Adalberto Benito

Nod2, a Nod1/Apaf-1 Family Member That Is Restricted to Monocytes and Activates NF-κB

Apaf-1 and Nod1 are members of a protein family, each of which contains a caspase recruitment domain (CARD) linked to a nucleotide-binding domain, which regulate apoptosis and/or NF-κB activation. Nod2, a third member of the family, was identified. Nod2 is composed of two N-terminal CARDs, a nucleotide-binding domain, and multiple C-terminal leucine-rich repeats. Although Nod1 and Apaf-1 were broadly expressed in tissues, the expression of Nod2 was highly restricted to monocytes. Nod2 induced nuclear factor κB (NF-κB) activation, which required IKKγ and was inhibited by dominant negative mutants of IκBα, IKKα, IKKβ, and IKKγ. Nod2 interacted with the serine-threonine kinase RICK via a homophilic CARD-CARD interaction. Furthermore, NF-κB activity induced by Nod2 correlated with its ability to interact with RICK and was specifically inhibited by a truncated mutant form of RICK containing its CARD. The identification of Nod2 defines a subfamily of Apaf-1-like proteins that function through RICK to activate a NF-κB signaling pathway.

Erythropoietin can induce the expression of Bcl-x(L) through Stat5 in erythropoietin-dependent progenitor cell lines

Erythropoietin (Epo) initiates its cellular response by binding to the Epo receptor, which triggers the activation of signal transducer and activator of transcription (Stat) 5 protein. Cell culture studies of erythroid progenitors have suggested that Epo functions as a survival factor by repressing apoptosis at least in part through Bcl-xL, an anti-apoptotic protein of the Bcl-2 family. In this report, we examine whether Stat5 can induce transactivation of the bcl-x gene in response to Epo. Two Epo-responsive progenitor cell lines, HCD-57 and Bcl-2-transfected Ba/F3-Epo receptor (Ba/F3-EpoR-Bcl-2), were used in this study. After Epo stimulation, we observed a correlation between expression ofbcl-x L and activation of Stat5 as assessed by the expression of oncostatin M, a direct target of Stat5, and the phosphorylation and nuclear translocation of Stat5. Moreover, a Stat binding element in the bcl-x promoter was found to be active in response to Epo, a finding that was further confirmed because mutagenesis of this sequence motif abrogated its promoter activity and overexpression of a dominant negative Stat5 protein blocked transactivation. When DNA-protein binding analyses were performed, we found that Stat5, not Stat1 or Stat3, was the protein bound to the bcl-x promoter in response to Epo. These data suggest that Epo-dependent activation of Stat5 is a transcriptional pathway that can be used by Epo-responsive progenitor cells to induce the expression of bcl-xL and consequently to inhibit apoptosis.

Expression of Bcl-x in Erythroid Precursors from Patients with Polycythemia Vera

BACKGROUND Deregulating the expression of Bcl-xL, an inhibitor of apoptosis, in an erythropoietin-dependent erythroblast cell line averts apoptosis induced by the withdrawal of erythropoietin. Since in polycythemia vera an abnormal clone of erythroid progenitors is independent of erythropoietin, we investigated whether the endogenous expression of Bcl-xL was deregulated in these cells. METHODS Erythroid colonies from patients with polycythemia vera and normal subjects were cultured in the presence and absence of erythropoietin and assessed by immunocytochemical and flow-cytometric analysis with anti-Bcl-x antibodies that recognize the two species of Bcl-x (Bcl-xL and Bcl-xS). Reverse-transcriptase-polymerase-chain-reaction analysis was used to determine which one of the two species was responsible for anti-Bcl-x staining. Bone marrow mononuclear cells from 8 healthy bone marrow donors, 14 patients with polycythemia vera, 19 patients with other myeloproliferative syndromes, and 12 patients with secondary erythrocytosis were analyzed by flow cytometry with antibodies against Bcl-x and glycophorin A, an erythroid marker. RESULTS Erythroid cells from patients with polycythemia vera survived in vitro without erythropoietin, and this finding correlated with the expression of Bcl-x protein (Bcl-xL messenger RNA was the main species of Bcl-x found), even in mature erythroblasts that normally do not express Bcl-x. The mean (+/-SD) percentage of cells positive for both glycophorin A and Bcl-x in the 14 patients with polycythemia vera (21.8+/-3.6 percent) was significantly higher than that in 8 normal donors (6.62+/-1.58 percent), 12 patients with secondary erythrocytosis (6.87+/-1.95 percent), 9 patients with essential thrombocythemia (3.81+/-0.97 percent), and 10 patients with chronic myeloid leukemia (2.7+/-0.41 percent). CONCLUSIONS Deregulated expression of Bcl-x may contribute to the erythropoietin-independent survival of erythroid-lineage cells in polycythemia vera and thereby contribute to the pathogenesis of this disease.

