Kathleen M. Dickson

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.

Excitotoxic Death of Retinal Neurons In Vivo Occurs via a Non-Cell-Autonomous Mechanism

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 κB (NF-κB) activation in Müller glia but not in retinal neurons. Intriguingly, NMDA-induced death of retinal neurons is effectively blocked by inhibitors of NF-κB activity. We demonstrate that tumor necrosis factor α (TNFα) protein produced in Müller glial cells via an NMDA-induced NF-κB-dependent pathway plays a crucial role in excitotoxic loss of retinal neurons. This cell loss occurs mainly through a TNFα-dependent increase in Ca2+-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.

Nuclear Factor-κB Induced by Doxorubicin Is Deficient in Phosphorylation and Acetylation and Represses Nuclear Factor-κB–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.

Astrocyte reactivity in neonatal mice : Apparent dependence on the presence of reactive microglia/macrophages

In neonatal mice, an acute injury produced by a stab wound to the cortex results in minimal astrocyte reactivity, as has been observed by others. However, if the source of the stab wound, a piece of nitrocellulose (NC) membrane, were now implanted in the cortex for a period of time (chronic NC implant injury), then extensive astroglial reactivity in the neonatal brain ensues. The astrogliosis is manifested by increased mRNA, protein content, and immunoreactivity for GFAP, and by ultrastructural changes. Given the previous reports that inflammatory cytokines are possible mediators of astrocyte reactivity (e.g., Balasingam et al: J Neurosci 14:846, 1994), we examined the brain parenchyma of neonatal mice following an NC stab or implant injury, with minimal or extensive astrogliosis, respectively, for a possible differential representation of inflammatory cells. A significant correlation (r = 0.87, P < 0.05) was observed between the occurrence of astrogliosis and the presence of reactive microglia/macrophages; no other inflammatory cell type was detected in the brain parenchyma of neonatal mice following NC implant injury. We suggest that reactive microglia/macrophages are required for the evolution of cells into reactive astrocytes following insults to the neonatal brain.

Hypo-Osmolar Stress Induces p75NTR Expression by Activating Sp1-Dependent Transcription

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 hypo-osmolarity, 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.

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 β‐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. Developmental Dynamics 231:122–127, 2004.

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

J. Neurochem. (2011) 116, 396–405.