Expression And Function Of Pigment Epithelium-derived Factor In Skin

[Background]Pigment epithelium-derived factor (PEDF) is a 50 kDa secreted glycoprotein that belongs to the non-inhibitory serpin group. PEDF is widely expressed in human tissues, including the adult brain, spinal cord, plasma, liver, bone, eye, heart, and lung. In cultured cell lines, PEDF was found to be expressed in human lung, skin, and endometrial stromal fibroblasts, vascular smooth muscle cells, cardiac cells, retinal-pigmented epithelial cells, and T lymphocytes.PEDF has been demonstrated to function as a potent and broadly acting neurotrophic factor that induce cell differentiation and protects neurons in the brain, eye, and spinal cord against a wide range of neurodegenerative insults. In addition, PEDF has been described as a potent endogenous inhibitor of angiogenesis, more active than endostatine and thrombospondin-1. More recent investigations suggest that PEDF could play a role in immunomodulation and in the protection/survival against oxidative stress.In normal skin, PEDF was detected primarily in the dermis and keratinocytes in the epidermis expressed very little PEDF. However, Abe and colleagues have shown the presence of PEDF in both epidermis and dermis in normal skin and PEDF expression in dermis was less intense than in the epidermis. Further investigations should be done to elucidate the expression and function of PEDF.Part 1 The Expression and function of pigment epithelium-derived growth on HaCaT Cells[Objective]To investigate the expression of PEDF and its roles in proliferation, adhesion and migration of HaCaT cells, a human keratinocyte cell line.[Methods]RT-PCR and Western-blot were used to determine the mRNA and protein expression of the PEDF and its receptor on HaCaT cell line.Indirect immunofluorescent staining was used to detect the location of PEDF. MTT assays were used to determine the effect of different concentrations of PEDF on the proliferation of HaCaT cells.Wound healing assay and transwell assay were used to determine the effect of different concentrations of PEDF on the migration of HaCaT cells. Crystal violet assays were used to determine the effect of different concentrations of PEDF on the cell adhesion of HaCaT cells. The effect of PEDF on signal pathways of extracellular signal-regulated kinase (ERK) 1/2, p38, JNK and AKT was studied by using Western-blot.[Results]Our results have shown that both PEDF and its receptor were expressed in HaCaT cells at both mRNA and protein levels. PEDF signal mainly localizes in the cytoplasm of HaCaT cells. Furthermore, expression of PEDF is decreased by 50 ng/ml of VEGF165. Proliferation and migration of HaCaT cells are decreased by PEDF, while adhesion of HaCaT cells is upregulated. In addition, phosphorylation of ERK1/2, not p38, JNK and AKT, is decreased by PEDF.[Conclusion]PEDF is expressed in HaCaT cells. PEDF may function through the ERK1/2 pathway to decrease cell proliferation and chemotactic migration.Part 2 The expression of pigment epithelium-derived growth in normal human and psoriatic skin[Objective]To investigate the expression of PEDF in normal human and psoriatic skin tissues, epidermal keratinocytes and dermal fibroblasts.[Methods]Immunohistochemistry staining was used to examine the expression of PEDF in normal skin and psoriatic skin tissues. Isolate epidermal keratinocytes and dermal fibroblasts from normal people and psoriasis patients. The expression of PEDF in psoriatic epidermal keratinocytes and dermal fibroblasts at mRNA. and protein levels was determined by RT-PCR and Western-blot analysis.[Results]Through Immunohistochemistry, PEDF strongly expressed in stratum basal and spinosum adjacent to basal in normal skin. Lesional psoriatic skin had a higher staining for PEDF compared to normal, nonlesional or perilesional psoriatic skins. The mRNA level of PEDF in nonlesional, perilesional and lesional keratinocytes was significantly increased compared with that of normal keratinocytes. There are no obvious defference among mRNA level of PEDF in nonlesional, perilesional and lesional fibroblasts.The protein levels of PEDF in cultured normal and psoriatic keratinocytes and fibroblasts revealed a similar pattern as we observed at the mRNA level.[Conclusion]PEDF was overexpressed by psoriatic lesional keratinocytes which may participate in pathogenesis of psoriasis.Part 3 Expression of pigment epithelium-derived growth factor in human epidermal appendages[Objective]To investigate the expression of PEDF in epidermal appendages and its related function.[Methods]Immunohistochemistry staining was used to detect the expression of PEDF in human epidermal appendages. RT-PCR, Western-blot and indirect immunofluorescent staining were used to determine the mRNA and protein expression of the PEDF on ORS cells. Wound healing assay was used to determine the effect of different concentrations of PEDF on the migration of ORS cells.[Results]PEDF was shown to be expressed in epidermal matrix cells, the inner and outer root sheaths, fibrous sheath cells of hair follicle, sebaceous gland and eccrine sweat gland. Expression of PEDF is detected at RNA and protein levels and localized in the cytoplasm and nucleus of ORS cells. IL-4 and IL-17, especially IL-4, upregualted the expression of PEDF of ORS cells at a concentration of 25ng/ml. Furthermore, PEDF at 50ng/ml decreased migration of ORS cells.[Conclusion]PEDF is expressed in hair follicles, sebaceous glands, eccrine sweat glands and cultured ORS cells and may be an important agent in ORS cells function.