Analysis Of Epithelial-mesenchymal Transition Markers In Psoriatic Epidermal Keratinocytes

BACKGROUNDEpithelial mesenchymal transition (EMT) is a basic process during normal embryogenesis, and it has been implicated in wound healing, organ fibrosis and cancer invasion and metastasis. Research EMT is significant to understanding the pathophysiological mechanisms of inflammatory diseases.Psoriasis is a chronic inflammatory skin disease characterized by excessive proliferation of epidermis, abnormal proliferation of vascular endothelial cells and lymphocytic infiltration.During the process of cell proliferation and migration,the phenotype of epidermal keratinocytes transformed into fibroblast-like cells,and promote the ability of resistance to apoptosisis and migration at the same time.This process is similar to occur epithelial mesenchymal transition.OBJECTIVETo define the immunolocalization and expression of the molecular epithelial and mesenchymal markers in normal and psoriatic skin.Analysis of epithelial-mesenchymal transition markers in psoriatic epidermal keratinocytes and the cytokines and signalling mechanisms.METHODSPrimary cultured keratinocytes and skin samples were investigated in vitro. To define the immunolocalization and expression of the molecular epithelial and mesenchymal markers by immunohistochemisrty, RNAseq and quantitative Real-Time PCR (qRT-PCR). To further analysis the epithelial-mesenchymal transition markers expression in psoriatic epidermal keratinocytes by incubating with different psoriasis associated cytokines.To further elucidate the role of ERK,Rho,GSK3 in EMT.the specific inhibitor U0126, Y27632,SB216763 were included to incubate with psoriatic keratinocytes with or without Dexamethasone.RESULTS1.Expression of the epithelial and mesenchymal markers in normal and psoriatic skin1.The epithelial markers E-cad and β-catenin are distributed in the whole epidermal layer in both normal and psoriatic epidermal keratinocytes; K10 and K16 localize in the middle and upper spinous epidermal keratinocytes, and no difference between normal and psoriatic epidermal.K14 localizes in the basal and suprabasal keratinocytes,but localizes the whole epidermal layers in psoriatic epidermis.2.In both normal and psoriatic dermis, no fluorescent signals of E-cad, K10, K14 and K.16 are detected.3.In normal epidermis,no signals of Vim, Snail, Slug, FNand N-cad are detected.4. In psoriatic dermis,Vim, Snail, Slug and FN are strongly expressed. N-cad is moderately expressed.2.Expression of epithelial and mesenchymal markers in normal and psoriatic epidermal keratinocytes.1.Compared with normal keratinocytes,the result of RNA sequencing show the level of K10 decreased and increased Vim, FN, PAI-1 and Snail in psoriatic epidermal keratinocytes.No obvious differences in E-cad, K14, K16, N-cad,β-catenin and Slug are detected.4.In both normal and psoriatic keratinocytes,there were significant differences of Vim, FN and PAI-1 (p<0.01)by the mRNA level. No statistically significant difference between the two groups was observed for E-cad, K10, K14, K16, N-cad, β-catenin, Snail and Slug.3.The regulation of epithelial and mesenchymal markers in psoriatic keratinocytes1.TNF-a can slightly increased the expression of E-cad. K10 was markedly reduced by IL-8, TGF-β,IL-13 and especially IL-17A and TNF-α.K14 was reduced by IL-17A,but up regulated by TGF-β1.TGF-β1 also regulated the expression of N-cad markedly reduced.2.IL-17A and IL-13 down regulated the mesenchymal markers Vim and FN,but TGF-β1 reduced them.3.TNF-a reduced FN,but markedly decreased Slug.TGF-β increased PAI-1, Snail decreased by IL-17A, but up regulated by IL-8.4.The role of ERK, Rho and GSK3 in EMT in psoriatic keratinocytes1.Y27632 completely blocks Rho can increased the expression of E-cad, P-catenin and Slug in both mRNA and protein levels.2.Single incubate with Dex at 0.2 μM decreases Slug but shows no effect on E-cad and b-catenin,when at 2μM can increases E-cad and downregulation the expression of Slug.3.Dex at 0.2μM attenuates Y27632-mediated accumulation of E-cad and β-catenin, and Slug slightly,but at 2 μM does not change except for β-catenin.4.SB216763-mediated accumulation of β-catenin and Slug was not changed by Dex at 0.2 μM, but decreased by Dex at 2.0 μM.CONCLUSION1.Epidermal keratinocytes were characterized by up regulation of mesenchymal markers, such as Vim, FN, and PAI-1, whereas epithelial markers E-cad, K10, and K14 were not significantly changed.2.In the EMT process in psoriatic keratinocytes,IL-17A, LI-13 are negative regulators, TGF-β1 is a positive regulator,TNF-a revealed a diverse regulator.3.ERK, Rho and GSK3 play active roles in the process of EMT in psoriatic keratinocytes.Dex play a protective role additionally.4.We may be define the EMT in psoriatic epidermal keratinocytes as an intermediate phenotype of type 2 EMT.