Mechanisms And Functions Of Impaired Nuclear Translocation Of Glucocorticoid Receptor In Psoriatic Epidermal Keratinocytes

[BACKGROUND]Psoriasis is a common chronic inflammatory and proliferative skin disorder. Now psoriasis is thought not only to be a kind of dermatosis, but a complex disease affecting the health of common people as the patients with psoriasis always suffer from diabetes, hypertension, coronary heart disease, obesity, depression and metabolic syndrome.Topical corticosteroids are potent agents to clear psoriatic lesions and suggested as the first-line treatment of psoriasis. The powerful glucocorticoid (GC) exerts its effects mainly via its receptor---glucocorticoid receptor (GR). GR belongs to the nuclear receptor family and is ubiquitously expressed nearly in all body cells.Numerous previous investigations have established a shuttling capacity of the GR between the nuclear and cytoplasmic compartments. Depending upon the receptor and cell type, untransformed GR may be predominantly nuclear or cytoplasmic location. Upon ligand binding, the untransformed Gris translocalized into the nucleus by using nuclear transport factors. In a pilot study of our group that investigated the distribution of GR in psoriatic skin, we detected a cytoplasmic accumulation of GR in psoriatic epidermal keratinocytes. Therefore, we hypothesized that the abnormal distribution of GR may play an important role in the pathogenesis of psoriasis and investigated possible reasons behind this impaired nuclear translocation in vitro and in vivo.[OBJECTIVE]Define the mechanisms of impaired nuclear translocation of GR in psoriatic epidermal keratinocytes and clarify the possible signal transduction pathways underlying the shuttling of GR between the nucleus and cytoplasm of keratinocytes. Explore novel therapy of psoriasis through explaining the meaning of impaired nuclear translocation of GR in psoriatic epidermal keratinocytes.[METHODS]In the first part:Indirect immunofluorescence was employed to detect the expression and distribution of GR in normal and psoriatic epidermis. Normal and psoriatic epidermal keratinocytes were isolated and cultured and determined the distribution of GR by indirect immunofluorescence. Western blot was used to compare the expression level of GR between normal and psoriatic epidermal keratinocytes. The levels of ACTH and cortisol in peripheral blood of psoriasis patients and normal controls were measured by ELISA.In the second part:Isolate and culture normal and psoriatic epidermal keratinocytes. The keratinocytes were incubated with inhibitors of GSK-3, p53and microtubule and then detected the distribution of GR by indirect immunofluorescence. After stimulating by exogenous cytokines including VEGF165and IFN-γ, normal and psoriatic keratinocytes were determined the distribution of GR by indirect immunofluorescence. In the third part:Balb/c mice were induced psoriasis-like lesions by topical imiquimod. Observe the changes of imiquimod-induced psoriasis-like lesions after intravenous injection of IL-13or microtubule inhibitor vincristine. The paraffin sections of back skin of Balb/c mice were performed Masson's trichrome, a three-color staining standard procedure to stain nuclei black, collagen blue and cytoplasm, muscle erythrocytes red.[RESULTS]Ⅰ. The expression and distribution of GR in normal and psoriatic epidermis:1. GR was expressed on all layers of normal and psoriatic epidermis except for stratum corneum.2. The distribution of GR was different in normal and psoriatic epidermal keratinocytes:in psoriatic keratinocytes GR was localized in cytoplasm, while in normal keratinocytes GR was in nuclei.3. The distribution of GR was consistent with the tissue staining in primary cultured normal and psoriatic keratinocytes:GR was cytoplasmic distribution in cultured psoriatic keratinocytes while localized in nuclei of cultured normal keratinocytes.4. There was no difference between the expression level of GR in normal and psoriatic keratinocytes.Ⅱ.The impact factors of nuclear translocation of GR in normal and psoriatic epidermal keratinocytes:1. The expression levels of ACTH and cortisol were no difference between the peripheral bloods of psoriasis patients and normal controls.2. VEGF165and IFN-γ induced impaired nuclear translocation of GR in normal keratinocytes. 3. Dexamethasone reversed VEGF165and IFN-y induced cytoplasmic distribution of GR in normal keratinocytes.4. Cytokines including ET-1, HGF, TGF, IL-10, IL-13, IL-17, PDGF-AA, PDGF-AB and PDGF-BB exerted no effects on nuclear translocation of GR in normal keratinocytes.5. Nuclear translocation of GR is ATP-independent in normal keratinocytes.Ⅲ.The mechanism of impaired nuclear translocation of GR in psoriatic keratinocytes:1. The inhibitor of p53restrained VEGF]65-and IFN-γ-induced nuclear export of GR in normal keratinocytes.2. Microtubule inhibitor vincristine inhibited uptake of GR in normal keratinocytes.3. Inhibitor of GSK-3had no effect on uptake of GR in normal keratinocytes.4. Dexamethasone promoted cytoplasmic GR re-entering into nucleus in psoriatic keratinocytes.5. Cytokines such as IL-13, PDGF-AA, PDGF-AB and PDGF-BB facilitated nuclear translocation of GR in psoriatic keratinocytes, while ET-1, HGF, TGF-β1, IL-10and IL-17did not.6. Discontinuation of dexamethasone induced cytoplasmic retention of GR in normal keratinocytes.Ⅳ.Effects of IL-13and microtubule inhibitor on imiquimod-induced Balb/c mice:1. Imiquimod-induced skin inflammations that phenotypically resembled psoriasis including clinical sign of erythema, scales and thickening of the back skin of Balb/c mice and histological features such as epidermal hyperplasia, infiltration of inflammatory cells. 2. IL-13ameliorated the erythema, scales, thickening of epidermis and inflammatory cells infiltration of imiquimod-induced mouse model.3. Microtubule inhibitor exacerbated erythema, scales, thickening of epidermis and inflammatory cells infiltration of imiquimod-induced mouse model.[CONCLUSION]Ⅰ. Impaired nuclear translocation of GR occurred in psoriatic keratinocytes.Ⅱ.The levels of ACTH and cortisol in peripheral blood of psoriasis patients did not impact the distribution of GR in psoriatic keratinocytes.Ⅲ.VEGF165-and IFN-γ-induced nuclear export of GR in normal keratinocytes, and dexamethasone reversed this effect.Ⅳ.Nuclear translocation of GR is ATP-independent in normal keratinocytes.Ⅴ. VEGF165-and IFN-γ-induced nuclear export of GR required p53participant in normal keratinocytes.Ⅵ.Microtubule mediated uptake of GR, p53participated nuclear export of GR while GSK-3inhibitor did not impact the shuttling of GR between cytoplasm and nucleus in keratinocytes.Ⅶ.Dexamethasone pushed cytoplasmic GR re-entering nucleus in psoriatic keratinocytes, which need the participant of microtubule.Ⅷ.IL-13, PDGF-AA, PDGF-AB and PDGF-BB facilitated nuclear translocation of GR in psoriatic keratinocytes.Ⅸ.Discontinuation of dexamethasone induced cytoplasmic retention of GR in normal keratinocytes. Ⅹ.IL-13ameliorated the psoriasis-like skin lesions in imiquimod-induced mouse model.Ⅺ.Microtubule inhibitor exacerbated psoriasis-like skin lesions in imiquimod-induced mouse model.