cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination.

The inhibitor of apoptosis (IAP) family of proteins enhances cell survival through mechanisms that remain uncertain. In this report, we show that cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive ubiquitination of the RIP1 adaptor protein. We demonstrate that AEG40730, a compound modeled on BIR-binding tetrapeptides, binds to cIAP1 and cIAP2, facilitates their autoubiquitination and proteosomal degradation, and causes a dramatic reduction in RIP1 ubiquitination. We show that cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells and demonstrate that constitutively ubiquitinated RIP1 associates with the prosurvival kinase TAK1. When deubiquitinated by AEG40730 treatment, RIP1 binds caspase-8 and induces apoptosis. These findings provide insights into the function of the IAPs and provide new therapeutic opportunities in the treatment of cancer.

Stress-induced expression of the p75 neurotrophin receptor is regulated by O-GlcNAcylation of the Sp1 transcription factor.

Injury-induced expression of p75 neurotrophin receptor (p75NTR) in the CNS induces neuronal apoptosis and prevents neuronal regrowth. The mechanisms regulating injury-induced p75NTR expression are poorly characterized but previous studies have established that reductions in extracellular osmolarity which mimic cytotoxic edema induce p75NTR gene expression through pathways that activate the Sp1 transcription factor. In this report, we examined how extracellular osmolarity converges on Sp1 to regulate p75NTR expression. We report that levels of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), the enzyme that mediates O-linked attachment of GlcNAc, are reduced by extracellular hypo-osmolarity and that global levels of protein O-GlcNAcylation and of Sp1 show a corresponding decline. We demonstrate that chemical and RNAi-based treatments that reduce cellular O-GlcNAcylation facilitate p75NTR induction by hypo-osmolarity, directly linking protein O-GlcNAcylation to p75NTR induction. To determine if Sp1 O-GlcNAc content regulates p75NTR expression, we replaced endogenous Sp1 with a Sp1 mutated at O-GlcNAc target residues. This O-GlcNAc-deficient form of Sp1-enhanced p75NTR expression, demonstrating that O-GlcNAcylation of Sp1 negatively regulates p75NTR expression. We conclude that a stress-induced decline in the O-GlcNAc content of Sp1 drives expression of p75NTR.

AEG3482 is an antiapoptotic compound that inhibits Jun kinase activity and cell death through induced expression of heat shock protein 70.

We describe a group of small-molecule inhibitors of Jun kinase (JNK)-dependent apoptosis. AEG3482, the parental compound, was identified in a screening effort designed to detect compounds that reduce apoptosis of neonatal sympathetic neurons after NGF withdrawal. We show that AEG3482 blocks apoptosis induced by the p75 neurotrophin receptor (p75NTR) or its cytosolic interactor, NRAGE, and demonstrate that AEG3482 blocks proapoptotic JNK activity. We show that AEG3482 induces production of heat shock protein 70 (HSP70), an endogenous inhibitor of JNK, and establish that HSP70 accumulation is required for the AEG3482-induced JNK blockade. We show that AEG3482 binds HSP90 and induces HSF1-dependent HSP70 mRNA expression and find that AEG3482 facilitates HSP70 production while retaining HSP90 chaperone activity. These studies establish that AEG3482 inhibits JNK activation and apoptosis by a mechanism involving induced expression of HSP proteins.

Nuclear factor-kappaB induced by doxorubicin is deficient in phosphorylation and acetylation and represses nuclear factor-kappaB-dependent transcription in cancer cells.

The primary goal of chemotherapy is to cause cancer cell death. However, a side effect of many commonly used chemotherapeutic drugs is the activation of nuclear factor-kappaB (NF-kappaB), a potent inducer of antiapoptotic genes, which may blunt the therapeutic efficacy of these compounds. We have assessed the effect of doxorubicin, an anthracycline in widespread clinical use, on NF-kappaB activation and expression of antiapoptotic genes in breast cancer cells. We show that doxorubicin treatment activates NF-kappaB signaling and produces NF-kappaB complexes that are competent for NF-kappaB binding in vitro. Surprisingly, these NF-kappaB complexes suppress, rather than activate, constitutive- and cytokine-induced NF-kappaB-dependent transcription. We show that doxorubicin treatment produces RelA, which is deficient in phosphorylation and acetylation and which blocks NF-kappaB signaling in a histone deacetylase-independent manner, and we show that NF-kappaB activated by doxorubicin does not remain stably bound to kappaB elements in vivo. Together these data show that NF-kappaB signaling induced by doxorubicin reduces expression of NF-kappaB-dependent genes in cancer cells.

A pro-apoptotic fragment of the p75 neurotrophin receptor is expressed in p75NTRExonIV null mice.

The p75 neurotrophin receptor (p75NTR) regulates neuronal survival, apoptosis, and growth. Recent studies have reported that disruption of Exon IV produces a null mouse lacking all p75NTR gene products (p75NTRExonIV-/-), whereas mice lacking p75NTR Exon III (p75NTRExonIII-/-) maintain expression of an alternatively spliced form of p75NTR (s-p75NTR). Here, we report that p75NTRExonIV-/- mice express a p75NTR gene product that encodes a truncated protein containing the extracellular stalk region together with the entire transmembrane and intracellular domains. The gene product is initiated from a cryptic Kozak consensus/initiator ATG sequence within a region of Exon IV located 3' to the pGK-Neo insertion site. Overexpression of this fragment in heterologous cells results in activation of Jun kinase and induces Pro-caspase-3 cleavage, indicating that it activates p75NTR signaling cascades. These results indicate that aspects of the p75NTRExonIV-/- phenotype may reflect a gain-of-function mutation rather than loss of p75NTR function.

TRAF6-dependent NF-kB transcriptional activity during mouse development.

Nuclear factor-kappa B (NF-kB) transcriptional activity is induced by numerous stimuli. To identify tissues exhibiting NF-kB transcriptional activity during development, we analyzed transgenic reporter mice that express beta-galactosidase from an NF-kB-responsive element. We report that NF-kB activation is widespread and present in numerous epithelial structures and within vasculature. Several regions of the developing central nervous system, including the roof plate and floor plate of the midbrain, show prominent NF-kB activation. To assess the role of the TRAF6 adaptor protein in developmental NF-kB activity, we analyzed NF-kB activation in reporter mice rendered null for TRAF6. Deletion of TRAF6 resulted in the loss of NF-kB activity in epithelia, in vasculature, and in roof and floor plate but had no effect on NF-kB activity developing telencephalon, choroid plexus, cochlear canal, and thymus. These data indicate that NF-kB transcriptional activity is present in a broad range of structures during development and that TRAF6 plays a critical role mediating developmental NF-kB activation in many but not all tissues.