Inhibitory Effect of the LY2109761 on the Development of Human Keloid Fibroblasts

Keloids are scars characterized by abnormal proliferation of fibroblasts and overproduction of extracellular matrix components including collagen. We previously showed that LY2109761, a transforming growth factor- (TGF-) β receptor inhibitor, suppressed the secretion of matrix components and slowed the proliferation of fibroblasts derived from human hypertrophic scar tissue. However, the exact mechanism underlying this effect remains unclear. Here, we replicated the above results in keloid-derived fibroblasts and show that LY2109761 promoted apoptosis, decreased the phosphorylation of Smad2 and Smad3, and suppressed TGF-β1. These results suggest that the development and pathogenesis of keloids are positively regulated by the Smad2/3 signaling pathway and the upregulation of TGF-β1 receptors. LY2109761 and other inhibitors of these processes may therefore serve as therapeutic targets to limit excessive scarring after injury.


Introduction
Keloids represent a dysregulated cutaneous wound healing process characterized by abnormal proliferation of fibroblasts and excessive deposition of collagen [1,2] and can form after burns or other skin injuries [3,4]. Although keloids share several features with hypertrophic scars, such as excessive collagen deposition, they present also distinct features: they can extend beyond the borders of the wound, rarely regress over time, and have a high rate of recurrence [5,6]. At present, the exact mechanisms underlying the formation and progression of keloids remain unclear.
Surgical treatment often results in the recurrence of keloids, which can be larger than the original lesion. Radiation, which is currently used as a postoperative adjuvant therapy and has shown significant efficacy in reducing recurrence, is associated with an increased risk of skin cancer [5]. Other treatments including laser therapy, cryotherapy, and occlusive dressings have shown limited efficacy and significant adverse effects.
Keloid fibroblasts are the primary cells involved in the formation of the wound healing scar, and their elevated proliferation and limited apoptosis lead to excess scarring [7][8][9]. LY2109761, a transforming growth factor-(TGF-) β receptor inhibitor, reduces tumor cell growth, intravasation, and the metastatic dissemination of hepatocellular carcinoma (HCC) cells through different molecular mechanisms [10]. We previously showed that LY2109761 inhibited human hypertrophic scar formation [11]; however, its effect on keloid formation has not been tested yet.
In a separate study, we showed that growth differentiation factor-(GDF-) 9 promoted fibroblast proliferation and migration in keloids via the Smad2/3 pathway [5], while others have demonstrated that TGF-β/Smad signaling plays an essential role in the development of keloids [12][13][14][15]. In the present study, we focused on changes in the Smad2/3 signaling pathway in response to LY2109761 treatment. Specifically, we isolated keloid-derived fibroblasts and treated them with two concentrations of LY2109761 (5 and 10 μM). We assessed the effect of LY2109761 on keloid-derived fibroblast proliferation and apoptosis, suppression of the TGF-β/Smad signaling pathway, and expression of key factors involved in keloid formation.

Materials and Methods
2.1. Human Subjects. Surgically excised keloids (n = 6) were donated from patients, who underwent plastic surgery without previous treatment for scarring ( Table 1). The handling of human tissue was approved by the Ethics Committee of Shanghai Ninth People's Hospital (Shanghai, China). Detailed information on the patients, lesion locations, and harvested areas is provided in Table 1.

Primary Cell Isolation and
In Vitro Culture. Fibroblasts derived from excised keloids were isolated as previously described with several modifications [12]. Briefly, keloids were immersed in 2.5% chloramphenicol solution, rinsed with phosphate-buffered saline (PBS), cut into small pieces (approximately 24 mm 2 ), and immersed in 0.2% dispase (Roche Diagnostics, Indianapolis, IN, USA) dissolved in Dulbecco's modified Eagle medium (DMEM; Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS). Keloids were incubated overnight at 4°C to enzymatically dissociate scar tissue. The next day, the dermis was mechanically isolated from the surrounding scar tissue with forceps and subjected to another round of enzymatic digestion with 0.2% collagenase II (SERVA, Heidelberg, Germany) in DMEM for 3 h at 37°C on a rotator. The digested cell suspension was filtered through a nylon mesh; washed in DMEM containing 10% FBS, penicillin (100 U/mL), and streptomycin (0.1 mg/mL); and centrifuged at 524 × g for 5 min. Pelleted fibroblasts were resuspended in DMEM supplemented with 10% FBS, seeded in 10 cm culture dishes (BD, Falcon, TX, USA) and cultured at 37°C with 5% CO 2 until they reached 80% confluence. Confluent cells were then subcultured at a ratio of 1 : 3. All subsequent experiments were performed using mixed fibroblasts extracted from human keloid samples mentioned above.

