Uric acid is the end product of purine metabolism in humans and great apes, which have lost hepatic uricase activity, leading to uniquely high serum uric acid concentrations (200-500 microM) compared with other mammals (3-120 microM). About 70% of daily urate disposal occurs via the kidneys, and in 5-25% of the human population, impaired renal excretion leads to hyperuricemia. About 10% of people with hyperuricemia develop gout, an inflammatory arthritis that results from deposition of monosodium urate crystals in the joint. We have identified genetic variants within a transporter gene, SLC2A9, that explain 1.7-5.3% of the variance in serum uric acid concentrations, following a genome-wide association scan in a Croatian population sample. SLC2A9 variants were also associated with low fractional excretion of uric acid and/or gout in UK, Croatian and German population samples. SLC2A9 is a known fructose transporter, and we now show that it has strong uric acid transport activity in Xenopus laevis oocytes.

DAZL proteins are germ-cell-specific RNA-binding proteins essential for gametogenesis. The precise molecular role of these proteins in germ-cell development remains enigmatic; however, they appear to function in the cytoplasm. In order to directly address the function of vertebrate DAZL proteins, we have used Xenopus laevis oocytes as a model system. Here we demonstrate that members of this family, including Xdazl, mouse Dazl, human DAZL, human DAZ and human BOULE, have the ability to stimulate translation and function at the level of translation initiation. We show that DAZL proteins interact with poly(A)-binding proteins (PABPs), which are critical for the initiation of translation. Mapping and tethered function experiments suggest that these interactions are physiologically important. This leads to an attractive hypothesis whereby DAZL proteins activate translationally silent mRNAs during germ cell development through the direct recruitment of PABPs.

We generated mice carrying a STAT3 allele amenable to Cre-mediated deletion and intercrossed them with Mx-Cre transgenic mice, in which the expression of Cre recombinase can be induced by type I interferon. Interferon-induced deletion of STAT3 occurred very efficiently (more than 90%) in the liver and slightly less efficiently (about 70%) in the bone marrow. Analysis of the induction of liver acute-phase genes in response to bacterial lipopolysaccharide unequivocally identifies STAT3 as a fundamental mediator of their induction. The different degrees of defectiveness displayed by the various genes allowed us to differentiate them into three separate groups according to their degree of dependence on STAT3. Induction was totally defective for group I genes, defective at 24 h but almost normal at earlier time points for group II genes, and only slightly defective for group III genes. This division was in good agreement with the known structures of the respective promoters. We also found that the overall induction of the transcription factors C/EBP beta and -delta was only minimally defective in the absence of STAT3. Finally, even though corticosterone levels and action were found to be normal in the conditional-mutant mice, production of both proinflammatory and antiinflammatory cytokines was increased and prolonged, probably as a result of STAT3 deletion in macrophages.

Poly(A)-binding proteins (PABPs) are central to the regulation of messenger RNA (mRNA) translation and stability; however, the roles and contributions of different PABP family members in controlling gene expression are not yet fully understood. In this paper, the current state of knowledge of the different cytoplasmic PABP proteins and their function in animal cells will be summarised, with particular reference to their roles in development. Possible regulatory mechanisms and potential new roles for these proteins in the control of specific mRNAs are also highlighted.

Prostaglandins are important mediators of activated macrophage functions, and their inducible synthesis is mediated by cyclooxygenase-2 (COX-2). Here, we make use of the murine macrophage cells RAW264 as well as of immortalized macrophages derived from mice deficient for the transcription factor CCAAT enhancer-binding protein ␤ (C/EBP␤) to explore the molecular mechanisms regulating COX-2 induction in activated macrophages. We demonstrate that lipopolysaccharide-mediated COX-2 mRNA induction is biphasic. The initial phase is independent of de novo protein synthesis, correlates with cAMP-response element-binding protein (CREB) activation, is inhibited by treatments that abolish CREB phosphorylation and reduce NF-B-mediated gene activation, and requires the presence of the transcription factor C/EBP␤. On the other hand, C/EBP␦ appears to be essential in addition to C/EBP␤ to effect the second phase of COX-2 gene transcription, which is important for maintaining the induced state and requires de novo protein synthesis. Indeed, both phases of COX-2 induction were defective in C/EBP␤؊/؊ macrophages. Moreover, the synthesis of C/EBP␦ was increased dramatically by treatment with lipopolysaccharide and, like COX-2 induction, repressed by combined inhibition of the MAPK and of the SAPK2/p38 cascades. Taken together, these data identify CREB, NF-B, and both C/EBP␤ and -␦ as key factors in coordinately orchestrating transcription from the COX-2 promoter in activated macrophages.

