RESUMO
Little is known about the cellular mechanisms modulating the shift in balance from a state of survival to cell death by caspase-mediated apoptosis in response to a lethal stress. Here we show that the RNA-binding protein HuR has an important function in mediating this switch. During caspase-mediated apoptosis, HuR is cleaved to generate two cleavage products (CPs). Our data demonstrate that the cleavage of HuR switches its function from being a prosurvival factor under normal conditions to becoming a promoter of apoptosis in response to a lethal stress. In the absence of an apoptotic stimuli, HuR associates with and promotes the expression of caspase-9 and prothymosin α (ProT) mRNAs, and pro- and antiapoptotic factors, respectively, both of which have been characterized as important players in determining cell fate. During the early steps of caspase-mediated apoptosis, however, the level of caspase-9 protein increases, while ProT remains unchanged. Under these conditions, the two HuR-CPs selectively bind to and stabilize caspase-9 mRNA, but do not bind to ProT. Hence, taken together, our data show that by maintaining a threshold of expression of proapoptotic factors such as caspase-9 in response to a lethal stress, the HuR-CPs help a cell to switch from resisting death to undergoing apoptosis.
Assuntos
Apoptose/fisiologia , Caspase 9/metabolismo , Caspases/metabolismo , Proteínas ELAV/fisiologia , Caspase 9/biossíntese , Caspase 9/genética , Sobrevivência Celular/fisiologia , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Células HeLa , Humanos , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Timosina/biossíntese , Timosina/genética , Timosina/metabolismo , TransfecçãoRESUMO
Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein involved in the regulation of messenger RNA stability and internal initiation of translation. We have used Unr-deficient murine embryonic stem (ES) cells to analyse Unr role in cell proliferation and response to stress. Disruption of both unr gene copies had no effect on ES cell proliferation. However, after ionizing radiation (IR), clonogenic survival of unr(-/-) ES cells was approximately 3-fold enhanced as compared to unr(+/+) cells. We further determined that IR-induced apoptosis was decreased in unr(-/-) ES cells, and that reintroduction of the unr gene in unr(-/-) cells restored normal IR-induced apoptosis. Three pro-apoptotic genes, p53, caspase-3 and Gadd45gamma, were downregulated in unr(-/-) ES cells, indicating that Unr, as other cytoplasmic RNA-binding proteins, regulates a complex genetic program, promoting cell death after IR. In contrast, in the human hepatoma cell line HuH7, Unr knockdown using unr-specific small interfering RNAs induced apoptosis, both in untreated and gamma-irradiated cells. Thus, our results establish that Unr acts as a positive or negative regulator of cell death, depending on the cell type. Manipulating the level of Unr may constitute a specific approach to sensitize cancer cells to anticancer treatments.