Emmanuelle Nicolas

The Dorsoventral Regulatory Gene Cassette spätzle/Toll/cactus Controls the Potent Antifungal Response in Drosophila Adults

The cytokine-induced activation cascade of NF-kappaB in mammals and the activation of the morphogen dorsal in Drosophila embryos show striking structural and functional similarities (Toll/IL-1, Cactus/I-kappaB, and dorsal/NF-kappaB). Here we demonstrate that these parallels extend to the immune response of Drosophila. In particular, the intracellular components of the dorsoventral signaling pathway (except for dorsal) and the extracellular Toll ligand, spätzle, control expression of the antifungal peptide gene drosomycin in adults. We also show that mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. Antibacterial genes are induced either by a distinct pathway involving the immune deficiency gene (imd) or by combined activation of both imd and dorsoventral pathways.

miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1.

These studies show that miR-122, a 22-nucleotide microRNA, is derived from a liver-specificnon-coding polyadenylated RNA transcribed from the gene hcr. The exact sequence of miR-122as well as the adjacent secondary structure within the hcr mRNA are conserved from mammalianspecies back to fish. Levels of miR-122 in the mouse liver increase to half maximal valuesaround day 17 of embryogenesis, and reach near maximal levels of 50,000 copies per averagecell before birth. Lewis et al (2003) predicted the cationic amino acid transporter (CAT-1 orSLC7A1) as a miR-122 target. CAT-1 protein and its mRNA are expressed in all mammaliantissues but with lower levels in adult liver. Furthermore, during mouse liver development CAT-1mRNA decreases in an almost inverse correlation with miR-122. Eight potential miR-122 targetsites were predicted within the human CAT-1 mRNA, with six in the 3’-untranslated region.Using a reporter construct it was found that just three of the predicted sites, linked in a 400-nucleotide sequence from human CAT-1, acted with synergy and were sufficient to stronglyinhibit protein synthesis and reduce mRNA levels. In summary, these studies followed theaccumulation during development of miR-122 from its mRNA precursor, hcr, through toidentification of what may be a specific mRNA target, CAT-1. Link to supplemental material: http://www.landesbioscience.com/supplement/changRNA1-2-sup.pdf

The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment

Development of immature T-cell precursors (thymocytes) to either the CD4 helper or CD8 killer T-cell lineages correlates precisely with their T-cell receptor specificity for major histocompatibility complex class II or class I molecules, respectively, indicating that the process is carefully regulated. Although intensively studied owing to its importance in determining the composition of the mature T-cell compartment and as a general model of binary lineage decisions, the underlying molecular pathways remain obscure. We have previously reported a spontaneous mouse mutant (HD (helper deficient) mice) in which lineage commitment is specifically perturbed without affecting positive selection. Here we show that a point mutation in the zinc finger transcription factor Th-POK (T-helper-inducing POZ/Krüppel-like factor) is responsible for redirection of class-II-restricted thymocytes to the CD8 lineage in HD mice. Furthermore, we demonstrate that constitutive expression of this factor during thymic development leads to redirection of class-I-restricted thymocytes to the CD4 lineage, indicating that Th-POK is a master regulator of lineage commitment.

The role of the Birt–Hogg–Dubé protein in mTOR activation and renal tumorigenesis

Birt–Hogg–Dubé (BHD) syndrome is a tumor-suppressor gene disorder characterized by skin tumors, cystic lung disease and renal cell carcinoma. Very little is known about the molecular pathogenesis of BHD. Clinical similarities between BHD and tuberous sclerosis complex (TSC) suggest that the BHD and TSC proteins may function within a common pathway. The TSC proteins inhibit the activity of the mammalian target of rapamycin complex 1 (TORC1), and in Schizosaccharomyces pombe, Bhd and Tsc1/Tsc2 have opposing roles in the regulation of amino-acid homeostasis. We report here that in mammalian cells, downregulation of BHD reduces the phosphorylation of ribosomal protein S6, an indicator of TORC1 activity. To determine whether folliculin, the product of the BHD gene, regulates mammalian target of rapamycin activity in vivo, we generated a mouse with targeted inactivation of the Bhd gene. The mice developed spontaneous oncocytic cysts and tumors composed of cells that resemble the renal cell carcinomas in BHD patients. The cysts and tumors had low levels of phospho-S6. Taken together, these data indicate that folliculin regulates the activity of TORC1, and suggest a new paradigm in which both inappropriately high and inappropriately low levels of TORC1 activity can be associated with renal tumorigenesis.

Estrogen promotes the survival and pulmonary metastasis of tuberin-null cells

Lymphangioleiomyomatosis (LAM) is an often fatal disease primarily affecting young women in which tuberin (TSC2)-null cells metastasize to the lungs. The mechanisms underlying the striking female predominance of LAM are unknown. We report here that 17-β-estradiol (E2) causes a 3- to 5-fold increase in pulmonary metastases in male and female mice, respectively, and a striking increase in circulating tumor cells in mice bearing tuberin-null xenograft tumors. E2-induced metastasis is associated with activation of p42/44 MAPK and is completely inhibited by treatment with the MEK1/2 inhibitor, CI-1040. In vitro, E2 inhibits anoikis of tuberin-null cells. Finally, using a bioluminescence approach, we found that E2 enhances the survival and lung colonization of intravenously injected tuberin-null cells by 3-fold, which is blocked by treatment with CI-1040. Taken together these results reveal a new model for LAM pathogenesis in which activation of MEK-dependent pathways by E2 leads to pulmonary metastasis via enhanced survival of detached tuberin-null cells.

Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time

In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17β-estradiol (E2) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E2 paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E2-induced apoptosis by analysis of gene expression across time (2–96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E2-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E2 in 5C cells compared with both WS8 and 2A cells and hence, were associated with E2-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E2 inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E2-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E2-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E2 interacted to superadditively induce apoptosis. Therefore, these data indicate that E2 induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.

Histone modifications silence the GATA transcription factor genes in ovarian cancer

Altered expression of GATA factors was found and proposed as the underlying mechanism for dedifferentiation in ovarian carcinogenesis. In particular, GATA6 is lost or excluded from the nucleus in 85% of ovarian tumors and GATA4 expression is absent in majority of ovarian cancer cell lines. Here, we evaluated their DNA and histone epigenetic modifications in five ovarian epithelial and carcinoma cell lines (human ‘immortalized’ ovarian surface epithelium (HIO)-117, HIO-114, A2780, SKOV3 and ES2). GATA4 and GATA6 gene silencing was found to correlate with hypoacetylation of histones H3 and H4 and loss of histone H3/lysine K4 tri-methylation at their promoters in all lines. Conversely, histone H3/lysine K9 di-methylation and HP1γ association were not observed, excluding reorganization of GATA genes into heterochromatic structures. The histone deacetylase inhibitor trichostatin A, but not the DNA methylation inhibitor 5′-aza-2′-deoxycytidine, re-established the expression of GATA4 and/or GATA6 in A2780 and HIO-114 cells, correlating with increased histone H3 and H4 acetylation, histone H3 lysine K4 methylation and DNase I sensitivity at the promoters. Therefore, altered histone modification of the promoter loci is one mechanism responsible for the silencing of GATA transcription factors and the subsequent loss of a target gene, the tumor suppressor Disabled-2, in ovarian carcinogenesis.

In vivo regulation of the IkappaB homologue cactus during the immune response of Drosophila

The dorsoventral regulatory gene pathway (spätzle/Toll/cactus) controls the expression of several antimicrobial genes during the immune response of Drosophila. This regulatory cascade shows striking similarities with the cytokine-induced activation cascade of NF-κB during the inflammatory response in mammals. Here, we have studied the regulation of the IκB homologue Cactus in the fat body during the immune response. We observe that the cactusgene is up-regulated in response to immune challenge. Interestingly, the expression of the cactus gene is controlled by thespätzle/Toll/cactus gene pathway, indicating that the cactus gene is autoregulated. We also show that two Cactus isoforms are expressed in the cytoplasm of fat body cells and that they are rapidly degraded and resynthesized after immune challenge. This degradation is also dependent on the Toll signaling pathway. Altogether, our results underline the striking similarities between the regulation of IκB and cactusduring the immune response.

Enzymatic mutation detection technologies.

Mutation is as necessary for life as fidelity is in DNA replication. The study of mutations reveals the normal functions of genes, messages, proteins, the causes of many diseases, and the variability of responses among individuals. Indeed, recent mutations that have not yet become polymorphisms are often deleterious and pertinent to the disease history of afflicted individuals. This review discusses the principles behind a variety of methods for the detection of mutations and factors that should be considered in future methods design. One enzymatic approach in particular using orthologs of the CEL I nuclease that show high specificity for all mismatches, appears to be easy and robust. Further developments of this and other methods will allow mutation detection to become an integral component of individualized medicine.

The Birt-Hogg-Dube and Tuberous Sclerosis Complex Homologs Have Opposing Roles in Amino Acid Homeostasis in Schizosaccharomyces pombe

Birt-Hogg-Dube (BHD) is a tumor suppressor gene disorder characterized by skin hamartomas, cystic lung disease, and renal cell carcinoma. The fact that hamartomas, lung cysts, and renal cell carcinoma can also occur in tuberous sclerosis complex (TSC) suggests that the BHD and TSC proteins may function within a common pathway. To evaluate this hypothesis, we deleted the BHD homolog in Schizosaccharomyces pombe. Expression profiling revealed that six permease and transporter genes, known to be down-regulated in Δtsc1 and Δtsc2, were up-regulated in Δbhd, and levels of specific intracellular amino acids known to be low in Δtsc1 and Δtsc2 were elevated in Δbhd. This “opposite” profile was unexpected, given the overlapping clinical phenotypes. The TSC1/2 proteins inhibit Rheb in mammals, and Tsc1/Tsc2 inhibit Rhb1 in S. pombe. Expression of a hypomorphic allele of rhb1+ dramatically increased permease expression levels in Δbhd but not in wild-type yeast. Loss of Bhd sensitized yeast to rapamycin-induced increases in permease expression levels, and rapamycin induced lethality in Δbhd yeast expressing the hypomorphic Rhb1 allele. In S. pombe, it is known that Rhb1 binds Tor2, and Tor2 inhibition leads to up-regulation of permeases including those that are regulated by Bhd. Our data, therefore, suggest that Bhd activates Tor2. If the mammalian BHD protein, folliculin, similarly activates mammalian target of rapamycin, it will be of great interest to determine how mammalian target of rapamycin inhibition in BHD patients and mammalian target of rapamycin activation in TSC patients lead to overlapping clinical phenotypes.