Blimp-1, a transcriptional repressor, drives the terminal differentiation of B cells to plasma cells. Using DNA microarrays, we found that introduction of Blimp-1 into B cells blocked expression of a remarkably large set of genes, while a much smaller number was induced. Blimp-1 initiated this cascade of gene expression changes by directly repressing genes encoding several transcription factors, including Spi-B and Id3, that regulate signaling by the B cell receptor. Blimp-1 also inhibited immunoglobulin class switching by blocking expression of AID, Ku70, Ku86, DNA-PKcs, and STAT6. These findings suggest that Blimp-1 promotes plasmacytic differentiation by extinguishing gene expression important for B cell receptor signaling, germinal center B cell function, and proliferation while allowing expression of important plasma cell genes such as XBP-1.

Iron chelators are pluripotent neuronal antiapoptotic agents that have been shown to enhance metabolic recovery in cerebral ischemia models. The precise mechanism(s) by which these agents exert their effects remains unclear. Recent studies have demonstrated that iron chelators activate a hypoxia signal transduction pathway in non-neuronal cells that culminates in the stabilization of the transcriptional activator hypoxia-inducible factor-1 (HIF-1) and increased expression of gene products that mediate hypoxic adaptation. We examined the hypothesis that iron chelators prevent oxidative stress-induced death in cortical neuronal cultures by inducing expression of HIF-1 and its target genes. We report that the structurally distinct iron chelators deferoxamine mesylate and mimosine prevent apoptosis induced by glutathione depletion and oxidative stress in embryonic cortical neuronal cultures. The protective effects of iron chelators are correlated with their ability to enhance DNA binding of HIF-1 and activating transcription factor 1(ATF-1)/cAMP response element-binding protein (CREB) to the hypoxia response element in cortical cultures and the H19-7 hippocampal neuronal cell line. We show that mRNA, protein, and/or activity levels for genes whose expression is known to be regulated by HIF-1, including glycolytic enzymes, p21(waf1/cip1), and erythropoietin, are increased in cortical neuronal cultures in response to iron chelator treatment. Finally, we demonstrate that cobalt chloride, which also activates HIF-1 and ATF-1/CREB in cortical cultures, also prevents oxidative stress-induced death in these cells. Altogether, these results suggest that iron chelators exert their neuroprotective effects, in part, by activating a signal transduction pathway leading to increased expression of genes known to compensate for hypoxic or oxidative stress.

Class II transactivator (CIITA), a coactivator required for class II major histocompatibility complex (MHC) transcription, is expressed in B cells but extinguished in plasma cells. This report identifies B lymphocyte-induced maturation protein I (BLIMP-I), a transcriptional repressor that is capable of triggering plasma cell differentiation, as a developmentally regulated repressor of CIITA transcription. BLIMP-I represses the B cell-specific promoter of the human gene that encodes CIITA (MHC2TA) in a binding site-dependent manner. Decreased CIITA correlates with increased BLIMP-I during plasma cell differentiation in cultured cells. Ectopic expression of BLIMP-I represses endogenous mRNA for CIITA and the CIITA targets, class II MHC, invariant chain and H2-DM (the murine equivalent of HLA-DM) in primary splenic B cells as well as 18-81 pre-B cells. Thus, the BLIMP-I program of B cell differentiation includes loss of antigen presentation via extinction of CIITA expression.

Plasma cell differentiation is orchestrated by the transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1), which silences the gene expression program of mature B cells. The molecular mechanism underlying Blimp-1 suppression of mature B-cell gene expression is not fully understood. Here we report that a proline-rich domain in Blimp-1 directly interacts with LSD1, a histone lysine demethylase. Both LSD1 knockdown and expression of Blimp-1 lacking the proline-rich domain derepressed the activities of Blimp-1-dependent luciferase reporters. Disruption of the Blimp-1 interaction with LSD1 or reduced LSD1 expression attenuated antibody production, demonstrating the biological significance of this interaction. Finally, using chromatin immunoprecipitation, we showed that Blimp-1 binding to its target sites is accompanied by LSD1 binding to those same sites and that LSD1 binding correlates with histone modifications of accessible chromatin. These findings provide further insights into the molecular mechanism of the silencing of mature B-cell genes by Blimp-1 in plasma cell differentiation.Nucleosomes contain the four canonical histones, H2A, H2B, H3, and H4 (26). The covalent posttranslational modification of histones, including acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, and ADP ribosylation, generates the epigenetic information of chromatin called the histone code, which is crucial for regulating DNA transcription (5,7,10,15,43). By recruiting enzymatic regulatory complexes or altering chromatin architecture, the combined effects of several posttranslational modifications of histones on specific amino acid residues determine the outcome of transcription, either activating or suppressing gene expression (5,7,10,15). For example, methylation of histone H3 on lysine residue 9 (K9) or K27 and methylation of histone H4 on K20 or K59 are associated with gene silencing, whereas methylation of histone H3 on K4 (H3K4) or H3K36 is linked to active gene transcription (20,28,44). Methylation of arginine residues of H3 or H4 is also linked to active transcription (50).The processes of addition and removal of acetyl groups to histones by histone acetyltransferases and histone deacetylases (HDACs), respectively, and histone methylation are both dynamic and reversible (15). The histone methyltransferases, which often contain a common SET domain (17), add one to three methyl groups to lysine residues of histones; histone demethylases remove methyl groups (18). Lysine-specific demethylase 1 (LSD1), a nuclear amine oxidase homolog, specifically demethylates mono-or dimethyl groups on H3K4 (41, 42). It is associated with gene repression through an interaction with corepressor for RE1 silencing transcription factor/neural restrictive silencing factor (CoREST) and HDAC1/2 in a multiprotein complex (42). LSD1 also interacts with androgen receptors and acts as a coactivator for transcriptional activation by removal of a dimethyl group from H3K9 (31).B-lymphocyte-induced maturation protein-1 (...

