedly synthesized less decorin than cells from papillary dermis.28 Since excessive scarring commonly develops in second-degree burns and other injuries that penetrate the dermis but where some deep dermal fibroblasts survive, it is possible that physical selection of cells with an intrinsically lower capacity for synthesis of decorin contributed to its delayed reappearance in these wounds.
Conclusion Decorin was found to inhibit cell proliferation and downregulate TGF-β1 production in keloid fibroblasts. Furthermore, decorin had a downregulatory effect on type I procollgen production. Further investigations are required to elucidate the role of decorin in vivo in the development of keloid and its possible modulation in an effort to control or prevent problematic keloid.
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| Wounds - ISSN: 1044-7946 - Volume 18 - Issue 8 - August 2006 - Pages: 203 - 212 | |
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