Role of Protein Tyrosine Phosphatase Epsilon (PTPε) in Leukotriene D4-Induced CXCL8 Expression.

Phosphorylation on tyrosine residues is recognized as an important mechanism for connecting extracellular stimuli to cellular events and defines a variety of physiologic responses downstream of G protein-coupled receptor (GPCR) activation. To date, few protein tyrosine phosphatases (PTPs) have been shown to associate with GPCRs, and little is known about their role in GPCR signaling. To discover potential cysteinyl-leukotriene receptor (CysLT1R)-interacting proteins, we identified protein tyrosine phosphatase ε (PTPε) in a yeast two-hybrid assay. Since both proteins are closely linked to asthma, we further investigated their association. Using a human embryonic kidney cell line 293 (HEK-293) cell line stably transfected with the receptor (HEK-LT1), as well as human primary monocytes, we found that PTPε colocalized with CysLT1R in both resting and leukotriene D4 (LTD4)-stimulated cells. Cotransfection of HEK-LT1 with PTPε had no effect on CysLT1R expression or LTD4-induced internalization, but it inhibited LTD4-induced CXC chemokine 8 (CXCL8) promoter transactivation, protein expression, and secretion. Moreover, reduced phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), but not of p38 or c-Jun-N-terminal kinase 1 or 2 mitogen-activated protein kinases (MAPKs), was observed upon LTD4 stimulation of HEK-LT1 coexpressing cytosolic (cyt-) PTPε, but not receptor (R) PTPε The increased interaction of cyt-PTPε and ERK1/2 after LTD4 stimulation was shown by coimmunoprecipitation. In addition, enhanced ERK1/2 phosphorylation and CXCL8 secretion were found in LTD4-stimulated human monocytes transfected with PTPε-specific siRNAs, adding support to a regulatory/inhibitory role of PTPε in CysLT1R signaling. Given that the prevalence of severe asthma is increasing, the identification of PTPε as a new potential therapeutic target may be of interest.

Differential signaling defects associated with the M201V polymorphism in the cysteinyl leukotriene type 2 receptor.

The cysteinyl-leukotrienes (cysLTs) LTC(4), LTD(4), and LTE(4), are involved in a variety of inflammatory diseases, including asthma, and act on at least two distinct receptors, CysLT(1) and CysLT(2). Specific antagonists of CysLT(1) are currently used to control bronchoconstriction and inflammation in asthmatic patients. The potential role of CysLT(2) in asthma remains poorly understood. A polymorphism in the CysLT(2) gene, resulting in a single amino acid substitution (M201V), was found to be associated with asthma in three separate population studies. Here, we investigated whether the M201V mutation affected the affinity of CysLT(2) for its natural ligands and its signaling efficiency. Human embryonic kidney 293 cells were stably transfected with either wild-type (wt) or mutant (M201V) CysLT(2). Affinity of the M201V receptor for LTC(4) was reduced by 50%, whereas affinity for LTD(4) was essentially lost. LTC(4)-induced production of inositol phosphates (IPs) in M201V-expressing cells was significantly decreased at suboptimal concentrations of the ligand, but no difference was observed at high concentrations. In contrast, LTD(4)-induced IP production was 10- to 100-fold less in M201V- than in wt-expressing cells. Similar results were also observed with the transactivation of the interleukin-8 promoter induced by LTC(4) or LTD(4). Moreover, in contrast to wt-expressing cells, phosphorylation of nuclear factor κB p65 was absent in LTD(4)-stimulated M201V-expressing cells. Likewise, phosphorylation of c-Jun N-terminal kinase was not induced in LTD(4)-stimulated M201V cells, whereas activation of extracellular response kinase and p38 was maintained, at least at higher LTD(4) concentrations. Our results indicate that the M201V polymorphism drastically affects CysLT(2) responses to LTD(4) and less to LTC(4).

Caveolae facilitate but are not essential for platelet-activating factor-mediated calcium mobilization and extracellular signal-regulated kinase activation.