AICAR induces apoptosis independently of AMPK and p53 through up-regulation of the BH3-only proteins BIM and NOXA in chronic lymphocytic leukemia cells

5-Aminoimidazole-4-carboxamide riboside or acadesine (AICAR) induces apoptosis in chronic lymphocytic leukemia (CLL) cells. A clinical study of AICAR is currently being performed in patients with this disease. Here, we have analyzed the mechanisms involved in AICAR-induced apoptosis in CLL cells in which it activates its only well-known molecular target, adenosine monophosphate-activated protein kinase (AMPK). However, AMPK activation with phenformin or A-769662 failed to induce apoptosis in CLL cells and AICAR also potently induced apoptosis in B lymphocytes from Ampkα1(-/-) mice, demonstrating an AMPK-independent mechanism of cell death. Importantly, AICAR induced apoptosis irrespective of the tumor suppressor TP53 or ataxia telangiectasia mutated (ATM) status via induction of the mitochondrial pathway. Apoptosis was preceded by an increase in mRNA and protein levels of proapoptotic BCL-2 family proteins of the BH3-only subgroup, including BIM, NOXA, and PUMA in CLL cells. Strikingly, B lymphocytes from Noxa(-/-) or Bim(-/-) mice were partially protected from the cytotoxic effects of AICAR. Consistently, B cells from Noxa(-/-)/Bim(-/-) mice resisted induction of apoptosis by AICAR as potently as B lymphocytes overexpressing transgenic BCL-2. These findings support the notion that AICAR is an interesting alternative therapeutic option for CLL patients with impaired p53 function and resistance to conventional chemotherapy.

Critical Role for DP5/Harakiri, a Bcl-2 Homology Domain 3-Only Bcl-2 Family Member, in Axotomy-Induced Neuronal Cell Death

The survival of neurons is maintained primarily by neurotrophic factors that suppress the apoptotic program. Axotomy or removal of peripheral targets causes neuronal cell death, but the mechanisms involved in the induction of this type of cell death remain poorly understood. Here, we show that DP5/Harakiri, a Bcl-2 homology domain 3-only member of the Bcl-2 family, is induced in motoneurons after transection of the hypoglossal nerve in mice and in sympathetic neurons after nerve growth factor (NGF) withdrawal. To assess the role of DP5 in neuronal cell death, mutant mice deficient in DP5 were generated by gene targeting. DP5–/– mice were viable and exhibited normal postnatal development. Notably, motoneurons from DP5–/– mice were highly protected from cell death induced by resection of the hypoglossal nerve compared with motoneurons from DP5+/+ littermate mice. In addition, deficiency of DP5 in superior cervical ganglia (SCG) neurons resulted in delayed neuronal cell death triggered by NGF withdrawal. Analysis of SCG neurons from DP5–/– mice revealed increased preservation of mitochondrial membrane potential and reduced activation of caspase-3 compared with neurons from wild-type mice. These results indicate that DP5 plays an important role in neuronal cell death induced by axotomy and NGF deprivation through the regulation of mitochondrial function and caspase-3 activation.

Embryonic loss due to exposure to polycyclic aromatic hydrocarbons is mediated by Bax

The high miscarriage rates observed in women smokers raises the possibility that chemicals in cigarette smoke could be detrimental to embryo development. Previous studies have established that polycyclic aromatic hydrocarbons (PAHs), transactivate the arylhydrocarbon receptor (AhR), leading to cell death. Herein we show that PAH exposure results in murine embryo cell death, acting as a potential mechanism underlying cigarette-smoking-induced pregnancy loss. Cell death was preceded by increases in Bax levels, activation of caspase-3 and decreased litter size. Chronic exposure of females to PAHs prior to conception impaired development, resulting in a higher number of resorptions. This embryonic loss could not be prevented by the disruption of Hrk, but was diminished in embryos lacking Bax. We conclude that exposure of early embryos to PAHs reduces the allocation of cells to the embryonic and placental lineages by inducing apoptosis in a Bax-dependent manner, thus compromising the developmental potential of exposed embryos.

