RESUMO
The composition of IL-23R complex is similar to that of the IL-12 receptor (IL-12R) complex with a shared IL-12R-ß1 chain. The IL-12R-ß1 heterodimerizes with IL-23R and IL-12R-ß2 to form IL-23R and IL-12R complexes, respectively. The IL-12R-ß2 has been shown to function as a tumor suppressor gene and apoptotic inducer. However, whether IL-23R also functions in cell apoptosis is currently unknown. In this study, we demonstrate for the first time that overexpression of IL-23R markedly induces cell apoptosis in both 293ET and HeLa cells. The activations of caspase 3 and caspase 9 are induced by IL-23R. Mechanistic study reveals that IL-23R markedly inhibits RAS/MAPK and STAT3 but not STAT1 and PI-3K/Akt signaling pathways in both 293ET and HeLa cells. Overexpression of IL-23R significantly up-regulates IL-12Rß1 expression but not IL-23α and IL-12ß expressions in both cell lines. Therefore, our data strongly indicates that IL-23R is able to induce cell apoptosis by activating the intrinsic mitochondrial pathways associated with the inhibition in RAS/MAPK and STAT3 activations in mammalian cells.
Assuntos
Apoptose , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores de Interleucina/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas ras/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Regulação para Baixo , Células HEK293 , Células HeLa , Humanos , Subunidade beta 1 de Receptor de Interleucina-12/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Interleucina/genética , Transdução de SinaisRESUMO
Neurofibromatosis type 1 (NF1) microdeletion is a large genomic deletion that embraces at least 11 continuous genes at human chromosome 17q11.2. To date, most of these genes' functions still remain undefined. In this study, we report an unknown cytokine receptor like molecule (p48.2) that is frequently deleted in patients with type-1 and type-2 NF1 microdeletions in the neurofibromin locus. The cloned gene has 1317 base pair long that encodes a 438aa intracellular protein. The gene was subsequently named p48.2 based on its predicted molecular weight. A typical fibronectin type III (FNIII) domain was identified in p48.2 between Arg(176) and Pro(261) in which a palindromic Arg-Gly-Asp (RGD) repeat plus a putative Trp-Ser-X-Trp-Ser (WSXWS) motif were found at the domain's C-terminus. p48.2 mRNAs were abundant in many tumor cell lines and normal human tissues and up-regulated in some freshly isolated lung cancer and leukemia cells. Interestingly, over-expression of p48.2 in human embryo kidney 293T cells could significantly cause G0/G1 arrest and prevented S phase entry. In contrast, repressing endogenous p48.2 gene expression by specific siRNA markedly reduced G0/G1 population. Importantly, over-expression of p48.2 could significantly up-regulate rather than down-regulate cyclin D1 and cyclin D3 expressions. We further showed that the induction of cyclin D1 expression was directly due to the activation of signal transducers and activators of transcription 3 (STAT3), but was independent of RAS/mitogen-activated protein kinase (RAS/MAPK) signaling pathway. Thus, p48.2 may represent a novel type of intracellular protein functioning as a negative regulator at the G0/G1 phase.
Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular , Espaço Intracelular/metabolismo , Receptores de Citocinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Clonagem Molecular , Biologia Computacional , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina D3/genética , Ciclina D3/metabolismo , Regulação para Baixo/genética , Fase G1 , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma Humano/genética , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno , Receptores de Citocinas/química , Receptores de Citocinas/metabolismo , Fase de Repouso do Ciclo Celular , Fator de Transcrição STAT3/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
AIM: Cytokine receptor-like factor 3 (CRLF3) is a novel gene cloned from K562 cell line. The aim of the current study is to observe CRLF3 expression and subcellular localization in 293T cells and to express and purify the CRLF3 fusion protein in E.coli. METHODS: The plasmid pCMV-myc-CRLF3 was transiently transfected into 293T cell. The expression and localization of CRLF3 protein was observed by immunostaining approach. CRLF3 was also cloned into pGEX-4T-1 vector. The GST-CRLF3 fusion protein was expressed in E.coli and purified by GST affinity chromatography. RESULTS: CRLF3 protein was highly expressed in transfected 293T cells. CRLF3 protein was distributed in both cytoplasm and cell membrane. The GST-CRLF3 fusion protein was expressed as a M(r) 74 000 protein and then successfully purified from E.coli cells. CONCLUSION: Overexpressed CRLF3 is a cytoplasmic protein that distributes in both cytoplasm and cell membrane in mammalian cells. The recombinant GST-CRLF3 fusion protein had been isolated and could be used for further antibody production and functional characterization.