Certain proteins, including receptors and signaling molecules, are known to be enriched in caveolae and lipid rafts. Caveolin-1, the major structural protein of caveolae, specifically interacts with many signaling molecules and, thus, caveolae and lipid rafts are often seen as preassembled signaling platforms. A potential binding site for caveolin-1 is present in the platelet-activating factor receptor (PAFR) sequence, and many downstream signaling components of PAFR activation preferentially localize in caveolae. The aim of this study was to investigate whether the PAFR was localized in caveolae/lipid raft domains and, if so, what would be the significance of such localization for PAFR signaling. In this study, we demonstrate that PAFR localizes within membrane microdomains, in close proximity to caveolin-1 in living cells, with potential interaction through a caveolin-1-binding sequence in the PAFR C terminus. Caveolin-1, however, is not essential for PAFR localization in lipid rafts. Disruption of caveolae/lipid rafts with methyl-beta-cyclodextrin markedly reduced PAF-triggered inositol phosphate production and cytosolic calcium flux, suggesting that PAFR signaling through the Galphaq protein was critically dependent on integrity of lipid rafts and/or caveolae. Interestingly, whereas in caveolin-1-expressing cells lipid raft disruption markedly decreased PAFR-mediated activation of the ERK/MAPK pathway, in cells lacking caveolae, such as leukocytes, lipid raft disruption had either the same inhibitory effect (Ramos B cells) or no effect (monocytes) on PAFR capacity to signal through the ERK/MAPK pathway. In conclusion, PAFR appears to localize within caveolae or lipid rafts in different cell types, and this location may be important for specific signaling events.

Regulation of platelet-activating factor-mediated interleukin-6 promoter activation by the 48 kDa but not the 45 kDa isoform of protein tyrosine phosphatase non-receptor type 2.

BACKGROUND: An underlying state of inflammation is thought to be an important cause of cardiovascular disease. Among cells involved in the early steps of atherosclerosis, monocyte-derived dendritic cells (Mo-DCs) respond to inflammatory stimuli, including platelet-activating factor (PAF), by the induction of various cytokines, such as interleukin 6 (IL-6). PAF is a potent phospholipid mediator involved in both the onset and progression of atherosclerosis. It mediates its effects by binding to its cognate G-protein coupled receptor, PAFR. Activation of PAFR-induced signaling pathways is tightly coordinated to ensure specific cell responses. RESULTS: Here, we report that PAF stimulated the phosphatase activity of both the 45 and 48 kDa isoforms of the protein tyrosine phosphatase non-receptor type 2 (PTPN2). However, we found that only the 48 kDa PTPN2 isoform has a role in PAFR-induced signal transduction, leading to activation of the IL-6 promoter. In luciferase reporter assays, expression of the 48 kDa, but not the 45 kDa, PTPN2 isoform increased human IL-6 (hIL-6) promoter activity by 40% after PAF stimulation of HEK-293 cells, stably transfected with PAFR (HEK-PAFR). Our results suggest that the differential localization of the PTPN2 isoforms and the differences in PAF-induced phosphatase activation may contribute to the divergent modulation of PAF-induced IL-6 promoter activation. The involvement of PTPN2 in PAF-induced IL-6 expression was confirmed in immature Mo-DCs (iMo-DCs), using siRNAs targeting the two isoforms of PTPN2, where siRNAs against the 48 kDa PTPN2 significantly inhibited PAF-stimulated IL-6 mRNA expression. Pharmacological inhibition of several signaling pathways suggested a role for PTPN2 in early signaling events. Results obtained by Western blot confirmed that PTPN2 increased the activation of the PI3K/Akt pathway via the modulation of protein kinase D (PKD) activity. WT PKD expression counteracted the effect of PTPN2 on PAF-induced IL-6 promoter transactivation and phosphorylation of Akt. Using siRNAs targeting the individual isoforms of PTPN2, we confirmed that these pathways were also active in iMo-DCs. CONCLUSION: Taken together, our data suggest that PTPN2, in an isoform-specific manner, could be involved in the positive regulation of PI3K/Akt activation, via the modulation of PKD activity, allowing for the maximal induction of PAF-stimulated IL-6 mRNA expression.

Cellular signalling of cysteinyl leukotriene type 1 receptor variants CysLT1-G300S and CysLT1-I206S.

Cysteinyl-leukotrienes are pro-inflammatory lipid mediators, involved in allergic asthma, that bind the G-protein-coupled receptors CysLT1, CysLT2 and GPR99. A polymorphism in one of these receptors, CysLT1-G300S was strongly associated with atopy, whereas the CysLT1-I206S polymorphism was not. In the present work, our aim was to characterize these two variants by studying their cellular signalling. Cell surface expression of mutant receptors in transfected HEK-293 cells was comparable to that of the wild-type receptor. Compared to CysLT1-WT, production of inositol phosphates as well as IL-8 and IL-13 promoter transactivation in response to either LTD4 or LTC4 was significantly increased in CysLT1-G300S-transfected cells. Moreover, LTD4-induced phosphorylation of the signalling effector Erk, but not p38, p65 or c-Jun was higher in CysLT1-G300S-transfected cells. On the other hand, the variant CysLT1-I206S did not show a significant difference in its signal transduction compared to the wild-type receptor. Taken together, our results indicate that the variant CysLT1-G300S can induce a greater signal than the CysLT1-WT receptor, a feature that may be relevant to its association with atopy.

