P2Y receptors are G-protein-coupled receptors activated by extracellular nucleotides. The P2Y 6 receptor is selectively activated by UDP, and its transcript has been detected in numerous organs, including the spleen, thymus, intestine, blood leukocytes, and aorta. To investigate the biological functions of this receptor, we generated P2Y 6 -null mice by gene targeting. The P2Y 6 knockout (KO) mice are viable and are not distinguishable from the wild-type (WT) mice in terms of growth or fertility. In thioglycollate-elicited macrophages, the production of inositol phosphate in response to UDP stimulation was lost, indicating that P2Y 6 is the unique UDP-responsive receptor expressed by mouse macrophages. Furthermore, the amount of interleukin-6 and macrophage-inflammatory protein-2, but not tumor necrosis factor-␣, released in response to lipopolysaccharide stimulation was significantly enhanced in the presence of UDP, and this effect was lost in the P2Y 6 KO macrophages. The endothelium-dependent relaxation of the aorta by UDP was abolished in KO P2Y 6 mice. The contractile effect of UDP on the aorta, observed when endothelial nitric-oxide synthase is blocked, was also abolished in P2Y 6 -null mice. In conclusion, we generated P2Y 6 -deficient mice and have shown that these mice have a defective response to UDP in macrophages, endothelial cells, and vascular smooth muscle cells. These observations might be relevant to several physiopathological conditions such as atherosclerosis or hypertension.

The oppositely-imprinted genes insulin-like growth factor-II (IGF2) and H19, a putative tumor suppressor, often show coordinate, reciprocal regulation and are believed to play a role in carcinogenesis. To explore the possible interactions between these genes, we stably transfected diHepG2 cells with a plasmid containing either the sense or the antisense H19 cDNA sequences and verified their expression by Northern analysis and by RNase protection analysis. Levels of H19, IGF2 and g-actin mRNA were quantified by competitive RT-PCR analysis. Although H19 sense transgene overexpression (n 24 clones) did not decrease the low, basal levels of IGF2 mRNA compared to control cells, levels of IGF2 mRNA were positively correlated with the levels of H19 antisense mRNA (P , 0.0001, n 40 clones). Furthermore, the increase in IGF2 mRNA level was accompanied by an elevation of IGF-II peptide in conditioned media. To see if H19 mRNA had a specific effect on transcription, we also performed transient transfections with reporter gene constructs containing IGF2 promoter 3 in the presence of sense or antisense H19 cDNA sequences under control of a cytomegalovirus promoter. We show a lower reporter gene activity from reporter gene constructs in the presence of sense H19 cDNA than from those with antisense or neomycin. Our results suggest that H19 participates in the repression of IGF2, at least in part through effects on IGF2 transcription, an effect which may contribute to its action as a tumor suppressor.Keywords: insulin-like growth factor-II; gene regulation; H19; tumor suppressor genes.Human insulin-like growth factor (IGF-II) is an important mitogen which plays a role in normal fetal and postnatal growth, and also in tumorogenesis [1]. Regulation of the human IGF2 gene is very complex, both at the transcriptional and posttranscriptional level. Firstly, this gene is directed by four promoters which exhibit a tissue and development-specific expression pattern. Secondly, it includes sequence elements that are recognized by transcription factors (such as p53, WT-1 or C/EBPa), some of which are known to have tumor suppressor activity [2±4]. Thirdly, the minisatellite DNA polymorphism consisting of a variable number of tandem repeats (VNTR) at the human INS (insulin gene) 5 H -flanking region, has recently been shown to affect IGF2 transcription [5]. Finally, IGF-II mRNAs are subjected to differential polyadenylation, alternative splicing and site-specific endonucleolytic cleavage [4].IGF2 is also epigenetically regulated [6], so that only the paternal allele is expressed in most tissues prenatally and in some tissues during postnatal life, a phenomenon referred to as genomic imprinting. Preferential loss of maternal alleles (loss of heterozygosity) and relaxation of parental imprinting (loss of imprinting) of IGF2 occur frequently in certain pediatric tumors, such as Wilms' tumors, adrenocortical carcinomas, hepatoblastomas and rhabdomyosarcomas, and in malignant adulthood tumors (including uterine, lung and testicular tumors), sugg...