Nod2, a member of the Apaf1/Nod protein family, confers responsiveness to bacterial products and activates NF-B, a transcription factor that plays a central role in innate immunity. Recently, genetic variation in Nod2 has been associated with susceptibility to Crohn's disease. Here, we report that expression of Nod2 is induced upon differentiation of CD34؉ hematopoietic progenitor cells into granulocyte or monocyte/macrophages. In peripheral blood cells, the highest levels of Nod2 were observed in CD14 ؉ (monocytes), CD15 ؉ (granulocytes), and CD40؉ /CD86 ؉ (dendritic cells) cell populations. Notably, stimulation of myeloblastic and epithelial cells with bacterial lipopolysaccharide or TNF␣ resulted in up-regulation of Nod2. A search for consensus sites within the Nod2 promoter revealed a NF-B binding element that was required for transcriptional activity in response to TNF␣. Moreover, ectopic expression of p65 induced transactivation, whereas that of dominant-negative IB␣ blocked the transcriptional activity of the Nod2 promoter. Upon stimulation with TNF␣ or lipopolysaccharide, both p50 and p65 subunits of NF-B were bound to the Nod2 promoter. Thus, Nod2 expression is enhanced by proinflammatory cytokines and bacterial components via NF-B, a mechanism that may contribute to the amplification of the innate immune response and susceptibility to inflammatory disease.

There are two COUP-TF genes, COUP-TFI and COUP-TFII (for a review, see reference 58), which share a high degree of homology in their DBDs and putative LBDs. In addition, COUP-TFI and COUP-TFII are highly conserved among different species, suggesting that they play important roles in development. Although the expression of COUP-TFI and COUP-TFII is highly overlapping in mice (24, 43), COUP-TFI is abundantly expressed in the central and peripheral nervous system, while COUP-TFII is highly expressed in the mesenchymal component of the developing organs (46). Based on this expression pattern, we have proposed that COUP-TFI is important for neural development and that COUP-TFII is important in organogenesis through the regulation of mesenchymal cell-epithelial cell interactions. Indeed, the targeted disruption of either of these two genes in mice results in a lethal phenotype. COUP-TFI-deficient mice show defects in the development of the central and peripheral nervous systems (47; unpublished observations), whereas mutation of the COUP-TFII gene results in defects in the heart and vasculature formation (unpublished observations).Vasculature and organ formation requires very tight communication between epithelial cell and mesenchymal cell populations (7). This communication is essential for the differentiation of these two cell types (14). Since COUP-TFII is highly expressed in the mesenchyme but is undetectable in the epithelium of most organs, it is likely that COUP-TFII plays an important role in mesenchymal cell-epithelial cell interactions during organogenesis. This process has been well studied for the prostate, where several growth factors and the extracellular matrix are involved in this communication between the mesenchyme and the epithelium (11,19).COUP-TFs are able to bind to a variety of dispositions of the basic AGGTCA motif, including those recognized by the receptors of retinoic acid (RAR), thyroid hormone, and vitamin D (13). This ability makes COUP-TFs capable of competing for the response elements of these receptors, thus acting as passive repressors of the transcriptional activation induced by them (12,13,56). Another mechanism of passive repression by COUP-TFs involves their ability to heterodimerize with the 9-cis retinoic acid receptor (RXR), reducing its availability for other nuclear receptors that use it as a partner. In addition, COUP-TFs contain an active repression domain within their putative LBDs (1,31). This repression domain is capable of interacting with corepressors such as SMRT and N-CoR (50), molecules that can recruit histone deacetylase activities to the DNA to suppress transcription (2,23,39).

