RESUMO
Human antigen R (HuR) is a RNA-binding protein, which binds to the AU-rich element (ARE) in the 3'-untranslated region (3'-UTR) of certain mRNA and is involved in the export and stabilization of ARE-mRNA. HuR constitutively relocates to the cytoplasm in many cancer cells, however the mechanism of intracellular HuR trafficking is poorly understood. To address this question, we examined the functional role of the cytoskeleton in HuR relocalization. We tested the effect of actin depolymerizing macrolide latrunculin A or myosin II ATPase activity inhibitor blebbistatin for HuR relocalization induced by the vasoactive hormone Angiotensin II in cancer and control normal cells. Western blot and confocal imaging data revealed that both inhibitors attenuated the cytoplasmic HuR in normal cells but no such alteration was observed in cancer cells. Concomitant with changes in intracellular HuR localization, both inhibitors markedly decreased the accumulation and half-lives of HuR target ARE-mRNAs in normal cells, whereas no change was observed in cancer cells. Furthermore, co-immunoprecipitation experiments with HuR proteins revealed clear physical interaction with ß-actin only in normal cells. The current study is the first to verify that cancer cells can implicate a microfilament independent HuR transport. We hypothesized that when cytoskeleton structure is impaired, cancer cells can acquire an alternative HuR trafficking strategy.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Neoplasias/metabolismo , Regiões 3' não Traduzidas , Actinas/efeitos dos fármacos , Actinas/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Células HeLa , Células Hep G2 , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Miosinas/antagonistas & inibidores , Neoplasias/genética , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tiazolidinas/farmacologiaRESUMO
HuR is an RNA-binding protein of the embryonic lethal abnormal vision (ELAV) family, which binds to the AU-rich element (ARE) in the 3'-untranslated region (UTR) of certain mRNAs and is involved in the nucleo-cytoplasmic export and stabilization of ARE-mRNAs. The cytoplasmic relocalization of ARE-mRNAs with several proteins such as HuR and pp32 increases in cells transformed by the adenovirus oncogene product E4orf6. Additionally, these ARE-mRNAs were stabilized and acquired the potential to transform cells. Although, the relocalization of HuR and the stabilization of ARE-mRNAs are crucial for cell transformation, evidence regarding the relationship of HuR and ARE-mRNAs with adenovirus replication is lacking. In this report, we demonstrate that adenovirus infection induces the relocation of HuR to the cytoplasm of host cells. Analysis using the luciferase-ARE fusion gene and the tetracycline (tet)-off system revealed that the process of stabilizing ARE-mRNAs is activated in adenovirus-infected cells. Heat shock treatment or knockdown-mediated depletion of HuR reduced adenovirus production. Furthermore, expression of ARE-including viral IVa2 mRNA, decreased in HuR-depleted infected cells. These results indicate that HuR plays an important role in adenovirus replication, at least in part, by up-regulating IVa2 mRNA expression and that ARE-mRNA stabilization is required for both transformation and virus replication.
Assuntos
Infecções por Adenovirus Humanos/metabolismo , Infecções por Adenovirus Humanos/virologia , Proteína Semelhante a ELAV 1/metabolismo , Regiões 3' não Traduzidas , Elementos Ricos em Adenilato e Uridilato , Infecções por Adenovirus Humanos/genética , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Transformação Celular Viral/genética , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Proteína Semelhante a ELAV 1/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Transporte Proteico , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Virais/genética , Replicação Viral/genéticaRESUMO
The adhesion of circulating cancer cells to vascular endothelial cells is an initial and critical step in distant metastases. Amphoterin-induced gene and open reading frame 2 (AMIGO2) was found to regulate tumor cell adhesion to hepatic endothelial cells and act as a driver gene for liver metastasis in mouse cell lines. However, whether the role of AMIGO2 observed in mouse tumor cells can be extrapolated to human cancer cells in vivo has not been verified. In this study, AMIGO2 expression in various human gastric and colorectal cancer cells was found to be closely associated with their adhesion to human hepatic sinusoidal endothelial cells (HHSECs). Constitutive AMIGO2-knockdown clones of human gastric (MKN-45) and colorectal cancer cell lines (DLD-1) were established to examine whether AMIGO2 expression in cancer cells is involved in the adhesion to HHSECs in vitro and the formation of liver metastasis in vivo. All AMIGO2-knockdown cells showed significantly attenuated adhesion to HHSECs. In vivo analysis revealed that intrasplenic inoculation of AMIGO2-knockdown clones could engraft in the spleen but significantly suppressed liver metastasis in nude mice. This study demonstrated that the role of AMIGO2 as a driver gene of liver metastasis in mouse tumor cells can be extrapolated to human cancer cells.