We found that two of the most up regulated genes after NGF withdrawal, trib3 and ddit3, are associated with the ER unfolded protein response and CEP 11004 prevented their increase in expression suggesting that they are potential MLK JNK c Jun targets. Furthermore, functional analysis revealed Inhibitors,Modulators,Libraries that the ER unfolded protein response annota tion was the most overrepresented gene category after NGF withdrawal suggesting that an ER stress response occurs in sympathetic neurons under these conditions. The exact role of these genes in ER stress induced apoptosis remains unclear, however, it has been shown that CHOP10, a known AP 1 target gene, is induced by both ER stress and oxidative stress. A propapopto tic role for CHOP10 has been reported since its overex pression can lead to apoptosis, whilst MEFs derived from CHOP10 mice are resistant to ER stress induced cell death.

However, the mechanism by which CHOP induces apoptosis still remains unclear. It has been shown that CHOP induced cell death is associated with the translocation of Bax from the cytosol to the mitochondria and that CHOP induced cell death can be prevented by the overexpression Inhibitors,Modulators,Libraries of Bcl 2 or the knock down of Bax. The link between CHOP and Bax translocation could involve a novel ER stress inducible gene, trib3. It has been shown that trib3 is induced via the ATF4 CHOP pathway through the identification of a CHOP binding site in the proximal portion of the pro moter. Also, ER stress can activate bim through CHOP C EBPa dependent transcriptional activation and in other studies CHOP has been found to bind to the promoter of the proapoptotic Bcl 2 family mem ber puma.

The relationship between ER stress, CHOP, Trib3 and BH3 only proteins may suggest an important role in the apoptotic pathway after NGF with Cilengitide drawal. Inhibitors,Modulators,Libraries Furthermore, we also identified a conserved ATF site 14 bp upstream of Exon 1 in the rat trib3 gene which is identical to the reverse complement of the ATF site in the dp5 promoter. This potential c Jun ATF2 binding site in the promoter of the rat trib3 gene suggests that this gene might also be a direct target of the MLK JNK c Jun pathway. We also found evidence of an increase in Trib3 and Ddit3 CHOP10 protein levels after NGF withdrawal and this increase was prevented by CEP 11004. NGF withdrawal leads to a decrease in PI3K and Akt activity resulting in FOXO activation.

FOXO3a translo cates into the nucleus and has been shown to trigger apoptosis Inhibitors,Modulators,Libraries by activating the transcription of genes necessary for cell death, such as bim. The mxi1 gene is also a target of FOXO3a, which binds to sites in the first intron downstream of the Mxi1 SRa promoter. Mxi1 dimerises with Max and binds to E boxes and represses c Myc and MycN target genes by recruiting co repressors to their promoters. Interestingly, the mxi1 mRNA increases in level after NGF withdrawal whereas c myc and mycn mRNA levels decrease.