RESUMO
Interleukin (IL)-15 is an important proinflammatory cytokine that can protect epidermal keratinocytes (KCs) from ultraviolet-induced apoptosis and plays a role in the pathogenesis of psoriasis. However, the impact of IL-15 on KC differentiation remains unknown. In this study, isolated human primary epidermal KCs were treated with various concentrations of IL-15 for different times, and the expression of differentiation markers (keratin 1, involucrin and loricrin) and p53 as well as the activation of ERK, AKT and Notch induced by IL-15 in the absence or presence of Ca(2+) was detected by real-time PCR and Western blot. The results showed that stimulation with Ca(2+) alone increased the expression of KC differentiation markers and p53 and promoted the activation of Notch1. Pretreatment with IL-15 resulted in a decrease in the Ca(2+) -induced expression of KC differentiation markers and p53. Additionally, Ca(2+) continually inhibited the phosphorylation of ERK1/2 and activated AKT, and IL-15 reduced the effect of Ca(2+) on ERK1/2 and AKT. FR180204, a specific inhibitor of ERK1/2 phosphorylation, slightly attenuated the effect of Ca(2+) on the expression of differentiation markers and p53 and the activation of Notch1. In contrast, MK-2206, an inhibitor of pAKT, strongly blocked the expression of the differentiation markers and p53 and the activation of Notch1. An anti-IL-15 antibody neutralized the effect of IL-15 on KC differentiation. These results indicate that IL-15 inhibits the Ca(2+) -induced differentiation of KCs, mainly via the attenuation of Ca(2+) -stimulated PI3K-AKT signalling.
Assuntos
Cálcio/metabolismo , Interleucina-15/metabolismo , Queratinócitos/metabolismo , Diferenciação Celular , Expressão Gênica , Humanos , Queratinócitos/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/metabolismoRESUMO
Toll-like receptors (TLRs) play important roles in activation of immunoreaction and tumor development. Toll-like receptor 7 (TLR7), one of the TLRs binding with single-stranded RNA, activates intracellular pathways and stimulates the release of proinflammatory cytokines, chemokines. In this study, we investigated the impact of the TLR7-signaling pathway on the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 1 (TIMP1), interleukin 6 (IL-6), and interleukin 15 (IL-15), which have been testified to refer to the immunomodulating and tumor progression. We confirmed that the TLR7 was expressed by Hela cells, despite the abundance was weak. Gardiquimod, one of the TLR7 ligands, can promote these five genes expression in varying degrees. After stimulating with gardiquimod, the expression of the IL-15V1, 3 increased about 4.5 times on RNA level, the other expression was only up-regulated about 2 times. We also discovered that gardiquimod could activate the MAPK/ERK- and PI3K/AKT-signaling pathways, and the specific inhibitors studies indicate that, the effect of gardiquimod on these genes expression is mainly or partially dependent on the activation of these two signaling pathways. To sum up, the activation of TLR7 signaling pathway may modulate some genes expression in Hela cells and may contribute to the pathogenesis of the cervical cancer.
Assuntos
Expressão Gênica/genética , Interleucina-15/genética , Interleucina-6/genética , Metaloproteinase 2 da Matriz/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Receptor 7 Toll-Like/genética , Fator A de Crescimento do Endotélio Vascular/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Transdução de Sinais/genéticaRESUMO
Toll-like receptor 7 (TLR7) is an important member in pattern recognition receptors families. TLR7 signal pathway is involved in the physiological process in many type cells, but the impact of TRL7 on differentiation in the human keratinocytes is still unknown. In this study, we investigated the expression of TLR7 in keratinocytes, and the effect of TLR7 agonist gardiquimod on the expression of calcium (Ca(2+))-induced keratinocytes differentiation markers in HaCaT cells. Immunohistochemistry and western-blotting analysis showed that TLR7 is expressed in basal keratinocytes of normal skin and in the human keratinocyte cell line HaCaT, but not expressed in the keratinocytes of psoriasis lesions. Pretreatment with gardiquimod could down-regulate Ca(2+)-induced differentiation marker expression and activate Raf-MEK-ERK and PI3K-AKT signal pathways in HaCaT cells. However, specific inhibitors studies showed that the down-regulation of the differentiation markers expression by gardiquimod was not dependent on the activation of these two pathways. TLR7 may play an important role in the pathogenesis of psoriasis through regulating the differentiation of the keratinocytes, and will give a new insight into the psoriasis.
Assuntos
Aminoquinolinas/farmacologia , Antígenos de Diferenciação/genética , Cálcio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Linhagem Celular , Humanos , Queratina-1/genética , Queratina-1/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Psoríase/genética , Psoríase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/metabolismoRESUMO
Toll-like receptors (TLRs) are critical in the induction of the immune response in tumor development. TLR7 has previously been demonstrated to be associated with the development of pancreatic cancer, and the release of cytokines and chemokines from other types of cancer cell; however, the specific expression induced by TLR7 agonists in pancreatic cancer cells remains to be elucidated. The present study aimed to investigate the effects of the TLR7 agonist, gardiquimod, on ERK1/2 signaling pathway, and on the expression of genes involved in the pathogenesis of cancer, including phosphatase and tensin homolog deleted on chromosome 10 (PTEN), p53, type â ¢ interferon (IFN-λ1), vascular endothelial growth factor (VEGF), matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1). The results demonstrated that activation of TLR7 upregulated the expression levels of certain genes to varying degrees; the expression levels of IFN-λ1 and MMP-9 were increased by ~3 fold, whereas other genes (p53, PTEN, TIMP-1) were upregulated by ~2 fold, and VEGF was marginally upregulated after 10 min. Furthermore, gardiquimod increased the expression levels of phosphorylated-extracellular signal-regulated kinase (ERK)1/2. In addition, PD98059, a specific inhibitor of ERK phosphorylation, inhibited the ability of gardiquimod to activate ERK1/2; consequently weakening the effect of gardiquimod on gene regulation. These findings indicated that the effect of TLR7 agonists, including gardiquimod, on gene expression in BxPC-3 pancreatic cancer cells was partly associated with the mitogen-activated protein kinase-ERK1/2 signaling pathway.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucinas/genética , Metaloproteinase 9 da Matriz/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias Pancreáticas/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Receptor 7 Toll-Like/metabolismo , Proteína Supressora de Tumor p53/genética , Fator A de Crescimento do Endotélio Vascular/genética , Aminoquinolinas/farmacologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Humanos , Imidazóis/farmacologia , Interferons , Interleucinas/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Pancreatic cancer is one of the most malignant types of tumor and has a poor prognosis. Tolllike receptor 7 (TLR7) has been found to be present and have different roles in different types of cancer cells. In the present study, the roles of TLR7 in BxPC3 cells, a human pancreatic adenocarcinoma cell line, were investigated. The cells were treated with gardiquimod, an agonist of TLR7, following which the properties of the cells, including proliferation, migration, cell cycle and apoptosis, were analyzed. It was revealed that activation of TLR7 by gardiquimod inhibited cell proliferation and migration, and induced apoptosis of the cells. In addition, gardiquimod downregulated the expression levels of cyclin B1, cyclin E and Bcell lymphoma 2, while upregulating the expression of Bcellassociated X protein. These results suggested that the activation of TLR7 suppresses the progression of pancreatic cancer.