Slit1 Promotes Hypertrophic Scar Formation Through the TGF-β Signaling Pathway

Abstract

Background and objectives: Slit1 is a secreted protein that is closely related to cell movement and adhesion. Few studies related to fibrosis exist, and the preponderance of current research is confined to the proliferation and differentiation of neural systems. Hypertrophic scars (HTSs) are delineated by an overproduction of the extracellular matrix (ECM) by activated fibroblasts, leading to anomalous fibrosis, which is a severe sequela of burns. However, the functionality of Slit1 in HTS formation remains unknown. We aimed to investigate whether Slit1 regulates fibroblasts through a fibrosis-related mechanism derived from post-burn HTS tissues and normal patient tissues. Methods: Human normal fibroblasts (HNFs) and hypertrophic scar fibroblasts (HTSFs) were extracted from normal skin and post-burn HTS tissues, with settings grouped according to the patient of origin. Cell proliferation was evaluated using a CellTiter-Glo Luminescent Cell Viability Assay Kit. Cell migration experiments were carried out using a μ-Dish insert system. Protein and mRNA expression levels were quantified by Western blot and quantitative real-time polymerase chain reaction. Results: We found increased expressions of Slit1 in HTS tissues and HTSFs compared to normal tissues and HNFs. The treatment of human recombinant Slit1 protein (rSlit1) within HNFs promoted cell proliferation and differentiation, leading to an upregulation in ECM components such as α-SMA, type I and III collagen, and fibronectin. The treatment of rSlit1 in HNFs facilitated cell migration, concurrent with enhanced levels of N-cadherin and vimentin, and a diminished expression of E-cadherin. Treatment with rSlit1 resulted in the phosphorylation of SMAD pathway proteins, including SMAD2, SMAD3, and SMAD1/5/8, and non-SMAD pathway proteins, including TAK1, JNK1, ERK1/2, and p38, in HNFs. Conclusions: Exogenous Slit1 potentiates the epithelial-mesenchymal transition and upregulates SMAD and non-SMAD signaling pathways in HNFs, leading to the development of HTS, suggesting that Slit1 is a promising new target for the treatment of post-burn HTS.

Keywords: Slit1; fibroblast; post-burn hypertrophic scar.

Figure 1

Tissue morphology and expression of Slit1 in tissues and fibroblasts. (A) H&E staining in normal skin and hypertrophic scar (HTS). The thickness of the epidermis in HTS appears to be greater than that of normal skin. The arrow marked out the epithelial layer of tissue. Images were acquired at ×10 magnification, scale bar = 50 μm. (B,C) Significantly increased levels of both mRNA and protein of Slit1 were observed in HTS tissue compared to those in normal tissues. ** p < 0.01, vs. Normal. (D,E) Significantly increased levels of both mRNA and protein of Slit1 were observed in HTSFs compared to those in HNFs. HNFs and HTSFs were extracted from normal skin tissues and post-burn HTS tissues obtained from the same patients. ** p < 0.01, vs. HNFs. Data represent the mean ± SD; n = 3.

Figure 2

Effects of rSlit1 treatment on the proliferation and differentiation of HNFs. (A) Significantly increased proliferation of HNFs was observed following treatment with 10 and 100 ng/mL compared to DPBS-treated cells. (B,C) Significantly increased levels of both mRNA and protein of α-SMA (ACTA2) were observed in HNFs treated with 10 and 100 ng/mL of rSlit1 compared to DPBS-treated cells. DPBS was used as the control. * p < 0.05, vs. DPBS. Data represent the mean ± SD; n = 3.

Figure 3

Effects of rSlit1 treatment on the expression of ECM components in HNFs. Significant increases of both mRNA and protein levels of (A,B) type Ⅰ collagen (COL1AⅠ), (C,D) type Ⅲ collagen (COL3AⅠ), and (E,F) fibronectin (FN1) were observed in HNFs treated with 10 and 100 ng/mL of rSlit1 compared to DPBS-treated cells. DPBS was used as the control. * p < 0.05, vs. DPBS. Data represent the mean ± SD; n = 3.

Figure 4

Effects of rSlit1 treatment on the EMT phenotype of HNFs. The mRNA and protein levels exhibited significant increases in the expression of (A,B) vimentin (VIM) and (C,D) N-cadherin (CDH2), whereas a notable decrease in (E,F) E-cadherin (CDH1) expression was observed in HNFs treated with 10 and 100 ng/mL rSlit1, compared to those treated with DPBS. (G,H) Cell imaging demonstrated enhanced migration of HNFs treated with rSlit1 at concentrations of 10 and 100 ng/mL compared to the DPBS-treated controls. Enlarged images belong to the green box. * p < 0.05, vs. DPBS. Data represent the mean ± SD; n = 3.

Figure 5

Effects of rSlit1 treatment on expression of SMAD signaling in HNFs. Significantly increased phosphorylated protein expression of (A,B) SMAD2, (A,C) SMAD, and (A,D) SMAD1/5/8 was observed in HNFs treated with 10 and 100 ng/mL rSlit1, compared to DPBS-treated cells. DPBS was used as the control. * p < 0.05, vs. DPBS. Data represent the mean ± SD; n = 3.

Figure 6

Effects of rSlit1 treatment on expression of non-SMAD signaling in HNFs. Significantly increased phosphorylated protein expression of (A,B) TAK1, (A,C) JNK1, (A,D) ERK1/2, and (A,E) p38 was observed in HNFs treated with 10 and 100 ng/mL rSlit1 compared to DPBS-treated cells. DPBS was used as the control. * p < 0.05, vs. DPBS. Data represent the mean ± SD; n = 3.