Caspase activating and recruitment domain 8 (CARD8) potently inhibits NF-κB signaling, which plays a key role in inflammation, and may contribute to avoid a pathologic activation of NF-κB; however, the transcriptional mechanisms regulating CARD8 expression and the relevance of this protein in inflammatory diseases are poorly understood. We found a NF-κB-binding element within the human CARD8 promoter that was required for transcriptional activity in response to TNF-α and the p65 subunit of NF-κB. Moreover, TNF-α and overexpression of p65 induced the formation of NF-κB-CARD8 promoter complexes. Thus, CARD8 may control NF-κB activation through a regulatory loop. To study the relevance of CARD8 in chronic inflammatory disorders, we functionally characterized a deleterious polymorphism (p.C10X) and studied its association with rheumatoid arthritis (RA). Transfection of cell lines with the allelic variants of CARD8 revealed that full-length (CARD8-L) but not truncated (CARD8-S) protein inhibits NF-κB transcriptional activity, and abrogates the binding of NF-κB to its consensus site. Furthermore, in contrast to the full-length protein, CARD8-S did not modify the expression of NF-κB target genes (cIAP, A1), in response to TNF-α. We analyzed the p.C10X polymorphism in 200 patients with RA, and found that homozygous carriers of the CARD8-S allele have higher disease activity score (p = 0.014), more extra-articular manifestations (p = 0.03), and a lower probability of clinical remission (p = 0.03) than the CARD8-L allele carriers. Overall, our findings provide molecular insight into the expression of CARD8 by NF-κB, and suggest that a deleterious polymorphism of CARD8 may help predict the severity of RA.

Early growth response factor (Egr-1) is an inducible zinc finger transcription factor that binds specific GC-rich enhancer elements and impacts female reproduction. These studies document for the first time that FSH rapidly induces Egr-1 expression in granulosa cells of small growing follicles. This response is transient but is reinitiated in preovulatory follicles exposed to the LH analog, human chorionic gonadotropin. Immunohistochemical analysis also showed gonadotropin induced Egr-1 in theca cells. The Egr-1 gene regulatory region responsive to gonadotropin signaling was localized within -164 bp of the transcription initiation site. Binding of Sp1/Sp3 to a proximal GC-box at -64/-46 bp was enhanced by FSH in immature granulosa cells but reduced after human chorionic gonadotropin stimulation of preovulatory follicles despite constant protein expression. This dynamic regulation of Sp1 binding was dependent on gonadotropin-regulated mechanisms that modulate Sp1/3-DNA binding activity. Serum response factor was active in granulosa cells and bound a consensus CArG-box/serum response element site, whereas two putative cAMP response elements within the -164-bp region bound cAMP regulatory element (CRE) binding protein (CREB) and a second cAMP-inducible protein immunologically related to CREB. Transient transfection analyses using Egr-1 promoter-luciferase constructs and site-specific mutations show that the serum response element, GC-box, and CRE-131 are involved in gonadotropin regulation of Egr-1 expression in granulosa cells. Specific kinase inhibitors of Erk or protein kinase A antagonized this induction while exogenously expressed Egr-1 enhanced reporter expression. These observations indicate that the Egr-1 gene is a target of both FSH and LH action that may mediate molecular programs of proliferation and/or differentiation during follicle growth, ovulation, and luteinization.

BH3-only proteins are required for execution of apoptotic cell death. We have found that one of these proteins, Bik, is strongly induced in cancer cells treated with chemotherapeutic agents. Furthermore, we showed that chemotherapy-induced expression of bik is independent of p53. Consistent with its pro-apoptotic activity, blockade of bik expression reduces the adriamycin-mediated apoptotic cell death. We also found that the bik gene is transcriptionally activated by E2F proteins. Consistently, adriamycin induces the E2F-bik pathway. In addition, E2Fs transactivate bik by a p53-independent mechanism. Thus, our data indicate that transcriptional regulation of bik contributes to the efficient apoptotic response to chemotherapeutic agents.

