RESUMO
The transcription factor LIM-only protein 4 (LMO4) is overexpressed in the psoriatic epidermis and regulates keratinocyte proliferation and differentiation. High LMO4 expression levels are induced by interleukin-23 (IL-23) to activate the AKT/STAT3 signaling pathway. Interleukin-6 (IL-6) is mainly involved in regulating T cell functions and development in patients with psoriasis. However, whether LMO4 expression is regulated by IL-6 remains unclear. Therefore, the purpose of this study is to explore the role and molecular mechanisms of IL-6 in regulating LMO4 expression. The interleukin-6 (IL-6) levels in human plasma were determined using a chemiluminescence immunoassay system. A psoriasis-like mouse model was established using imiquimod induction. Epidermal keratinocytes (HaCaT) were cultured in defined keratinocyte-serum-free medium and stimulated by IL-6 alone or with inhibitors. The proteins of interest were detected using western blot analysis, immunofluorescence, and immunohistochemistry. The 5-ethynyl-2'-deoxyuridine assay was used to detect cell proliferation. The results revealed that IL-6 levels were markedly increased in the plasma of patients with psoriasis, compared to healthy control. The high expression of LMO4 was consistent with high levels of IL-6, p-AKT, and p-STAT3 in the lesions of both psoriasis patients and imiquimod-induced psoriasis-like mice. IL-6 activates the AKT/STAT3 signaling pathway, followed by LMO4 high-expression in HaCaT cells. IL-6 induces HaCaT proliferation and differentiation via AKT/STAT3 signaling pathway activation. We think that the high expression of LMO4 in psoriatic keratinocytes requires IL-6 to activate the AKT/STAT3 signaling pathway and leads to epidermal keratinocytes abnormal proliferation and differentiation.
Assuntos
Interleucina-6 , Psoríase , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citocinas/metabolismo , Imiquimode/efeitos adversos , Queratinócitos , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Proteínas Proto-Oncogênicas c-akt , Fator de Transcrição STAT3/metabolismoRESUMO
The adeno-associated virus (AAV) genome can be stably integrated into the AAVS1 region of human chromosome 19 (19q13.4-qter) with the assistance of Rep68/78 protein. In the current models of AAV integration in a locus-specific manner, the foreign genes were randomly inserted into the AAVS1 region, which contains several functional genes. As random integration in this region may lead to insertion mutations and disrupt normal gene expression or critical signaling pathways of the host cells, it is necessary to find a precise insertion site in the AAVS1 region. Homologous recombination is the most accurate and versatile mechanism for such site-specific integration. To investigate site-specific integration in the AAVS1 region, a targeted vector containing two homologous arms derived from AAVS1 and a reporter gene was transfected into HeLa cells with or without Rep68/78 mRNA. The results indicated that transient expression of Rep68/78 in HeLa cells improved integration of the gene of interest at the AAVS1 locus in a site-specific manner. Compared with locus-specific integration reported in previous studies, site-specific integration may minimize the risk associated with random DNA integration in the AAVS1 region, which might be helpful for gene therapy.
Assuntos
Proteínas de Ligação a DNA/genética , Dependovirus/genética , Recombinação Genética , Proteínas Virais/genética , Integração Viral , Sítios de Ligação/genética , Cromossomos Humanos Par 19/genética , Proteínas de Ligação a DNA/metabolismo , Dependovirus/metabolismo , Citometria de Fluxo , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Modelos Genéticos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução Genética/métodos , Proteínas Virais/metabolismoRESUMO
Psoriasis is an autoimmune disease characterized by abnormal differentiation and hyperproliferation of epidermal keratinocytes. LIM-domain only protein 4 (LMO4) is a transcription factor coregulator that promotes the assembly of multiprotein complexes to regulate mammary epithelium and keratinocyte differentiation and proliferation during embryogenesis. In this study, LMO4 has been found to be abundantly expressed in psoriatic epidermis. LMO4 expression is increased in human keratinocytes induced to differentiate by calcium ex vivo, and LMO4 overexpression induces spontaneous differentiation and growth acceleration of human keratinocytes in the absence of calcium. IL-23, a cytokine highly expressed in psoriatic skin lesions, induces differentiation and promotes proliferation of human keratinocytes. The IL-23-mediated effects are accompanied by an increase in LMO4 expression mediated by signal transducer and activator of transcription 3 through an IL-23/acutely transforming retrovirus AKT8 in rodent T-cell lymphoma/signal transducer and activator of transcription 3 pathway in keratinocytes. Knockdown of LMO4 effectively inhibits differentiation and growth of keratinocytes both ex vivo and in IL-23-injected ears of mice. LMO4 appears to mediate IL-23-related responses in psoriatic keratinocytes and is a potential therapeutic target in psoriasis.