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.

The central hypothesis of excitotoxicity is that excessive stimulation of neuronal NMDA-sensitive glutamate receptors is harmful to neurons and contributes to a variety of neurological disorders. Glial cells have been proposed to participate in excitotoxic neuronal loss, but their precise role is defined poorly. In this in vivo study, we show that NMDA induces profound nuclear factor B (NF-B) activation in Müller glia but not in retinal neurons. Intriguingly, NMDA-induced death of retinal neurons is effectively blocked by inhibitors of NF-B activity. We demonstrate that tumor necrosis factor ␣ (TNF␣) protein produced in Müller glial cells via an NMDA-induced NF-B-dependent pathway plays a crucial role in excitotoxic loss of retinal neurons. This cell loss occurs mainly through a TNF␣-dependent increase in Ca 2ϩ -permeable AMPA receptors on susceptible neurons. Thus, our data reveal a novel non-cell-autonomous mechanism by which glial cells can profoundly exacerbate neuronal death following excitotoxic injury.

The function of PMP-22 is unknown, but roles in cell growth, apoptosis, higher order macromolecular structure, and intracellular signaling have been proposed (1). Intrachromosomal duplications (2) and deletions (3) encompassing PMP-22 cause Charcot-MarieTooth disease and hereditary neuropathy with liability to pressure palsies, respectively. In addition, point mutations have been found in patients with Charcot-Marie-Tooth disease, hereditary neuropathy with liability to pressure palsies, and DejerineSottas syndrome (4). Indeed, the first missense mutations to be identified in the PMP-22 gene were found in the Tr (5) and Tr-J (6) mice with identical point mutations shared between humans and mice (7, 8).Many PMP-22 mutations are retained intracellularly (9-12) and appear to belong to a growing class of mutations termed ''endoplasmic reticulum (ER) retention mutations'' that are recognized by ER resident-folding proteins, molecular chaperones, and͞or other enzymes that serve to monitor the fidelity of protein synthesis and macromolecular assembly (13,14). Among ER retention diseases, however, heritable neuropathies caused by PMP-22 mutation or overexpression are unique because they are dominant gain-of-function diseases (15).Here, transient and glucosylation-dependent association of PMP-22 with the ER chaperone calnexin (CNX) was observed. PMP-22 associated only with CNX. Formation of intracellular myelin-like figures (IMLFs) in transfected cells coincided with the cosequestration of CNX in a glucosylation dependent fashion. Similar intracellular myelin-like structures were present in the sciatic nerves of homozygous Tr-J mice. These results provide a mechanistic explanation for the human Charcot-Marie-Tooth disease secondary to the ER retention of mutant PMP-22 via the CNX cycle and provide an unexpected link between the gain-offunction phenotype of such diseases and sequestration of the resident ER lectin chaperone, CNX. Materials and MethodsMetabolic Labeling, Immunoprecipitations, and Western Blot Analysis.Metabolic labeling and pulse-chase of mouse sciatic nerves and immunoprecipitations for PMP-22 (16) as well as nondenaturing CNX immunoprecipitations, BiP immunoprecipitation, and ATP depletion of cell lysates have been described (17, 18). For sequential PMP-22 immunoprecipitations, lysates were first CNX immunoprecipitated as described (17) followed by resolubilization in 0.5% SDS in 50 mM Tris (pH 8.0) and PMP-22 immunoprecipitated in modified radioimmunoprecipitation assay buffer (50 mM Tris, pH 8.0͞150 mM NaCl͞1% deoxycholate containing 0.5% Nonidet P-40). Samples were electrophoresed, transferred to nitrocellulose membranes, and exposed to film by using the Kodak Biomax Transcreen LE system (NEN). For some experiments, sciatic nerves were pretreated with 1 mM castanospermine (SigmaAldrich) for 1 h at 37°C before and during metabolic labeling. Samples were then homogenized in 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate lysis buffer containing 5 mM iodoacetamide and gels processed for fluo...

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-KB (NF-KB), 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-KB activation and expression of antiapoptotic genes in breast cancer cells. We show that doxorubicin treatment activates NF-KB signaling and produces NF-KB complexes that are competent for NF-KB binding in vitro. Surprisingly, these NF-KB complexes suppress, rather than activate, constitutiveand cytokine-induced NF-KB-dependent transcription. We show that doxorubicin treatment produces RelA, which is deficient in phosphorylation and acetylation and which blocks NF-KB signaling in a histone deacetylase-independent manner, and we show that NF-KB activated by doxorubicin does not remain stably bound to KB elements in vivo. Together these data show that NF-KB signaling induced by doxorubicin reduces expression of NF-KB-dependent genes in cancer cells.

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ExonIIIϪ/Ϫ) maintain expression of an alternatively spliced form of p75NTR (s-p75NTR). Here, we report that p75NTR ExonIVϪ/Ϫ 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 3Ј 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 p75NTR ExonIVϪ/Ϫ phenotype may reflect a gain-of-function mutation rather than loss of p75NTR function.

Injury-induced expression of the p75 neurotrophin receptor (p75NTR) in the CNS facilitates neuronal apoptosis and prevents neuronal regrowth, but the mechanisms regulating p75NTR expression are poorly characterized. In this study, we showed that hypo-osmolarity induces p75NTR expression in primary neurons, and, using a comparative genomics approach, we identified conserved elements in the 25 kb upstream sequences of the rat, mouse, and human p75NTR genes. We found that only one of these, a proximal region rich in Sp1 sites, responds to changes in hypo-osmolarity. We then showed that Sp1 DNA binding activity is increased in cells exposed to hypoosmolarity, established that hypo-osmolarity enhanced Sp1 binding to the endogenous p75NTR promoter, and showed that Sp1 is required for p75NTR expression induced by hypo-osmolarity. We examined how Sp1 is regulated to effect these changes and established that Sp1 turnover is strongly inhibited by hypo-osmolarity. We propose that stress-induced Sp1 accumulation that results from reductions in Sp1 turnover rate contributes to injury-induced gene expression.

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.