Comparative Investigation Of Involucrin And ECM In Psoriatic And Scleroderma Epidermal Keratinocytes And Dermal Fibroblasts

[BACKGROUND]Psoriasis is a chronic inflammatory and proliferative skin disorder characterized by epidermal hyperplasia, abnormal keratinization, angiogenesis, immune activation and inflammatory response. Systemic scleroderma is a connective tissue disease characterized by collagen fibers hyperplasia, vascular changes and resistance to a variety of autoantibodies. The pathogenesis of the two diseases are not yet clear, but it is indicated that they are related with pro-inflammationm cytokines, such as interleukins, vascular endothelial growth factor, transfer growth factor, tumor necrosis factor and so on.As we all know, hyperkeratosis and abnormal cornification exist in psoriasis keratinocytes, but the exact mechanism is not clear. At the same time, the latest research shows that cornification delays in scleroderma keratinocytes. Involucrin is an early marker of terminal differentiation of keratinocytes. In normal human epidermis, Involucrin is expressed in the upper prickle cell layer and granular layer. While in psoriatic patients, Involucrin is expressed in all epidermis except the basal cell layer and the stratum corneum. Meanwhile, the expression is also increased significantly in psoriatic keratocytes. Till now, few reports concerned the expression and distribution of involucrin in scleroderma epidermis.On the other hand, the scleroderma fibroblasts activation is the main reason for the deposition of collagen in the dermis. As a result, researchs have been focus on the fibroblasts of scleroderma. TGF-β is one of the most powerful fibrogenic cytokines involved in the activation of fibroblasts and collagen synthesis, and play a key role in chronic fibrotic diseases. VEGF involves in the pathogenesis of scleroderma disease, and it is reported VEGF is elevated in the of scleroderma serum and lesions. However, the mechanisms of these pro-inflammatory cytokines are still unknow.Epidermal keratinocytes are the main target cells of psoriasis research. It is reported that the fibroblast-keratinocyte interaction plays a role in the pathogenesis of psoriasis. VEGF and angiopoietin secreted by vascular endothelial cells play a key role in psoriatic angiogenesis. Psoriatic patients overexpressed of TGFβ1in vivo. The wild-type transgenic mice (K5.TGFβ1), which TGF-β1is overexpressed, developed psoriasis-like inflammation lesions. But few research has been done to investigate the role and mechanism of pro-inflammation cytokines in psoriatic fibroblasts.Therefore, we conduct this research to to explore the role of inflammatory cytokines in the pathogenesis of psoriasis and scleroderma in keratinocytes and fibroblasts.[OBJECTIVE]Define the machanism of abnormal cornification in psoriatic and scleroderma keratinoyctes;clarify the cytokins in the dysfunction of psoriatic fibroblasts and abnormal activation of the scleroderma fibroblasts and the possible signal transduction pathways underlied.[METHODS]In the first part:Indirect immunofluorescence was employed to detect the expression and distribution of Involucrin in normal and psoriatic epidermis. Western blot was used to compare the expression level of Involucrin between normal and psoriatic epidermal keratinocytes. The keratinocytes were incubated with IL-13, IL-17A, TNF-α, ET-1, IFN-γ and inhibitors of ERK1/2and GSK-3β, and then western blot was used to compare the expression level of Involucrin between normal and psoriatic epidermal keratinocytes.Normal and psoriatic epidermal keratinocytes were isolated and cultured and determined the distribution of GR by indirect immunofluorescenceIn the second part:Indirect immunofluorescence was employed to detect the expression and distribution of Involucrin in normal and scleroderma epidermis. Normal and scleroderma epidermal keratinocytes were isolated and cultured and determined the distribution and expression of Involucrin by indirect immunofluorescence. The keratinocytes were incubated with IL-13, ET-1, IFN-γ and the inhibitor of Rho and then western blot was used to compare the expression level of Involucrin between normal and scleroderma epidermal keratinocytes.In the third part:Normal, psoriatic and scleroderma fibroblasts were isolated and cultured. and determined. The fibroblasts were incubated with VEGF165, IFN-γ, TGF-β1and inhibitors of ERK1/2and GSK-3β, and then western blot was used to compare the expression level of FN, CTGF and pAKT between normal, psoriatic and scleroderma fibroblasts. [RESULTS]1The expression and regulation of Involucrin in normal and psoriatic epidermis:1) Involucrin was expressed in the upper prickle cell layer and granular layer in normal epidermis. While in psoriasis patients, Involucrin is expressed in all epidermis except the basal cell layer and the stratum corneum.2) The expression of Involucrin was increased in cultured psoriatic keratinocytes compared with normal keratinocytes.3) The expression of Involucrin was increased after ERK1/2and GSK-3β signaling pathways were inhibited in cultured normal and psoriatic keratinocytes.4) IL-13, IL-17A, ET-1, TNF-aand IFN-y increased the expression of Involucrin in cultured normal and psoriatic keratinocytes. However, they acted differently to different cytokines.5) After ERK1/2signaling pathway was inhibited, IL-13(only in normal keratinocytes), IL-17A, ET-1, TNF-aand IFN-γ have no effect on the up-regulation of involucrin expression in normal and psoriatic keratinocytes. Moreover, IL-13even decreased involucrin in psoriatic keratinocytes.6) After GSK-3p signaling pathway was inhibited, IL-13, ET-1and TNF-α could still increase involucrin expression in normal keratinoyctes, while IL-17A and IFN-γ decreased involucrin. In psoriatic keratinocytes, IL-17A and IFN-γ could significantly decrease involucrin, while IL-13, ET-1and TNF-α showed no effect in involucrin expression. 