The transcription factor C/EBPβ is believed to play a fundamental role in regulating activated macrophage functions. However, the molecular mechanisms and the target genes involved have been, so far, poorly characterized, partly due to the difficulty of reproducibly obtaining homogeneous and abundant primary macrophage populations. In this study, we describe the generation and characterization of immortalized macrophage-like cell lines from C/EBPβ-deficient and wild-type mice. Using these cells, we were able to identify a number of genes involved in activated macrophage functions whose induction was affected in the C/EBPβ−/− cells. IFN-γ/LPS-dependent induction of IL-6, IL-1β, TNF-α, inducible NO synthase, and plasminogen activator inhibitor-1 mRNAs was variably impaired, while IL-12 p40, RANTES and macrophage inflammatory protein-1β mRNAs were up-regulated in the absence of C/EBPβ. The differential mRNA expression correlated with differential transcription levels of the corresponding genes, and was in most cases confirmed in primary macrophage populations. Moreover, in sharp contrast to the enhanced induction of IL-12 p40 mRNA, C/EBPβ−/− primary macrophages derived from both the bone marrow and the peritoneal cavity displayed totally defective expression of IL-12 p35 mRNA. Therefore, the IL-12 p35 gene represents a novel obligatory target for C/EBPβ in macrophages and this may explain the defective production of bioactive IL-12 and the impaired Th1 responses of C/EBPβ-deficient mice to Candida albicans infection observed in previous work.

Cyclooxygenase-2 (COX-2) is the rate-limiting enzyme for the inducible synthesis of prostaglandins, and its up-regulated activity is thought to play a pathological role in diseases such as inflammatory bowel disease, rheumatoid arthritis, and cancer. Regulation of COX-2 expression is complex and appears to involve diversified mechanisms in different cell types and conditions. Here we make use of immortalized macrophages and fibroblasts that we have generated from C/EBP␤-deficient mice to directly test and compare the specific role played by this factor in inducible COX-2 expression in these two cell types. We could demonstrate that COX-2 mRNA induction and promoter activity were profoundly impaired in C/EBP␤ ؊/؊ macrophages and could be rescued by expression of C/EBP␤. The obligatory role of C/EBP␤ in COX-2 expression appeared to be mediated exclusively by the C/EBP element located at positions ؊138/؊130 of the murine cox-2 promoter, and did not involve altered activity at the level of the other promoter elements described previously (the ؊402/؊392 NF-B site, the ؊59/؊48 CRE/E box element, and a potential second C/EBP site located at positions ؊93/؊85). In contrast, COX-2 induction was completely normal in C/EBP␤-deficient fibroblasts, thus highlighting the diversity of cell-specific molecular mechanisms in determining inducible COX-2 expression and prostaglandins production.

Metazoan replication-dependent histone mRNAs do not have a poly(A) tail but end instead in a conserved stem-loop structure. Efficient translation of these mRNAs is dependent on the stem-loop binding protein (SLBP). Here we explore the mechanism by which SLBP stimulates translation in vertebrate cells, using the tethered function assay and analyzing protein-protein interactions. We show for the first time that translational stimulation by SLBP increases during oocyte maturation and that SLBP stimulates translation at the level of initiation. We demonstrate that SLBP can interact directly with subunit h of eIF3 and with Paip1; however, neither of these interactions is sufficient to mediate its effects on translation. We find that Xenopus SLBP1 functions primarily at an early stage in the cap-dependent initiation pathway, targeting small ribosomal subunit recruitment. Analysis of IRES-driven translation in Xenopus oocytes suggests that SLBP activity requires eIF4E. We propose a model in which a novel factor contacts eIF4E bound to the 5 0 cap and SLBP bound to the 3 0 end simultaneously, mediating formation of an alternative end-to-end complex.