Françoise Wilkin

The P2Y11 Receptor Mediates the ATP-Induced Maturation of Human Monocyte-Derived Dendritic Cells

Recently, it has been shown that ATP and TNF-α synergize in the activation and maturation of human dendritic cells (DC); the effect of ATP was reproduced by hydrolysis-resistant derivatives of ATP and was blocked by suramin, suggesting the involvement of a P2 receptor, but the particular subtype involved was not identified. In this report we confirm that ATP and various derivatives synergize with TNF-α and LPS to induce the maturation of human monocyte-derived DC, as revealed by up-regulation of the CD83 marker and the secretion of IL-12. The rank order of potency of various analogs (AR-C67085 > adenosine 5′-O-(3-thiotriphosphate) = 2′- and 3′-O-(4-benzoyl-benzoyl) ATP > ATP > 2-methylthio-ATP) was close to that of the recombinant human P2Y11 receptor. Furthermore, these compounds activated cAMP production in DC, in a xanthine-insensitive way, consistent with the involvement of the P2Y11 receptor, which among P2Y subtypes has the unique feature of being dually coupled to phospholipase C and adenylyl cyclase activation. The involvement of the P2Y11/cAMP/protein kinase A signaling pathway in the nucleotide-induced maturation of DC is supported by the inhibitory effect of H89, a protein kinase A inhibitor. Taken together, our results demonstrate that ATP activates DC through stimulation of the P2Y11 receptor and subsequent increase in intracellular cAMP.

Extracellular adenine nucleotides modulate cytokine production by human monocyte‐derived dendritic cells: dual effect on IL‐12 and stimulation of IL‐10

To clarify the functional consequences of adenine nucleotides action on human monocyte‐derived dendritic cells (DC), we have systematically compared the effects of adenosine 5′‐O‐(3‐thiotriphosphate) (ATPγS), an ATP analog active on the P2Y11 receptor, on the responses to three DC stimuli, TNF‐α, LPS, sCD40L, tested at various concentrations, using two different IL‐12 assays. We observed that ATPγS potentiated the IL‐12p40 release induced by TNF‐α, but also by lipopolysaccharides (LPS) and soluble CD40 ligand (sCD40L). This potentiation was observed as long as the IL‐12p40 concentration under agonist stimulation remained below a threshold value close to 10 ng/ml; inhibition was observed above this value. The combinations ATPγS‐TNF‐α and ATPγS‐sCD40L were unable to induce detectable bioactive IL‐12p70 production and at concentrations of LPS that induced a significant stimulation of IL‐12p70, the effect of ATPγS was purely inhibitory. Our results also show that ATPγS synergized with LPS and sCD40L, but not TNF‐α, to stimulate IL‐10 production. In conclusion, we have clarified the discrepancies in the literature concerningthe action of adenine nucleotides on DC and our study supports the concept that, like prostaglandin E2 and other agents increasing cyclic AMP, they favor either a Th2 response or tolerance.

Profiling of olfactory receptor gene expression in whole human olfactory mucosa.

Olfactory perception is mediated by a large array of olfactory receptor genes. The human genome contains 851 olfactory receptor gene loci. More than 50% of the loci are annotated as nonfunctional due to frame-disrupting mutations. Furthermore haplotypic missense alleles can be nonfunctional resulting from substitution of key amino acids governing protein folding or interactions with signal transduction components. Beyond their role in odor recognition, functional olfactory receptors are also required for a proper targeting of olfactory neuron axons to their corresponding glomeruli in the olfactory bulb. Therefore, we anticipate that profiling of olfactory receptor gene expression in whole human olfactory mucosa and analysis in the human population of their expression should provide an opportunity to select the frequently expressed and potentially functional olfactory receptors in view of a systematic deorphanization. To address this issue, we designed a TaqMan Low Density Array (Applied Biosystems), containing probes for 356 predicted human olfactory receptor loci to investigate their expression in whole human olfactory mucosa tissues from 26 individuals (13 women, 13 men; aged from 39 to 81 years, with an average of 67±11 years for women and 63±12 years for men). Total RNA isolation, DNase treatment, RNA integrity evaluation and reverse transcription were performed for these 26 samples. Then 384 targeted genes (including endogenous control genes and reference genes specifically expressed in olfactory epithelium for normalization purpose) were analyzed using the same real-time reverse transcription PCR platform. On average, the expression of 273 human olfactory receptor genes was observed in the 26 selected whole human olfactory mucosa analyzed, of which 90 were expressed in all 26 individuals. Most of the olfactory receptors deorphanized to date on the basis of sensitivity to known odorant molecules, which are described in the literature, were found in the expressed olfactory receptors gene set.

Identification and Characterization of Novel Genes Modulated in the Thyroid of Dogs Treated with Methimazole and Propylthiouracil

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific stimulation or repression of a large number of genes. To better understand differentiated epithelial cell growth regulation, we have initiated a study to identify genes which are regulated by the thyrotropin-dependent mitogenic pathway in dog thyroid cells. A thyroid cDNA library was prepared from a methimazole and propylthiouracil-treated dog and differentially screened with probes derived from control or stimulated thyroids. Among 19 clones isolated, 6 encode known proteins (inwardly rectifying potassium channel, nucleosome assembly protein, ribosomal protein L7, elongation factor 1α, non-muscle myosin light chain, and heat shock protein 90β). The 13 others correspond to proteins whose function is unknown. Among them, 5 correspond to mRNAs whose expression was modulated by mitogenic stimulation of thyrocytes in primary culture. A preliminary characterization of two of these cDNAs is reported: clone 5, which might represent a novel, atypical protein kinase, and clone 3, which contains ankyrin-like repeats, suggesting that it might interact with other proteins.

Deorphanization and characterization of human olfactory receptors in heterologous cells.

Olfaction plays an indispensable role in human and animals in self and environmental recognition, as well as intra‐ and interspecific communication. Following the discovery of a family of olfactory receptors (ORs) by Buck and Axel in 1991, it has been established that the sense of smell begins with the molecular recognition of a chemical odorant by one or more ORs expressed in the olfactory sensory neurons. Therefore, characterization of the molecular interactions between odorant molecules and ORs is a key step in the elucidation of the general properties of the olfactory system and in the development of applications, i.e., design of new odorants, search for blockers, etc. The process putted in place at ChemCom to improve the expression of ORs at the cytoplasmic membrane of the HEK293 cell and assays enabling large‐scale deorphanization, and to characterize the interaction between chemical odorants and ORs is described. The family of human ORs includes ca. 400 putatively functional ORs which are GPCRs (G protein‐coupled receptors); to date over 100 human ORs have been deorphanized.

Cloning of cDNA Specifically Involved in the Thyroid cAMP Mitogenic Pathway

The activation of the cyclic AMP cascade in dog and human thyroid cells in primary culture induces the expression of differentiated gene expression, hyperfunction and proliferation. These programs are developed simultaneously in quiescent dedifferentiated cells. In this paper the strategy followed by our group to define the genes involved in the cAMP mitogenic cascade is outlined.