2The expression and regulation of Involucrin in normal and scleroderma epidermi:1) Involucrin was expressed in the upper prickle cell layer and granular layer in normal and scleroderma epidermis..2) The expression of Involucrin was decreased in cultured scleroderma keratinocytes compared with normal keratinocytes.3) Involucrin was detected around the nuclei of normal keratinocytes, while in scleroderma keratinocytes, it is detected in the nuclei. Meanwhile, involucrin expression decreased in scleroderma keratinocytes determined by indirect immunofluorescence.4) In normal keratinocytes, IL-6, IL-10, IL-17A, TNF-a, especially TGF-β1, ET-1, IFN-γ, VEGF165and PDGF-BB could increase involucrin. While in scleroderma keratinocytes, IL-1β, IL-6, IL-10, TGF-β1, ET-1, IFN-γ, VEGF165and PDGF-BB decreased involucrin, and IL-4,IL-17A and TNF-increased involucrin expression.5) Rho signaling pathway has no effect on the expression of involucrin in cultured normal and scleroderma keratinocytes.6) The pro-involucrin effect of IFN-γ was reversed after Rho signaling pathway was inhibited in normal keratinocytes. While in sclerderma keratinocytes, it is IL-13. In addition, IFN-γ could further increase involucrin after Rho signaling pathway was inhibited. 3The expression and regulation of ECM in normal, psoriatic and scleroderma fibroblasts:1) TGF-(31increased FN expression in normal, psoriatic and scleroderma fibroblasts, and ERK1/2and GSK-3(3signaling pathways have no effect in it.2) VEGF165increased FN expression in normal and psoriatic fibroblasts, but have no effect on scleroderma fibroblasts. After ERK1/2signaling pathway was inhibited, VEGF165could increase FN expression in scleroderma fibroblasts. And after GSK-3β signaling pathway was inhibited, the pro-FN effect of VEGF165was reversed after GSK-3β signaling pathway was inhibited in normal fibroblasts3) IFN-γ increased FN expression in normal fibroblasts, but have no effect on psoriatic and scleroderma fibroblasts. After ERK1/2signaling pathway was inhibited, IFN-y could significantly increase FN expression in normal fibroblasts. And after GSK-3(3signaling pathway was inhibited, IFN-γ decreased instead of increase FN expression in normal keratinocytes. Meanwhile, IFN-γ could promote FN in psoriatic fibroblasts.4) TGF-β1increased CTGF expression in normal, psoriatic and scleroderma fibroblasts, while VEGF165and IFN-γ show no effect on CTGF expression. And after ERK1/2and GSK-3(3signaling pathways were inhibited, TGF-β1could still increase CTGF expression, while VEGF165and IFN-y have no effect.5)-VEGF165increased AKT phosphorylation in normal fibroblasts, but has no effect in psoriatic and scleroderma fibroblasts. And after ERK1/2signaling pathway was inhibited, VEGF165increased AKT phosphorylation in psoriatic and scleroderma fibroblasts. In addition, after GSK-3β signaling pathway was inhibited, VEGF165decreased AKT phosphorylation in normal and psoriatic fibroblasts.6) IFN-y increased AKT phosphorylation in normal and psoriatic fibroblasts. After ERK1/2signaling pathway was inhibited, IFN-y could increase AKT phosphorylation in scleroderma fibroblasts. And after GSK-3β signaling pathway was inhibited, IFN-γ decrease AKT phosphorylation in psoriatic fibroblasts.7) TGF-β1could greatly increased AKT phosphorylation in normal, psoriatic and scleroderma fibroblasts. In addition, ERK1/2and GSK-3β signaling pathways have no effect in it.[CONCLUSION]1、The expression of Involucrin is increased in psoriatic epidermis and cultured psoriatic keratinocytes;2、ERK1/2and GSK-3β signaling pathways could inhibit abnormal epidermal cornification;3、IL-13, IL-17A, ET-1, TNF-α and IFN-γ could regulate involucrin expression in normal and psoriatic keratinocytes through ERK1/2and GSK-30signaling pathways;4、ERK1/2and GSK-3β signaling pathways act differently in IL-13-, IL-17A-, ET-1-, TNF-α-and IFN-γ-regulated involucrin expression in normal and psoriatic keratinocytes;5、The cornification is delayed in scleroderma epidermis; 6、The delayed cornification in scleroderma epidermis may related with IL-4, IL-6, IL-10, TGF-β1, ET-1, IFN-γ, VEGF165and PDGF-BB;7、Rho signaling pathway has no effect on involucrin expression in normal and scleroderma keratinocytes, but it could infuence IFN-y and IL-13in the regulation of involucrin;8、VEGF165, IFN-y and TGF-β1could regulate FN expression and AKT phosphorylation through ERK1/2and GSK-3β signaling pathways in inormal, psoriatic and scleroderma fibroblasts;9、TGF-β1could increase CTGF expression in normal, psoriatic and scleroderma fibroblasts, but not through ERK1/2and GSK-3β signaling pathways;10. The same kind of cells, if they are in different phenotype, may act differently to same cytokines and signaling pathway inhibitors.