In the developing spinal cord, regional and combinatorial activities of Hox transcription factors are critical in controlling motor neuron fates along the rostrocaudal axis, exemplified by the precise pattern of limb innervation by more than fifty Hox-dependent motor pools. The mechanisms by which motor neuron diversity is constrained to limb levels are, however, not well understood. We show that a single Hox gene, Hoxc9, has an essential role in organizing the motor system through global repressive activities. Hoxc9 is required for the generation of thoracic motor columns, and in its absence, neurons acquire the fates of limb-innervating populations. Unexpectedly, multiple Hox genes are derepressed in Hoxc9 mutants, leading to motor pool disorganization and alterations in the connections by thoracic and forelimb-level subtypes. Genome-wide analysis of Hoxc9 binding suggests that this mode of repression is mediated by direct interactions with Hox regulatory elements, independent of chromatin marks typically associated with repressed Hox genes.

Thyroid hormone modulates the expression of numerous genes that in turn regulate lipoprotein metabolism in vivo. We have examined the thyroid hormone-dependent regulation of apolipoprotein B (apoB) RNA editing in a strain of congenitally hypothyroid mice (Pax8(-/-)) that lacks thyroid follicular cells. Neonatal Pax8(-/-) mice demonstrate an approximately 10-fold increase in hepatic triglyceride content associated with a decrease in hepatic apoB RNA editing. Thyroid hormone administration resulted in hepatic triglyceride mobilization in conjunction with an increase in hepatic, but not intestinal, apoB RNA editing and without changing total apoB RNA abundance. ApoB RNA editing is mediated by a multicomponent enzyme complex whose catalytic core contains two proteins, apobec-1 and apobec-1 complementation factor (ACF). Hepatic ACF mRNA and protein abundance decreased in Pax8(-/-) mice, with restoration after thyroid hormone administration, whereas apobec-1 mRNA and protein abundance were unchanged. Immunohistochemical analysis revealed increased staining intensity of ACF within hepatocyte nuclei of treated mice, findings confirmed by Western analysis of isolated nuclei. In vitro RNA editing assays demonstrated that supplementation with recombinant ACF alone restored enzymatic activity of S100 extracts from hypothyroid, Pax8(-/-) mice. These data demonstrate that thyroid hormone modulates murine hepatic lipoprotein metabolism in association with tissue-specific effects on apoB RNA editing mediated through alterations in ACF gene expression.

BACKGROUND & AIMS:This study aimed to determine the role of the RNA binding protein apobec-1 in radioprotection of the intestine.METHODS:Apobec-1-deleted mice (APOBEC-1(-/-)) and wild-type controls were treated with 12 Gy of whole-body gamma-irradiation in a cesium irradiator. The number of surviving intestinal crypts was assessed 3.5 days after irradiation by using a clonogenic assay. Cyclooxygenase-2 messenger RNA and protein expression were determined by real-time polymerase chain reaction and Western blot, respectively. RNA stability was studied by examining the turnover of a chimeric transcript containing the cyclooxygenase-2 3' untranslated region cloned downstream of luciferase complementary DNA. Apobec-1 binding to the cyclooxygenase-2 3' untranslated region was studied by electrophoretic mobility shift and UV crosslinking assays.RESULTS:After gamma-irradiation, the survival of intestinal stem cells decreased significantly in APOBEC-1(-/-) mice. In wild-type mice treated with lipopolysaccharide before gamma-irradiation, intestinal stem cells were protected by marked increases in prostaglandin E 2 mediated by cyclooxygenase-2. No such effect was observed in the APOBEC-1(-/-) mice. The mechanism of this radioprotective effect involves the binding of apobec-1 to AU-rich sequences in the first 60 nucleotides of the 3' untranslated region of cyclooxygenase-2. Upon binding to the AU-rich sequences, apobec-1 stabilizes cyclooxygenase-2 messenger RNA. This stabilization process does not seem to be mediated by p38 mitogen-activated protein kinase pathways.CONCLUSIONS:Lipopolysaccharide increases intestinal stem cell survival through apobec-1-mediated regulation of cyclooxygenase-2 messenger RNA stability.

Damage to intestinal epithelium limits the use of ionizing radiation (IR) in cancer therapy. Prostaglandins (PGs), generated through the action of cyclooxygenase-1 (COX-1) and COX-2 protect the intestinal stem cells from IR. In previous studies, we demonstrated that the RNA-binding protein CUGBP2 regulates the stability and translation of COX-2 mRNA by interacting with AU-rich sequences in 3' UTR. Here, we demonstrate a dynamic antagonistic relationship between CUGBP2 and COX-2. Both CUGBP2 and COX-2 are rapidly induced after IR in intestinal crypt epithelial cells in mice, but CUGBP2 protein expression is observed immediately and COX-2 protein expression is delayed. In contrast, administration of bacterial lipopolysaccharide induced COX-2 expression and PGE(2), resulting in the inhibition of CUGBP2 expression and radioprotection of the intestine. These effects were reversed by NS398, a COX-2-specific inhibitor, suggesting that lipopolysaccharide-mediated inhibition of CUGBP2 is a PG-dependent mechanism. Furthermore, CUGBP2 expression is higher in COX-1(-/-) and COX-2(-/-) mice than wild-type controls at basal conditions, which is further increased after IR.