Proline-and acid-rich (PAR) basic region leucine zipper (bZIP) proteins thyrotroph embryonic factor (TEF), D-sitebinding 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.Two genes, cell death specification protein 1 (ces-1) and ces-2, control the decisions of neuro-secretory motor sister cells in Caenorhabditis elegans to undergo apoptosis. A genetic approach showed that these factors regulate the genes required for apoptosis and that a gain of ces-1 function or a reduction of ces-2 function prevents these cells from dying (1). The pro-apoptotic CES-2 protein negatively regulates CES-1, which prevents the death of the neuro-secretory motor sister cells by transcriptional silencing of Egl-1, a BH3-only protein required for apoptosis in C. elegans (2, 3). CES-2 is similar to members of the proline-and acid-rich (PAR) 4 subfamily of basic region leucine zipper (bZIP) transcription factors, and both share the DNA binding specificity (4).Mammalian homologs of CES-2 include thyrotroph embryonic factor (TEF), albumin D-site-binding protein (DBP), and hepatic leukemia factor (HLF) (5-7). These PAR bZIP proteins have recently been shown to be involved in amino acid and neurotransmitter metabolism through transcriptome profiling analyses in both liver and brain (8). Thus, despite the pro-apoptotic activity described for CES-2 promoting the induction of Egl-1 through an indirect pathway, none of its human homologs have been associated with the transcriptional regulation of BH3-only genes or other executors of apoptosis.BH3-only proteins are a pro-apoptotic subgroup of the Bcl-2 family of apoptosis regulators, which share only the short BH3 region with the rest of the family (9). Genetic experiments have shown that these proteins are essential initiators of programmed cell death in species as distantly related as mice and C. elegans. They are r...

NGFI-A is an immediate early gene that is rapidly activated in quiescent cells by mitogens or in postmitotic neurons after depolarization. We have previously shown that the expression of NGFI-A in the developing rat brain is under the control of thyroid hormone. Now we report, by means of in situ hybridization histochemistry, the differential effect of thyroid hormone on NGFI-A expression in distinct brain regions depending on the developmental stage. NGFI-A messenger RNA (mRNA) content was analyzed in the piriform cortex, striatum, hippocampus, and cerebral cortex of control, hypothyroid, and T3-injected hypothyroid rats at birth and on postnatal days 5 and 15. In the newborn rats, experimental hypothyroidism is associated with reduced levels of NGFI-A mRNA in most of the brain regions studied. On postnatal day 15, the difference in NGFI-A expression between control and hypothyroid rats is less apparent in the striatum or no longer present in the piriform cortex and the hippocampus. In the cerebral cortex, hypothyroidism is associated with reduced levels of NGFI-A mRNA on postnatal day 15. The dentate gyrus is always insensitive to the thyroidal state. Administration of T3 accelerates the recovery of NGFI-A mRNA in 5- and 15-day-old rats. However, in newborn rats, the effect of the hormone is noticeable only in the piriform cortex. We also show that the reduced level of NGFI-A mRNA in hypothyroidism is accompanied by a reduction in the protein level. Convulsions induced by pentylenetetrazole administration resulted in an increased expression of the NGFI-A gene, which is of similar magnitude in control and hypothyroid rats.

FA (Fanconi anaemia) is a hereditary disease characterized by congenital malformations, progressive bone marrow failure and an extraordinary elevated predisposition to develop cancer. In the present manuscript we describe an anomalous high level of the proinflammatory cytokine IL-1beta (interleukin-1beta) present in the serum of FA patients. The elevated levels of IL-1beta were completely reverted by transduction of a wild-type copy of the FancA cDNA into FA-A (FA group A) lymphocytes. Although the transcription factor NF-kappaB (nuclear factor-kappaB) is a well established regulator of IL-1beta expression, our experiments did not show any proof of elevated NF-kappaB activity in FA-A cells. However, we found that the overexpression of IL-1beta in FA-A cells is related to a constitutively activated PI3K (phosphoinositide 3-kinase)-Akt pathway in these cells. We provide evidence that the effect of Akt on IL-1beta activation is mediated by the inhibition of GSK3beta (glycogen synthase kinase 3beta). Finally, our data indicate that the levels of IL-1beta produced by FA-A lymphoblasts are enough to promote an activation of the cell cycle in primary glioblastoma progenitor cells. Together, these results demonstrate that the constitutive activation of the PI3K-Akt pathway in FA cells upregulates the expression of IL-1beta through an NF-kappaB-independent mechanism and that this overproduction activates the proliferation of tumour cells.