Mechanistic insight into WEB-2170-induced apoptosis in human acute myelogenous leukemia cells: the crucial role of PTEN.

OBJECTIVE: This study aimed to investigate the mechanisms of action of WEB-2170, an inverse agonist of platelet-activating factor receptor, capable of inducing apoptosis in human acute myelogenous leukemia (AML) cells. MATERIAL AND METHODS: Gene expression profiling followed by cytofluorimetric, morphologic, and biologic analyses were used to monitor WEB-2170 effects in AML cell lines (ie, NB4, KG1, NB4-MR4, THP1, and U937) and blasts from patients with different AML (M0-M5) subtypes. PTEN silencing with small interfering RNA was also performed. RESULTS: We have demonstrated that drug-mediated cytostasis/apoptosis in NB4 cells is characterized by upregulation of cyclin G2, p21/WAF1, NIX, TNF-alpha, and PTEN expression, and downregulation of cyclin D2 and BCL2 expression. We observed an increase in PTEN protein accompanied by a decrease in phospho-extracellular signal-regulated kinase 2 (ERK2) and phospho-AKT, and by forkhead box O3a (FOXO3a) cytoplasmic-nuclear translocation; the mitochondrial cytochrome C release and PARP cleavage marked the late apoptotic steps. We have found that WEB-2170 triggered apoptosis in NB4, KG1, and NB4-MR4 cells where PTEN was expressed, but not in THP1 and U937 cells where PTEN was absent. Finally, we show that PTEN silencing in NB4 cells by PTEN-specific small interfering RNA resulted in a significant reduction of drug-induced apoptosis. CONCLUSION: We demonstrated that WEB-2170 is a powerful antileukemic agent with interesting translational opportunities to treat AML and described mechanisms of drug-induced intrinsic and extrinsic apoptosis both in AML cell lines and blasts from AML patients by addressing PTEN as the master regulator of the whole process.

Deletion of many yeast introns reveals a minority of genes that require splicing for function.

Splicing regulates gene expression and contributes to proteomic diversity in higher eukaryotes. However, in yeast only 283 of the 6000 genes contain introns and their impact on cell function is not clear. To assess the contribution of introns to cell function, we initiated large-scale intron deletions in yeast with the ultimate goal of creating an intron-free model eukaryote. We show that about one-third of yeast introns are not essential for growth. Only three intron deletions caused severe growth defects, but normal growth was restored in all cases by expressing the intronless mRNA from a heterologous promoter. Twenty percent of the intron deletions caused minor phenotypes under different growth conditions. Strikingly, the combined deletion of all introns from the 15 cytoskeleton-related genes did not affect growth or strain fitness. Together, our results show that although the presence of introns may optimize gene expression and provide benefit under stress, a majority of introns could be removed with minor consequences on growth under laboratory conditions, supporting the view that many introns could be phased out of Saccharomyces cerevisiae without blocking cell growth.

Large-scale essential gene identification in Candida albicans and applications to antifungal drug discovery.

Candida albicans is the primary fungal pathogen of humans. Despite the need for novel drugs to combat fungal infections [Sobel, J.D. (2000) Clin Infectious Dis 30: 652], antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. A functional genomics approach based on the diploid C. albicans genome sequence, termed GRACETM (gene replacement and conditional expression), was used to assess gene essentiality through a combination of gene replacement and conditional gene expression. In a systematic application of this approach, we identify 567 essential genes in C. albicans. Interestingly, evaluating the conditional phenotype of all identifiable C. albicans homologues of the Saccharomyces cerevisiae essential gene set [Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., et al. (2002) Nature 418: 387-391] by GRACE revealed only 61% to be essential in C. albicans, emphasizing the importance of performing such studies directly within the pathogen. Construction of this conditional mutant strain collection facilitates large-scale examination of terminal phenotypes of essential genes. This information enables preferred drug targets to be selected from the C. albicans essential gene set by phenotypic information derived both in vitro, such as cidal versus static terminal phenotypes, as well as in vivo through virulence studies using conditional strains in an animal model of infection. In addition, the combination of phenotypic and bioinformatic analyses further improves drug target selection from the C. albicans essential gene set, and their respective conditional mutant strains may be directly used as sensitive whole-cell assays for drug screening.

Functional profiling of the Saccharomyces cerevisiae genome.

Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.