Hepatoma-derived growth factor and its role in keloid pathogenesis

Abstract

Hepatoma-derived growth factor (HDGF) is a novel mitogenic growth factor that has been implicated in many different carcinomas. Its role in keloid biology has not yet been investigated. The present study is aimed at examining the role of HDGF in keloid pathogenesis. Immunohistochemical staining and Western blot analyses were used to examine in vivo localization and expression of HDGF in keloid and normal skin tissue. This was followed by the detection of HDGF expression in fibroblasts cultured in vitro and fibroblasts exposed to serum. To investigate the effect of epithelial-mesenchymal interactions, a two-chamber system was employed in which keratinocytes on membrane inserts were co-cultured with the fibroblasts. HDGF expression levels in all cell extracts and conditioned media were assayed through Western blot analysis. In another set of experiments, the effect of exogenous recombinant HDGF on keloid fibroblasts (KF) and normal fibroblasts (NF) was examined. Cell proliferation was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by quantifying proliferating cell nuclear antigen (PCNA) expression. Downstream targets of HDGF were identified by detecting their expression through Western blot analysis. Our results indicate that there was an increase in HDGF expression in the dermis of keloid compared with normal skin tissue. The application of serum and epithelial-mesenchymal interactions did not seem to have any effect on intracellular HDGF expression levels. However, co-culturing keloid keratinocytes with KFs resulted in increased HDGF secretion when compared with monoculture or normal controls. Furthermore, treatment with exogenous recombinant HDGF was found to increase the proliferation of KFs, activate the extracellular signal-regulated kinase (ERK) pathway and up-regulate the secretion of vascular endothelial growth factor (VEGF).

Fig 1

Expression and localization of HDGF in keloid and normal tissue. Paraffin sections of normal and keloid tissue were prepared and stained with antibodies against HDGF. Pictures were taken with magnification at 40× (A, B) and 10× (C). The dermis and the epidermis are represented by (D) and (E), respectively. In each panel, the inset shows the same tissue labelled with a non-immune mouse antibody of the appropriate immunoglobulin isotype as a negative control. HDGF was detected in both the epidermis (A) and the dermis (B) of normal and keloid tissue. Increased expression was observed in the dermis of keloid tissue compared with the dermis of normal tissue (B, C). (D) In total, 50 μg of tissue extracts from nine keloid tissue specimens and four normal skin specimens was subjected to Western blot analysis with antibodies against HDGF. The whole-tissue extracts include both the epidermis and the dermis. The blots were probed with anti-β-actin antibody to confirm equal loading. The bar graph represents the mean ± S.E.M. of HDGF levels in the normal and keloid samples, as quantified by gel densitometry. *indicates statistical significance as assessed by Welch’s t-test.

Fig 2

Effect of serum and epithelial–mesenchymal interactions on intracellular HDGF expression. Six different strains of keloid/normal fibroblasts were cultured with DMEM, 10% FCS or co-cultured with keloid/normal keratinocytes for 5 days. In total, 50 μg of total protein extracts was subjected to Western blot analysis with HDGF antibodies. Two representative strains are shown in (A). The bar graphs in (B) represent the normalized mean ± S.E.M. of HDGF levels in the different conditions. All blots were probed and normalized with β-actin.

Fig 3

Expression of HDGF in conditioned media of monocultured and co-cultured cells. (A) Conditioned media of keloid fibroblasts monoculture (KF) and keloid fibroblasts co-cultured with keloid keratinocytes (KK/KF) were collected at days 1, 3 and 5. (B) Conditioned media of normal fibroblast monoculture (NF) and normal fibroblast co-cultured with normal keratinocytes (NK/NF) were collected at days 1, 3 and 5. Experiments were performed in duplicates. (C) Conditioned media of seven samples of singly cultured keloid keratinocytes (KK) and normal keratinocytes (NK) were collected at day 5. Four millilitres of the conditioned media from (A), (B) and (C) was then concentrated and subjected to Western blot analysis with anti-HDGF antibody. Representative figures are shown. The bar graphs represent the mean ± S.E.M. of HDGF levels. * indicates statistical significance as determined by the paired t-test.

Fig 4

Increased proliferation of keloid fibroblasts treated with recombinant HDGF. Cultures of keloid or normal fibroblasts were grown until 50% confluence and then serum starved for 48 hrs. The fibroblasts were then treated with HDGF (10, 50, 100 and 300 ng/ml) for 72 hrs and then subjected to the MTT proliferation assay. Untreated samples were used as control. The bar graph in (A) represents the mean proliferative response of treated normal fibroblasts as a percentage of the control. The bar graph in (B) represents the mean proliferative response of treated keloid fibroblasts as a percentage of the control. * indicates statistical significance compared with DMEM control as assessed by Student’s t-test.

Fig 5

Effect of HDGF on the expression of downstream targets. Normal fibroblasts and keloid fibroblasts were treated with either DMEM or 250 ng/ml of recombinant HDGF, harvested after 48 hrs and lysed for Western blot analysis, as described under experimental procedures. Blots were incubated with anti-PCNA (A), anti-phospho-ERK 1/2 and total ERK 1/2 (B) antibodies. The blots were also incubated with anti-β-actin antibody to confirm equal loading. Four millilitres of conditioned media from the same set of experiments was concentrated and subjected to Western blot analysis. Blots were then incubated with anti-VEGF (C) antibodies. In another set of experiments, normal fibroblasts and keloid fibroblasts were treated with either DMEM or HDGF and harvested after 1 hr, 6 hrs and 24 hrs for Western blot analysis. Blots were incubated with anti-phospho-ERK 1/2 and total ERK 1/2 (D) antibodies. All experiments were performed in duplicates. Representative figures are shown. The bar graphs represent the mean ± S.E.M. of HDGF levels. Phospho-ERK 1/2 was normalized against total ERK1/2 expression. * indicates statistical significance as determined by the paired t-test.

Fig 6

Schematic representation of the role of HDGF in keloid pathogenesis. Epithelial–mesenchymal interactions result in an increased secretion of HDGF in keloids. Overproduction of extracellular HDGF leads to the phosphorylation of ERK 1/2 and increased proliferation of keloid fibroblasts, most likely through a receptor-mediated pathway. HDGF also stimulates the fibroblasts to produce VEGF.