RESUMO
The CEBPA gene is mutated in 10% of acute myeloid leukemia (AML) cases. We find that CEBPA and Bcl-2 RNA levels correlate highly in low-risk human AMLs, suggesting that inhibition of apoptosis via induction of bcl-2 by CCAAT/enhancer binding protein alpha (C/EBPalpha) or its mutant variants contributes to transformation. C/EBPalphap30, lacking a NH2-terminal transactivation domain, or C/EBPalphaLZ, carrying in-frame mutations in the leucine zipper that prevent DNA binding, induced bcl-2 in hematopoietic cell lines, and C/EBPalpha induced bcl-2 in normal murine myeloid progenitors and in the splenocytes of H2K-C/EBPalpha-Emu transgenic mice. C/EBPalpha protected Ba/F3 cells from apoptosis on interleukin-3 withdrawal but not if bcl-2 was knocked down. Remarkably, C/EBPalphaLZ oncoproteins activated the bcl-2 P2 promoter despite lack of DNA binding, and C/EBPalphap30 also activated the promoter. C/EBPalpha and the C/EBPalpha oncoproteins cooperated with nuclear factor-kappaB (NF-kappaB) p50, but not p65, to induce bcl-2 transcription. Endogenous C/EBPalpha preferentially coimmunoprecipitated with p50 versus p65 in myeloid cell extracts. Mutation of residues 297 to 302 in the C/EBPalpha basic region prevented induction of endogenous bcl-2 or the bcl-2 promoter and interaction with p50 but not p65. These findings suggest that C/EBPalpha or its mutant variants tether to a subset of NF-kappaB target genes, including Bcl-2, via p50 to facilitate gene activation and offer an explanation for preferential in-frame rather than out-of-frame mutation of the leucine zipper with sparing of the basic region in C/EBPalphaLZ oncoproteins. Targeting interaction between C/EBPalpha basic region and NF-kappaB p50 may contribute to the therapy of AML and other malignancies expressing C/EBPs.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Leucemia Mieloide Aguda/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Adulto , Animais , Apoptose/fisiologia , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Linhagem Celular , Células HL-60 , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteínas Oncogênicas/genética , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ativação TranscricionalRESUMO
During heart organogenesis, the spatiotemporal organization of the extracellular matrix (ECM) undergoes significant remodeling. Because matrix metalloproteinases (MMPs) are known to be key regulators of cell-matrix interactions, we analyzed the role(s) of MMPs, and specifically MMP-2, in early heart development. Both MMP-2 neutralizing antibody and the broad-spectrum MMP inhibitor Ilomastat in a temporal manner, when applied between chick embryonic stages 5 (primitive streak stage) to stage 12 ( approximately 16-somites), produced severe heart tube defects. Exposure to the MMP inhibitor at stage 5 produced various degrees of cardia bifida. At the seven-somite stage, MMP-2/Ilomastat inhibition caused a shift in normal left-right patterning of cell proliferation within the dorsal mesocardium and mesoderm of the anterior heart field that correlated with a change in looping direction. MMP inhibition at the 10- to 12-somite stage resulted in an arrest of heart tube bending by inhibiting the breakdown of the dorsal mesocardial ECM. The experimental observations suggest that MMP activity regulates the coordination of early heart organogenesis by affecting ventral closure of the heart and gut tubes, asymmetric cell proliferation in the dorsal mesocardium to drive looping direction, and ECM degradation within the dorsal mesocardium allowing looping to proceed toward completion.