role of NO in gastric epithelial cell apoptosin during Helicobacter infi:ction remains contrnversial The aim of our study was to determine the interaction between NO and Fas mediated apoptotic signaling.Methods, Rat gastric mucosal ceils (RGM-1) were stably transfected with the pMSCV-puro vector containing the full length mouse Fas Ag eDNA.Fas Ag expressing clones were isolated and ctuaracterized, and clones expressing moderate levels of Fas Ag and moderately susceptible to Fas mediated apoptosis were chosen for this study.The apoptotic response to FasL 50ng/ml was determined in control medium, medium + lmM L-arginine (NO donor) or medium + 5raM I-NMA (N c monomethyl-L-arginine -an NO inhibitor), DNA fragmentation assay and Annex-V / propidium iodide FACS was used to qnanritate apoptosis Caspase 3 activation and PARP cleavage was detected using Western hlot, ResuhsRGM-.1 Fas Ag cells grown under standard culture conditions had a 1 6 • 0.3% level of apoptosis as detected by DNA fragmentation assay, Addition of FasL 50 n~'ml for 24 hours produced a moderate increase in apoptosis; 17.37+_2,3%When an NO donor is made avadabIe to the ceils i6 hours prior to ligand addition, protection from apoptosis is noted and the level of apoptosin decreased dramatically to 6.7 • 1.4% Conversely, when an NO inhibitor is added (L-NMA), apoptosis significantly increases to 32,24 • 3. I%.Annex-V Rl staining FACS analysis confirmed apoptosis levels, and further verified viable cells at; 98.32%, 900%, 961%, 783% in the t~specrive treatment groups.Caspase 3 and PARP cleavage was e~qdent after treatment with FasL 50 ng/m/ for 4 hours, was inhibited by pretreatment wifh L-arginine, and enhanced by L-NiVLA.. Conclusions, Gastric epithelial ceils can use L-argmme as substrate to produce NO, NO inhibits Fas mediated apoptosis at the level of caspase 3 activation, potentially via nitrosylation of caspase 3. Decreasing the availability of NO enhances Fas mediated apoptosis in gastric mucosal cells Therefore, NO may fuoction to mothdate Fas mediated apoptosls during Hdicobacter intection.

Cyclooxygenase-2 (COX-2) expression is translationally silenced in epithelial cells undergoing radiation-induced apoptosis. CUGBP2, a predominantly nuclear protein, is also rapidly induced in response to radiation and translocates to the cytoplasm. Antisense-mediated suppression of CUGBP2 renders radioprotection through a COX-2-dependent prostaglandin pathway, providing an in vivo demonstration of translation inhibition activity for CUGBP2. CUGBP2 binds to two sets of AU-rich sequences (AREs) located within the first sixty nucleotides of the COX-2 3' untranslated region (3'UTR). Upon binding, CUGBP2 stabilizes a chimeric luciferase-COX-2 3'UTR mRNA but inhibits its translation. These findings identify a novel paradigm for RNA binding proteins in facilitating opposing functions of mRNA stability and translation inhibition and reveal a mechanism for inhibiting COX-2 expression in cancer cells.

Cyclooxygenases (COX) also known as prostaglandin (PG) synthases are found in two forms, COX-1 and COX-2. While COX-1 is responsible for cytoprotective functions in a number of organs, COX-2, which is normally absent at basal levels, is induced under certain conditions. Induction of COX-2 is found in many pathophysiological states including acute inflammation, arthritis, as well as in cancer and cancer-related angiogenesis. Overexpression of COX-2 is sufficient to cause tumorigenesis in animal models and inhibition of COX-2 is sufficient to reduce tumour incidence and progression. Availability of a mouse model in which the COX-2 gene is deleted has been very useful in this regard and has provided compelling evidence that presence of COX-2 is essential for an aggressive phenotype. Indeed, COX-2 may very well be a diagnostic marker for cancer and identification of mechanisms that regulate its expression may help in designing new therapies for the treatment of cancer. COX-2 mediated prostaglandin E 2 is the primary mechanism of promoting cancer development in colon tissue. It should be noted that there are other COX-2 mechanisms, independent of prostaglandins as well as other natural prostaglandins such as 15-deoxy-prostaglandin J2 that also contribute to tumour development in other tissues. However, in the current article, we will attempt to review the recent progress in understanding the role of COX-2 dependent prostaglandin E 2 in colon cancer and mechanisms that regulate COX-2 expression.

