Comparative Analysis Of Integrin And Extracellular Matrix In Normal Human Skin,Scleroderma And Psoriatic Keratinocytes And Fibroblasts

BACKGROUNDIn the past20years, integrin family and ECM component become the hot-spot in research field of dermatology. Expression of the two groups of molecules is variable during the different period in skin development, and in different parts of skin, in order to make the different adhesion environment, to utilize the different cells better. It is very important for maintaining the epidermal structure and stable function. Scleroderma and psoriasis are two common skin diseases, and their relation with integrin and ECM have been studied for years. However, some researchers figured out that the entire skin is abnormal in psoriasis patients, not only in epidermis. Have the epidermis also involved in mechanism of scleroderma?Psoriasis is a chronic skin inflammation characterized with epidermal hyper-proliferation, abnormal proliferation of vascular and lymphocyte infiltration. Fibrosis and vascular impairment are the main pathological characteristics of scleroderma. Considering the opposite manifestations of the two diseases, we assume that there may be reversed molecular alteration in keratinocytes and fibroblasts between scleroderma and psoriasis.OBJECTIVESTo investigate the expression of integrin α5,β4in normal, scleroderma and psoriatic epidermal keratinocytes, the regulation of integrin α5,β4by cytokines in keratinocytes under different status; the regulation of ECM component by Ca++and dexamethasone in normal, scleroderma and psoriatic fibroblasts and the probable cell signal pathways involved.METHODSIn the first part:skin biopsies were divided into two parts, one part was paraffin embedded, and the other part was used to culture keratinocytes. The total protein was extracted from cultured epidermal keratinocytes. The protein level of integrin α5,β4was determined by western blot. We further localized the integrin α5,β4in normal, scleroderma and psoriatic epidermis by immunohistological staining. The expression of integrin α5,β4was observed by western blot after adding exogenous IL-1β,IL-13, IL-17A, TNF-α, TGF-β1, ET-1, IFN-γ and VEGF165.In the second part:the culture fibroblast from normal, scleroderma and psoriatic dermis were treated with Ca++and dexamethasone at the presence or absence of U0126, Y27632, pifithrin-α. The total protein was extracted from cultured dermal fibroblasts, and the protein was determined by western blot to investigate the expression of fibronectin, CTGF and type I collagen. RESULTS1,Localization and expression of integrin α5,β4in normal, scleroderma and psoriatic epidermis:1) Integrin a5localized in the whole epidermis except for squamous layer both in normal and scleroderma epidermis. In normal and scleroderma epidermis, the distribution of a5was both distinguished in the basal layer. Integrin β4was distributed in the whole epidermis, but faint in the basal keratinocytes, both in normal and scleroderma epidermis. Furthermore, the cellular distribution of β4in normal keratinocytes was membranous and cytoplasmic, while predominantly membranous in scleroderma keratinocytes. Western blot demonstrated that integrin α5and β4was decreased in scleroderma keratinocytes, comparing with normal keratinocytes;2) α5localized in the whole skin, except for squamous layer in psoriatic epidermis. In normal epidermis, the expression of α5was very weak, but showed a relative concentration at the basal layer. In psoriasis, the distribution of α5was almost homogenous in basal and prickle keratinocytes, and the immunoreaction was much stronger than that in normal epidermis. Integrin β4was detected in the membrane of normal epidermal keratinocytes from basal to granular layers, while β4being absent in the basal and parakeratosis layer of psoriatic epidermis. Furthermore, expression of α5in normal keratinocytes was less than that in psoriatic keratinocytes significantly, while no significant difference of β4being observed;3) In normal keratinocytes, integrin a5was increased by TNF-α and IFN-γ, but not altered by IL-17A and ET-1. However, in scleroderma keratinocytes, integrin α5was upregulated by IL-17A, especially significantly by ET-1, TNF-α, and IFN-γ. That is, scleroderma keratinocytes response more sensitive to IL-17A, ET-1, TNF-α and IFN-y than in normal keratinocytes, in production of integrin α5. Integrin β4in normal keratinocytes was increased by IL-17A, ET-1, TNF-α and IFN-γ, while in scleroderma keratinocytes, no obvious difference was observed of β4between IL-17A-, TNF-α-, IFN-γ-, ET-1-treated samples and controls;4) In normal keratinocytes, α5was upregulated by IL-13, ET-1, especially by IL-17A, TNF-α and IFN-γ, not altered by TGF-β1and VEGF165, but decreased by IL-1β.β4was increased by IL-17A, ET-1, TNF-α, particularly by IL-13, IFN-y, IL-1β, TGF-β1and VEGF165in normal keratinocytes. However, in psoriatic keratinocytes, α5was decreased by IL-13, ET-1, TNF-α, IFN-γ, VEGF165, especially IL-17A, not being altered by IL-1/3and TGF-β1.β4was decreased by IL-17A and TNF-α, not being altered by IL-13, ET-1, IFN-γ, IL-1β, TGF-β1and VEGF155;5) In normal keratinocytes,10μM U0126prominently decreased a5, but not alter the expression of β4. On the contrary, in psoriatic keratinocytes, integrin α5was nearly not altered, but β4was decreased very obviously, by10,uM U0126. In normal keratinocytes, in the presence of U0126, α5was increased by IL-13, ET-1and especially, IL-17A, TNF-a and IFN-γ.β4was decreased by IL-13, IL-17A, and IFN-γ, but being increased by ET-1and TNF-α. In psoriatic keratinocytes, with addition of U0126, a5was strongly increased by IL-13and IL-17A, not being altered by ET-1and TNF-α, but being decreased by IFN-γ. β4was increased by IL-13and IL-17A, not being altered by ET-1, TNF-α and IFN-γ.2,Regulation of FN, CTGF and col-1A by Ca++and dexamethasone1) In normal fibroblasts, Ca++induced enhancement of FN and CTGF, and have no effect on col-1A production. However, in both scleroderma and psoriatic fibroblasts, Ca++increased FN, CTGF and col-1A production.2) ERK, Rho, p53and Smad2signal pathways were involved in the regulation of ECM component in fibroblasts by Ca++3) In normal fibroblasts, dexamethasone shows suppression of FN and co-1A production, but enhancement of CTGF. However, in both scleroderma and psoriatic fibroblasts, dexamethasone increased FN, CTGF and col-1A production.4) ERK, Rho, p53and Smad2signal pathways were involved in the regulation of ECM component in fibroblasts by dexamethasone.Conclusion1. Integrin α5was distributed diffusely in normal and scleroderma epidermis, and expressed strong immune-activity in basal layer and the adjacent prickle layer; and α5was strong positive and homogenous in basal and prickle layer in psoriatic epidermis;2.β4was distributed in normal and scleroderma epidermis diffusely, but weakly in basal layer.β4was expressed in cell membrane and cytoplasm in normal keratinocyte, but only in cell membrane in scleroderma keratinocyte. There was no β4expression in stratum corneum and basal layer in psoriatic epidermis;3,Comparing with normal keratinocyte, integrin α5expression was decreased in scleroderma keratinocyte, but increased in psoriatic keratinocyte significantly; 4,Comparing with normal keratinocyte, integrin β4expression was decreased in scleroderma keratinocyte significantly, and decreased in psoriatic keratinocyte. but not significantly;5,With the same stimulation, the regulation of integrin α5and β4in normal, scleroderma and psoriatic keratinocytes was different;6,Calcium may play an important role in etiology of scleroderma and psoriasis;7,Dexamethasone will induce ECM protein promotion in scleroderma and psoriatic fibroblasts.