Scratch Wound Healing Assay.
A scratch wound assay was used to evaluate cell migration as previously described [16]. Briefly, fibroblasts were seeded in 6-well plates coated with collagen (BD Biosciences, San Diego, CA, USA) and cultured until they reached 80% confluence. Cells were then treated with LY2109761 (dissolved in DMSO; Sigma-Aldrich, St. Louis, MO, USA) at 5 or 10 μM for 36 h. Control fibroblasts were treated with DMSO only. A pipette tip was used to make a "wound" of approximately 0.45-0.50 mm in width at a 90°angle to the bottom of the dish. Cell migration into the wound area was assessed immediately after wound generation and at 24 and 48 h postwound generation using a light microscope (Nikon, Tokyo, Japan). The percentage of cells migrating into the wound area was calculated. Three independent fields were analyzed in each well, and each experiment was performed in triplicate.
2.4. Enzyme-Linked Immunosorbent Assay (ELISA). Keloidderived fibroblasts were incubated with LY2109761 (5 or 10 μM) or vehicle control (DMSO) for 48 h. After treatment, the supernatant was collected and the concentration of TGFβ was measured using a human TGF-β ELISA kit (R&D, San Jose, CA, USA). Absorbance was measured at 450 nm using a microreader (Thermo Fisher Scientific, Waltham, MA, USA). The assay was performed in triplicate and repeated in three cell samples.

Quantitative
Real-Time PCR (qRT-PCR). Total RNA was extracted from cultured fibroblasts as previously described [5]. Briefly, cells were incubated with LY2109761 (5 or 10 μM) or vehicle control (DMSO) for 48 h, after which total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed using the AMV Reverse Transcription System according to the manufacturer's instructions (TaKaRa Bio, Shiga, Japan). qRT-PCR was performed using SYBR Green PCR mix (Applied Biosystems, Foster City, CA, USA) on an ABI Prism 7900HT thermocycler (Applied Biosystems) under the following conditions: 95°C for 10 min followed by a two-step PCR    2.8. Flow Cytometric Analysis of Apoptosis. Cells treated with 5 or 10 μM LY2109761 were assessed with a cell apoptosis kit containing Annexin V-FITC and propidium iodide (PI; eBioscience San Diego, CA, USA) according to the manufacturer's instructions. Briefly, cells were washed twice in PBS and adjusted to 1 × 10 6 cells/mL with 1× Annexin-binding buffer (eBioscience). Annexin V-FITC (10 μL) and PI (10 μL) were added to a 100 μL cell suspension and incubated for 15 min at room temperature. Stained cells were then analyzed using multiple-color flow cytometry on a FACS Aria cell sorter (BD Biosciences).
2.9. Statistical Analysis. All data are presented as the mean ± standard error of the mean (SEM). Statistical significance was measured using Student's t-test for differences between two groups or by one-way ANOVA for multiple group comparisons. P < 0:05 was considered statistically significant. All statistical analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA).

LY2109761 Promotes Keloid-Derived Fibroblast
Apoptosis. Annexin V-FITC/PI staining and fluorescent cell sorting were used to evaluate the effect of LY2109761 on fibroblast apoptosis. LY2109761 treatment significantly increased apoptosis in a dose-dependent manner compared with control treatment (P < 0:05; Figure 4). As the concentration increased, inhibition was enhanced, too, reaching more than 90%.

LY2109761 Suppresses the Smad2/Smad3
Signaling Pathway. To identify the signaling pathway involved in LY2109761-induced keloid fibroblast alterations, protein expression of Smad2 and Smad3, as well as that of their respective phosphorylated forms (p-Smad2 and p-Smad3), was assessed by western blotting. As shown in Figure 5, treatment with LY2109761 at increasing doses significantly downregulated the phosphorylated forms of Smad2 and Smad3 compared with control treatment (P < 0:05).

LY2109761 Upregulates the mRNA and Protein
Expression of GDF-9. To identify the effect of LY2109761the expression of GDF-9 of keloid fibroblast, mRNA and protein of GDF-9 were assessed. As shown in Figure 6, treatment with LY2109761 at increasing doses significantly upregulated the mRNA and protein expression of GDF-9 compared with control treatment (P < 0:05).