Blockade of epidermal growth factor receptors chemosensitizes breast cancer cells through up-regulation of Bnip3L

Epidermal growth factor receptor-1 (EGFR) and EGFR-2 (HER2) have become major targets for cancer treatment. Blocking antibodies and small-molecule inhibitors are being used to silence the activity of these receptors in different tumors with varying efficacy. Thus, a better knowledge on the signaling pathways activated by EGFR and HER2 may help unravel novel therapeutic targets and molecular markers of response. Here, we show that treatment of breast cancer cell lines with blocking antibodies against EGFR (cetuximab) or HER2 (trastuzumab) promotes the specific induction of proapoptotic Bnip3L and chemosensitization. Moreover, we found that the Bnip3L gene is transcriptionally activated by FoxO3a. Trastuzumab-mediated induction of Bnip3L and nuclear translocation of FoxO3a was also shown in pleural effusion cells from a breast cancer patient. Transfection of breast cancer cells with constitutively active FoxO3a or with Bnip3L promotes sensitization to chemotherapy-induced apoptosis. On the contrary, blockade of Bnip3L expression by a small interfering RNA strategy significantly diminished the chemosensitizing effect of cetuximab. We found also an inverse correlation between EGFR and Bnip3L expression in surgical specimens from patients with breast cancer. Therefore, blockading EGFR or HER2 specifically up-regulates Bnip3L, which is required for chemosensitization of breast cancer cells. This novel pathway provides also the rationale for therapeutic strategies aimed to induce the expression of Bnip3L.

Genetic variation and activity of mouse Nod2, a susceptibility gene for Crohn’s disease

Genetic variation in human Nod2 has been associated with susceptibility to Crohns disease. The mouse Nod2 locus is located at chromosome 8 and composed of 12 exons, 11 of which encode the Nod2 protein. Sequence analysis of Nod2 from 45 different strains of Mus musculus and Mus spretus revealed extensive polymorphism involving all exons of Nod2. Of the 140 polymorphic sites identified, 68 were located in the coding region, of which 28 created amino acid substitutions in Nod2. Expression of mouse Nod2 activated NF-kappaB and conferred responsiveness to bacterial components, an activity that was deficient in mutants corresponding to those associated with susceptibility to Crohns disease. These studies demonstrate a conserved role for Nod2 in the response to bacterial components and suggest that selective evolutionary pressure exerted by pathogens may have contributed to the high level of variability of Nod2 sequences in both humans and mice.

A Novel Role for Proline- and Acid-rich Basic Region Leucine Zipper (PAR bZIP) Proteins in the Transcriptional Regulation of a BH3-only Proapoptotic Gene

Proline- and acid-rich (PAR) basic region leucine zipper (bZIP) proteins thyrotroph embryonic factor (TEF), D-site-binding protein (DBP), and hepatic leukemia factor have been involved in neurotransmitter homeostasis and amino acid metabolism. Here we demonstrate a novel role for these proteins in the transcriptional control of a BH3-only gene. PAR bZIP proteins are able to transactivate the promoter of bcl-gS. This promoter is particularly responsive to TEF activation and is silenced by NFIL3, a repressor that shares the consensus binding site with PAR bZIP proteins. Consistently, transfection of TEF induces the expression of endogenous bcl-gS in cancer cells, and this induction is independent of p53. A naturally occurring variant of DBP (tDBP), lacking the transactivation domain, has been identified and shown to impede the formation of active TEF dimers in a competitive manner and to reduce the TEF-dependent induction of bcl-gS. Of note, treatment of cancer cells with etoposide induces TEF activation and promotes the expression of bcl-gS. Furthermore, blockade of bcl-gS or TEF expression by a small interfering RNA strategy or transfection with tDBP significantly reduces the etoposide-mediated apoptotic cell death. These findings represent the first described role for PAR bZIP proteins in the regulation of a gene involved in the execution of apoptosis.

Involvement of the intrinsic and extrinsic cell-death pathways in the induction of apoptosis of mature lymphocytes by the Escherichia coli heat-labile enterotoxin

Escherichia coli heat‐labile enterotoxin (LT) exhibits a broad range of immunomodulatory activities, including the induction of lymphocyte‐programmed cell death. In previous studies, we have demonstrated that in vivo LT promotes apoptosis of immature T and B cells through the stimulation of endogenous glucocorticoids. In the present study, we show that the extrinsic cell‐death pathway as well as the apoptosis‐inducing factor do not participate in the LT‐induced elimination of thymocytes. In contrast to developing lymphocytes, LT promotes the death of mature lymphocytes by both glucocorticoid‐ and Fas death receptor/Fas ligand‐dependent mechanisms. However, the dependency of these mechanisms in the LT‐induced cell‐death activity seems to be different among CD4+ and CD8+ T cells. Altogether, our study shows that the same bacterial toxin can induce apoptosis of lymphoid cells through several mechanisms depending on the status of differentiation of these cells.