Role of estrogen in wound healing through regulation of epithelial-mesenchymal transition in human keratinocyte

Abstract

Aging of the skin is associated with delayed wound healing. This undesirable effect of aging is exacerbated by declining estrogen levels in postmenopausal women. Replacement of estrogen can increase collagen synthesis and keratinocyte migration to accelerate wound healing. Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells gain a mesenchymal phenotype, leading to increased motility. Hence, the hypothesis that acceleration of keratinocyte migration by estrogen may be mediated by EMT was tested in this study. The goals of this study were to examine the relationship between estrogen-induced keratinocyte migration and EMT and to identify a novel protein associated with estrogen-induced EMT through stable isotope labeling by amino acids in cell culture (SILAC) analysis.HaCaT immortalized human skin keratinocytes and primary human keratinocytes (PHK) expressed only ERβ. The expression of mesenchymal markers Slug, Twist, and vimentin was increased while the expression of the epithelial marker E-cadherin was decreased by treatment with 0.1, 1, 10, 100 nM estrogen for 48 h. The altered expression of these proteins was reversed following treatment with ICI, an estrogen receptor antagonist. The number of migrating cells was significantly increased in the presence of estrogen compared to control cells in wound scratch assay and transwell assay. Knockdown of Slug blocked estrogen-induced keratinocyte migration and reversed the changes in vimentin and E-cadherin induced by estrogen treatment.SILAC-based mass spectrometry was performed on keratinocytes with or without estrogen treatment and 1085 proteins were identified. Thirty-two proteins were down-regulated and 60 proteins were up-regulated significantly. Among the 60 up-regulated proteins, 6 high-mobility group proteins were identified.HMGB1 expression was increased after estrogen treatment, and the increased HMGB1 was reversed following treatment with ICI. The migration of keratinocytes was increased after treatment with recombinant HMGB1 protein and the estrogen-induced migration of keratinocytes was reversed by HMGB1 siRNA transfection. Knockdown of Slug in keratinocytes also resulted in a significant decrease in HMGB1.These experiments demonstrate that estrogen contributes to keratinocyte migration and that this activity may be mediated by the EMT process. Increased expression of HMGB1 may also be important in estrogen-induced keratinocyte migration and there may be a link between the EMT process and HMGB1.