Abstract
Bim is a pro-apoptotic member of the Bcl-2 family that is induced and contributes to neuron death in response to nerve growth factor (NGF) deprivation. Past work has revealed that Bim is downstream of multiple independent transcriptional pathways in neurons, including those culminating in activation of the c-Jun, FoxO, and Myb transcription factors. This study addresses the issue of whether the three signaling pathways are redundant with respect to Bim induction or whether they act cooperatively. Examination of the proximal Bim promoter reveals binding sites for FoxO, Mybs, and, as shown here, c-Jun. We find that mutation of any one of these types of sites abolishes induction of a Bim promoter-driven reporter in response to NGF deprivation. Moreover, down-regulation of either c-Jun, FoxOs, or Mybs by short hairpin RNAs blocks induction of Bim promoter-reporter activity triggered by withdrawal of NGF. This was the case for reporters driven by either the proximal promoter or a promoter that also includes additional regulatory elements in the first intron of the Bim gene. Such short hairpin RNAs also suppressed the induction of endogenous Bim protein. These findings thus indicate that the Bim promoter acts as a coincidence detector that optimally responds to the simultaneous activation of three different pro-apoptotic transcriptional pathways. Such a mechanism provides a "fail-safe" that prevents neurons from dying by accidental activation of any single pathway. It also permits neurons to utilize individual pathways such as JNK signaling for other purposes without risk of demise.
Highlights
These are the “JNK-c-Jun” pathway that includes activation of the c-Jun N-terminal kinase and phosphorylation/activation of the transcription factor c-Jun [3]; the “cell cycle” pathway that includes events associated with the cell cycle in mitotic cells such as activation of cyclin-dependent kinase 4 (Cdk4), phosphorylation of retinoblastoma family members, and induction of the transcription factors B- and C-myb [4]; and the “FoxO” pathway in which FoxO family transcription factors move from cytoplasm to nucleus as a consequence of reduced phosphorylation resulting from shut down of the kinase AKT [5]
We recently reported that Bim is a direct target of a cell cycle pathway in which nerve growth factor (NGF) deprivation leads to activation of Cdk4, phosphorylation of the retinoblastoma family member p130 and consequent loss of p130-E2F4 complexes, de-repression of the transcription factors B- and C-Myb, and induction of Bim [4, 11, 12]
ShRNAs Targeted to c-Jun or FoxOs Block Induction of the repressed activation of the Proximal Bim Promoter to NGF Deprivation—As an additional extended Bim promoter-reporter in response to NGF deprivameans to further examine the roles of the multiple transcrip- tion (Fig. 4B)
Summary
We identified two Myb binding sites in the promoter region of the Bim gene that are required for Bim induction in response to activation of the cell cycle pathway and NGF deprivation [11]. In the findings presented here, we have addressed these issues and find that maximal induction of Bim in response to NGF deprivation requires simultaneous occupancy of the Bim promoter by Mybs, c-Jun, and FoxOs. Such findings indicate that the Bim promoter functions as a coincidence detector that is maximally driven only when all three death pathways are activated.
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