RESUMO
A recent report claimed that endoplasmic reticulum (ER) stress activates the ER trans-membrane receptor IRE1α, leading to increased caspase-2 levels via degradation of microRNAs, and consequently induction of apoptosis. This observation casts caspase-2 into a central role in the apoptosis triggered by ER stress. We have used multiple cell types from caspase-2-deficient mice to test this hypothesis but failed to find significant impact of loss of caspase-2 on ER-stress-induced apoptosis. Moreover, we did not observe increased expression of caspase-2 protein in response to ER stress. Our data strongly argue against a critical role for caspase-2 in ER-stress-induced apoptosis.
Assuntos
Caspase 2/metabolismo , Cisteína Endopeptidases/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Animais , Caspase 2/genética , Cisteína Endopeptidases/genética , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Timócitos/enzimologia , Timócitos/metabolismo , Regulação para Cima , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Hoxb8 overexpression immortalises haematopoietic progenitor cells in a growth-factor-dependant manner and co-operates with interleukin-3 (IL-3) to cause acute myeloid leukaemia. To further understand how Hoxb8 contributes to myeloid cell immortalisation, we generated IL-3-dependant myeloid cells expressing Hoxb8 under the control of an inducible promoter. Downregulation of Hoxb8, in the presence of IL-3, caused cell-cycle arrest and apoptosis in the majority of cells. Apoptosis was dependant on Bax and Bak and, in part, on Bim, which was repressed by Hoxb8. Deletion of the miR-17â¼92 seed sequences in the Bim 3'UTR abolished Hoxb8-dependant regulation of Bim reporter constructs. Expression of all six miRNAs from this cluster were elevated when Hoxb8 was overexpressed. The miR-17â¼92 cluster was required for repression of Bim in Hoxb8-immortalised cells and deletion of the miR-17â¼92 cluster substantially inhibited Hoxb8, but not Hoxa9, mediated survival and proliferation. Hoxb8 appears to promote miR-17â¼92 expression through c-Myc, a known transcriptional regulator of the miR-17â¼92 cluster. We have uncovered a previously unrecognised link between Hoxb8 expression and microRNAs that provides a new insight into the oncogenic functions of Hoxb8.
Assuntos
Proteínas de Homeodomínio/genética , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Morte Celular/genética , Diferenciação Celular/genética , Processos de Crescimento Celular/genética , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transfecção , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The activation of the Akt signalling in response to cytokine receptor signalling promotes protein synthesis, cellular growth and proliferation. To determine the role of Akt in interleukin-3 (IL-3) signalling, we generated IL-3-dependent myeloid cell lines from mice lacking Akt1, Akt2 or Akt3. Akt1 deletion resulted in accelerated apoptosis at low concentrations of IL-3. Expression of constitutively active Akt1 was sufficient to delay apoptosis in response to IL-3 withdrawal, but not sufficient to induce proliferation in the absence of IL-3. Akt1 prolonged survival of Bim- or Bad-deficient cells, but not cells lacking Puma, indicating that Akt1-dependent repression of apoptosis was in part dependent on Puma and independent of Bim or Bad. Our data show that a key role of Akt1 during IL-3 signalling is to repress p53-dependent apoptosis pathways, including transcriptional upregulation of Puma. Moreover, our data indicate that regulation of BH3-only proteins by Akt is dispensable for Akt-dependent cell survival.
Assuntos
Apoptose/fisiologia , Citocinas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Células HEK293 , Humanos , Interleucina-3/metabolismo , Isoenzimas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/citologia , Células Mieloides/enzimologia , Receptores de Interleucina-3/metabolismo , Transdução de SinaisRESUMO
P53-upregulated modifier of apoptosis (PUMA), a pro-apoptotic member of the Bcl-2 family, is transcriptionally activated by p53 and is a key effector of p53-dependent apoptosis. We show that PUMA protein is subject to rapid post-translational regulation by phosphorylation at a conserved residue, serine 10, following serum or interleukin-3 (IL-3) stimulation. Serine 10 is not within the Bcl-2 homology (BH3) domain, and PUMA phosphorylated at serine 10 retained the ability to co-immunoprecipitate with antiapoptotic Bcl-2 family members. However, phosphorylated PUMA was targeted for proteasomal degradation indicating that it is less stable than unphosphorylated PUMA. Importantly, we identified IKK1/IKK2/Nemo as the kinase complex that interacts with and phosphorylates PUMA, thereby also demonstrating that IL-3 activates NFκB signaling. The identification and characterization of this novel survival pathway has important implications for IL-3 signaling and hematopoietic cell development.