Recent studies have established cell type– specific, proapoptotic, or antiapoptotic functions for the transcription factor NF-κB. In each of these studies, inhibitors of NF-κB activity have been present before the apoptotic stimulus, and so the role of stimulus- induced NF-κB activation in enhancing or inhibiting survival could not be directly assessed. Sindbis virus, an alphavirus, induces NF-κB activation and apoptosis in cultured cell lines. To address whether Sindbis virus– induced NF-κB activation is required for apoptosis, we used a chimeric Sindbis virus that expresses a superrepressor of NF-κB activity. Complete suppression of virus-induced NF-κB activity neither prevents nor potentiates Sindbis virus–induced apoptosis. In contrast, inhibition of NF-κB activity before infection inhibits Sindbis virus–induced apoptosis. Our results demonstrate that suppression of steady-state, but not stimulus-induced NF-κB activity, regulates expression of gene products required for Sindbis virus–induced death. Furthermore, we show that in the same cell line, NF-κB can be proapoptotic or antiapoptotic depending on the death stimulus. We propose that the role of NF-κB in regulating apoptosis is determined by the death stimulus and by the timing of modulating NF-κB activity relative to the death stimulus.

No abstract

Catalase is an antioxidant enzyme that has been shown to inhibit apoptotic or necrotic neuronal death induced by hydrogen peroxide. We report the purification of a contaminating antiapoptotic activity from a commercial bovine liver catalase preparation by following its ability to inhibit apoptosis when applied extracellularly in multiple death paradigms. The antiapoptotic activity was identified by protein microsequencing as arginase, a urea cycle and nitric oxide synthase-regulating enzyme, and confirmed by demonstrating the presence of antiapoptotic activity in a >97% pure preparation of recombinant arginase. The pluripotency of recombinant arginase was demonstrated by its ability to inhibit apoptosis in multiple paradigms including rat cortical neurons induced to die by glutathione depletion and oxidative stress, by 100 nM staurosporine treatment, or by Sindbis virus infection. The protective effects of arginase in these apoptotic paradigms, in contrast to previous studies on excitotoxic neuronal necrosis, are independent of nitric oxide synthase inhibition. Rather, arginase-induced depletion of arginine leads to inhibition of protein synthesis, resulting in cell survival. Because inhibitors of nitric oxide synthesis and of protein synthesis have been shown to decrease necrotic and apoptotic death, respectively, in animal models of stroke and spinal cord injury, arginine-depleting enzymes, capable of simultaneously inhibiting protein synthesis and nitric oxide generation, may be propitious therapeutic agents for acute neurological diseases. Furthermore, our results suggest caution in attributing the cytoprotective effects of some catalase preparations to catalase.

We previously established that NF-kappaB DNA binding activity is required for Sindbis Virus (SV)-induced apoptosis. To investigate whether SV induces nuclear translocation of NF-kappaB via the proteasomal degradation pathway, we utilized MG132, a peptide aldehyde inhibitor of the catalytic subunit of the proteasome. 20 microM MG132 completely abrogated SV-induced NF-kappaB nuclear activity at early time points after infection. Parallel measures of cell viability 48 h after SV infection revealed that 20 microM MG132 induced apoptosis in uninfected cells. In contrast, a lower concentration of MG132 (200 nM) resulted in partial inhibition of SV-induced nuclear NF-kappaB activity and inhibition of SV-induced apoptosis without inducing toxicity in uninfected cells. The specific proteasomal inhibitor, lactacystin, also inhibited SV-induced death. Taken together, these results suggest that the pro-apoptotic and anti-apoptotic functions of peptide aldehyde proteasome inhibitors such as MG-132 depend on the concentration of inhibitor utilized and expand the list of stimuli requiring proteasomal activation to induce apoptosis to include viruses.