Discussion
The present study investigated the effect and the underlying mechanisms of LY2109761 treatment on keloid-derived fibroblasts. LY2109761 treatment at two concentrations (5 and 10 μM) inhibited the expression of key extracellular matrix factors secreted by fibroblasts and responsible for promoting keloid development. This included TGF-β, collagens I and III, fibronectin, and MMP-1, and MMP-3, at both mRNA and protein levels. Previous studies reported that  Figure 2: LY2109761 inhibits keloid-derived fibroblast proliferation. Cell proliferation was assessed using the CCK-8 assay in keloid-derived fibroblasts treated with increasing concentrations of LY2109761 on days 3, 5, 7, and 9. Data are presented as the mean ± SEM from three independent experiments. * P < 0:05 between LY2109761 groups and DMSO control; * * P < 0:05 between LY2109761 5 μM and 10 μM treatment groups. 4 Analytical Cellular Pathology  5 Analytical Cellular Pathology 10-30 μM peptide inhibitors or LY2109761 were sufficient to exert an inhibitory effect [11,17,18]. Here, we show that 5 μM LY2019761 was sufficient to significantly inhibit factors associated with keloid formation, suggesting that low doses may be sufficient to slow or prevent pathological scarring. LY2109761, a TGF-β inhibitor, prevents the release of connective tissue growth factors in tumors, thereby reducing the size and metastasis of tumors [19,20]. He et al. [10] reported that LY2109761 in combination with transcatheter arterial chemoembolization reduced liver tumor size and metastases in a Smad2-dependent manner in both a rabbit tumor model and an HCC model [10]. A study by Xu et al. [21] showed that LY2109761 reversed the antiapoptotic effect of TGF-β1 in myelomonocyte leukemia cells [21]. More recently, our group used human hypertrophic scar-derived fibroblasts and showed that LY2109761 inhibited the secretion of factors related to the development of hypertrophic scars and reduced the migration of the ensuing fibroblasts [5]. Present results are in agreement with these previous observations.
Although conclusive, our previous study did not elucidate the mechanism underlying the effect of LY2109761 treatment. Based on reports showing that LY2109761 inhibited the Smad signaling pathway in human and rabbit tumor models [10], we hypothesized that a similar mechanism was involved in the suppression of keloid-derived fibroblast secretion and proliferation, as well as in the activation of apo-ptosis. We report that the activated forms of Smad2 and Smad3, p-Smad2 and p-Smad3, were significantly downregulated at 48 h after LY2109761 treatment in a dose-dependent manner. We previously showed that silencing of the TGF-β family member GDF-9 in keloid-derived fibroblasts downregulated p-Smad2 and p-Smad3 [5]. However, silencing of GDF-9 had no effect on the expression of TGF-β1, whereas in the current study, LY2109761 clearly suppressed TGF-β1 expression but upregulated GDF-9 expression. We speculate that the possible reason for this is that GDF-9, as one of the ligands of TGF-β receptors, is produced more to antagonize the inhibitory effect of TGF-β signaling pathway. In other words, it is a negative feedback mechanism. The overall effect of LY2109761 on keloid fibroblasts is inhibition, even though GDF-9 was partially elevated. Existing evidence indicates that regulation of TGF-β receptors is a key factor associated with keloid pathogenesis [22]. TGF-β1 is involved in the formation of keloids, and normal fibroblasts produce up to 12fold higher levels of collagen following treatment with TGF-β1 [23,24]. Another study also showed that interactions between the TGF-β/Smad and Wnt/β-catenin pathways were associated with the formation of keloids and hypertrophic scars [25].
Here, we provide further evidence that Smad2/3 signaling and the upregulation of TGF-β receptors are directly involved in keloid pathogenesis [26,27]. More importantly, Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as an internal control. * P < 0:05 between LY2109761 groups and DMSO control; * * P < 0:05 between LY2109761 5 μM and 10 μM treatment groups.  Analytical Cellular Pathology we show that LY2109761, a TGF-β receptor inhibitor, negatively regulated the Smad2/3 signaling pathway and downregulated the expression of TGF-β1 in keloid-derived fibroblasts. These results suggest that inhibition of pathways/receptors involved in keloid formation and pathogenesis may offer potential therapeutic strategies to limit scarring after injury.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
There is no conflict of interest.