Posttranscriptional control of gene expression plays a key role in regulating gene expression in cells undergoing apoptosis. Cyclooxygenase-2 (COX-2) is a crucial enzyme in the conversion of arachidonic acid to prostaglandin E2 (PGE(2)) and is significantly upregulated in many types of adenocarcinomas. COX-2 overexpression leads to increased PGE2 production, resulting in increased cellular proliferation. PGE2 enhances the resistance of cells to ionizing radiation. Accordingly, understanding mechanisms regulating COX-2 expression may lead to important therapeutic advances. Besides transcriptional control, COX-2 expression is significantly regulated by mRNA stability and translation. We have previously demonstrated that RNA binding protein CUGBP2 binds AU-rich sequences to regulate COX-2 mRNA translation. In the current study, we have determined that expression of both COX-2 mRNA and CUGBP2 mRNA are induced in MCF-7 cells, a breast cancer cell line, following exposure to 12 Gy gamma-irradiation. However, only CUGBP2 protein is induced, but COX-2 protein levels were not altered. Silencer RNA (siRNA)mediated inhibition of CUGBP2 reversed the block in COX-2 protein expression. Furthermore, MCF-7 cells underwent apoptosis in response to radiation injury, which was also reversed by CUGBP2 siRNAs. These data suggest that CUGBP2 is a critical regulator of the apoptotic response to genotoxic injury in breast cancer cells.

Apobec-1 catalyzes C to U editing of apolipoprotein B (apoB) mRNA in the mammalian intestine. Rat apobec-1 is transcribed from three distinct promoters, which contain distinct 5' untranslated regions (5'UTRs) accompanied by variable numbers of in-frame upstream AUGs (uAUGs). We have observed a shift in apobec-1 promoter usage in an experimental model of colon carcinogenesis, resulting in transcripts loaded with 5'AUGs. In colon cancer, apobec-1 protein levels decreased by 90% in the cancer tissue as compared to normal tissue, suggesting an inhibitory effect of the 5'UTR on apobec-1 translation. We investigated the effects of these different 5'UTRs by site-directed mutagenesis coupled with in vitro translation studies. These studies established that the uAUGs within the 5'UTR of the alternative transcripts inhibit apobec-1 translation. This effect was independent of the length of the 5'UTR. Further analysis demonstrated that these uAUGs altered the polysome distribution, shifting the mRNA towards a denser, post-polyribosomal fraction. These findings were confirmed in transient transfection studies in vivo using HepG2 cells, where functional expression of apobec-1 was restored by mutagenesis of the uAUGs. Taken together, these data imply that rat apobec-1 gene expression is downregulated through alternative promoter usage. This dominant translational control of apobec-1 gene expression is most plausibly exerted through uAUGs.

C-->U RNA editing of neurofibromatosis 1 (NF1) mRNA changes an arginine (CGA) to a UGA translational stop codon, predicted to result in translational termination of the edited mRNA. Previous studies demonstrated varying degrees of C-->U RNA editing in peripheral nerve-sheath tumor samples (PNSTs) from patients with NF1, but the basis for this heterogeneity was unexplained. In addition, the role, if any, of apobec-1, the catalytic deaminase that mediates C-->U editing of mammalian apolipoprotein B (apoB) RNA, was unresolved. We have examined these questions in PNSTs from patients with NF1 and demonstrate that a subset (8/34) manifest C-->U editing of RNA. Two distinguishing characteristics were found in the PNSTs that demonstrated editing of NF1 RNA. First, these tumors express apobec-1 mRNA, the first demonstration, in humans, of its expression beyond the luminal gastrointestinal tract. Second, PNSTs with C-->U editing of RNA manifest increased proportions of an alternatively spliced exon, 23A, downstream of the edited base. C-->U editing of RNA in these PNSTs was observed preferentially in transcripts containing exon 23A. These findings were complemented by in vitro studies using synthetic RNA templates incubated in the presence of recombinant apobec-1, which again confirmed preferential editing of transcripts containing exon 23A. Finally, adenovirus-mediated transfection of HepG2 cells revealed induction of editing of apoB RNA, along with preferential editing of NF1 transcripts containing exon 23A. Taken together, the data support the hypothesis that C-->U RNA editing of the NF1 transcript occurs both in a subset of PNSTs and in an alternatively spliced form containing a downstream exon, presumably an optimal configuration for enzymatic deamination by apobec-1.

Mammalian apolipoprotein B (apoB) C to U RNA editing is catalyzed by a multicomponent holoenzyme containing a single catalytic subunit, apobec-1. We have characterized an apobec-1 homologue, ARCD-1, located on chromosome 6p21.1, and determined its role in apoB mRNA editing. ARCD-1 mRNA is ubiquitously expressed; phylogenetic analysis reveals it to be a distant member of the RNA editing family. Recombinant ARCD-1 demonstrates cytidine deaminase and apoB RNA binding activity but does not catalyze C to U RNA editing, either in vitro or in vivo. Although not competent itself to mediate deamination of apoB mRNA, ARCD-1 inhibits apobec-1-mediated C to U RNA editing. ARCD-1 interacts and heterodimerizes with both apobec-1 and apobec-1 complementation factor (ACF) and localizes to both the nucleus and cytoplasm of transfected cells. Together, the data suggest that ARCD-1 is a novel cytidine deaminase that interacts with apobec-1 and ACF to inhibit apoB mRNA editing, possibly through interaction with other protein components of the apoB RNA editing holoenzyme.