The proapoptotic dp5 gene is a direct target of the MLK-JNK-c-Jun pathway in sympathetic neurons

Abstract

The death of sympathetic neurons after nerve growth factor (NGF) withdrawal requires de novo gene expression. Dp5 was one of the first NGF withdrawal-induced genes to be identified and it encodes a proapoptotic BH3-only member of the Bcl-2 family. To study how dp5 transcription is regulated by NGF withdrawal we cloned the regulatory regions of the rat dp5 gene and constructed a series of dp5-luciferase reporter plasmids. In microinjection experiments with sympathetic neurons we found that three regions of dp5 contribute to its induction after NGF withdrawal: the promoter, a conserved region in the single intron, and sequences in the 3′ untranslated region of the dp5 mRNA. A construct containing all three regions is efficiently activated by NGF withdrawal and, like the endogenous dp5, its induction requires mixed-lineage kinase (MLK) and c-Jun N-terminal kinase (JNK) activity. JNKs phosphorylate the AP-1 transcription factor c-Jun, and thereby increase its activity. We identified a conserved ATF site in the dp5 promoter that binds c-Jun and ATF2, which is critical for dp5 promoter induction after NGF withdrawal. These results suggest that part of the mechanism by which the MLK-JNK-c-Jun pathway promotes neuronal apoptosis is by activating the transcription of the dp5 gene.

Egr-1 TransactivatesBimGene Expression to Promote Neuronal Apoptosis

“…Inhibiting the JNK pathway may suppress the proapoptotic activity of Bim by reducing the JNK-mediated phosphorylation of Bim at Ser65 or Thr112 (Putcha et al, 2003;Becker et al, 2004;Hübner et al, 2008), or it may block the c-Jun-dependent upregulation of Dp5, another proapoptotic BH3-only protein (Ma et al, 2007;Towers et al, 2009). IGF-1 may increase the Akt-mediated phosphorylation of Bim at Ser87 to reduce its proapoptotic potency (Qi et al, 2006), or it may inactivate caspase 9 downstream of Bim (Cardone et al, 1998).…”

Bag1-L Is a Phosphorylation-Dependent Coactivator of c-Jun during Neuronal Apoptosis

“…CGNs depend on potassium depolarization for survival and undergo apoptosis when deprived of potassium. Dp5 is upregulated in a JNK/c-Jun-dependent manner during apoptosis induced by potassium deprivation, and knockdown of Dp5 by small interfering RNA rescued neurons from apoptosis (32,46). Since Bag1-L cooperated with c-Jun in dp5 regulation (Fig.…”

“…Similarly, trophic factor deprivation-induced death was significantly delayed in junAA homozygous primary sympathetic neurons (6). Expression of a c-Jun dominant-negative mutant or the junAA knock-in mutation diminished the induction of bim and dp5 RNA and protein levels after NGF withdrawal (6,32,46,52). In addition, the mixed-lineage kinase inhibitor CEP-1347, which prevents JNK activation, also reduces the increase in bim and dp5 mRNA levels after NGF deprivation (32, 46).…”

“…PC12 cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% horse serum, 5% fetal calf serum (FCS), 2 mM glutamine, and penicillin-streptomycin. Cells were grown on collagencoated tissue culture dishes, and differentiation was induced by NGF (Promega) as previously described (46). Cells were transfected with Lipofectamine 2000 (Invitrogen).…”

“…5A). The proapoptotic dp5 gene is a direct target of the JNK-c-Jun pathway and contributes to c-Jun-mediated apoptosis (32,46). Bag1-L bound to the CRE element in the dp5 promoter, and binding of Bag1-L to both the c-jun and dp5 promoters was strongly increased when JNK activation was triggered by UV (Fig.…”