Production and function of pigment epithelium-derived factor in isolated skin keratinocytes

Abstract

Pigment epithelium-derived factor (PEDF) is a multifunctional factor with potent anti-angiogenic activity that may play a role in skin homoeostasis and wound healing. Analysis of PEDF levels demonstrated that PEDF levels are high in normal skin but quite low in early wounds. As previous studies have suggested that keratinocytes can produce PEDF, we investigated how conditions that mimic those found at sites of injury influence PEDF production by keratinocytes in vitro. Both injury by mechanical disruption (scratch assay) and treatment of human keratinocytes with inflammatory cytokines (IL-1β, IL-6 and TNF-α) inhibited PEDF expression. We next examined how PEDF affects keratinocyte functions that are important in tissue repair. Treatment of keratinocytes with exogenous PEDF enhanced keratinocyte adhesion, therefore impairing migration, while having no effect on cell proliferation. The results suggest that modulation of PEDF levels may play a pivotal role in skin homoeostasis and the response of keratinocytes to injury or inflammatory insults.

Keywords: cytokine; in vitro; keratinocyte; pigment epithelium-derived factor; wound.

Figure 1

PEDF expression decreases in injured and inflammatory cytokine treated NHEK. a) NHEK monolayers were wounded by scratch injury. PEDF mRNA expression at 1, 5, and 24h after injury was analyzed by real time PCR. (*p<0.01-0.001 compared to uninjured control, t test). b,c) NHEK monolayers were treated with a range of concentrations of inflammatory cytokines (0.02-50ng/ml, IL-1β, IL-6 or TNF-α. PEDF mRNA and protein levels were examined 24h after treatment by real time PCR and ELISA respectively (*p<0.01, #p<0.05 compared to untreated control, t test). Results are expressed as means ± standard deviations, n= 3. Similar results were obtained by using cells from another subject.

Figure 2

PEDF inhibits NHEK migration and increases adhesion. a,b) Scratch migration assay: NHEK monolayers were treated with mitomycin C for 2 hours followed by the production of mechanical scratches and incubation with or without PEDF (0.02-500ng/ml) for 24 hours. The percent of the unoccupied area in the wounds was calculated compared to the time 0 untreated value (*p<0.01-0.001 compared to untreated control/0ng/ml, t test). c) Transwell migration assay: NHEK (1×10⁵) were added in an upper chamber of 24-well transwell plate; lower chambers contained culture medium with a range of concentrations of PEDF. Following a 4 hour incubation period, the number of cells that had migrated was counted. d). Adhesion assay: NHEK (1x10⁵) were cultured in a 96-well plate treated PEDF for 4 hours. The unattached cells were washed and adherent cells were fixed in ethanol, stained by crystal violet and then lysed by Triton x-100. The optical density at 550 nm was measured. (*p<0.001 compared to untreated control using at test). e) Proliferation assay: 5×10³ NHEK cells/well were cultured in 96-well plate. After 24 hours, PEDF (0.02-500ng/ml) was added and the cells were then incubated for further 24 hours. Cell proliferation was examined using CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay kit (Promega). For all experiments of Figure 2, results are expressed as means ± standard deviations, n=3. Similar results were obtained by using cells from another subject.