The Experts below are selected from a list of 516 Experts worldwide ranked by ideXlab platform
Kathleen E. Sullivan - One of the best experts on this subject based on the ideXlab platform.
-
Noncoding RNAs and LRRFIP1 Regulate TNF Expression
Journal of immunology (Baltimore Md. : 1950), 2014Co-Authors: Lihua Shi, Asen Bagashev, Michael C. Fitzgerald, Kelly Maurer, Li Song, Kathleen E. SullivanAbstract:Noncoding RNAs have been implicated in the regulation of expression of numerous genes; however, the mechanism is not fully understood. We identified bidirectional, long noncoding RNAs upstream of the TNF gene using five different methods. They arose in a region where the repressors LRRFIP1, EZH2, and SUZ12 were demonstrated to bind, suggesting a role in repression. The noncoding RNAs were polyadenylated, capped, and chromatin associated. Knockdown of the noncoding RNAs was associated with derepression of TNF mRNA and diminished binding of LRRFIP1 to both RNA targets and chromatin. Overexpression of the noncoding RNAs led to diminished expression of TNF and recruitment of repressor proteins to the locus. One repressor protein, LRRFIP1, bound directly to the noncoding RNAs. These data place the noncoding RNAs upstream of TNF gene as central to the transcriptional regulation. They appear to serve as a platform for the assembly of a repressive complex.
-
Leucine-rich repeat (in Flightless I) interacting protein-1 regulates a rapid type I interferon response.
Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, 2010Co-Authors: Asen Bagashev, Michael C. Fitzgerald, David F. Larosa, Patrick P. Rose, Sara Cherry, Alfred C. Johnson, Kathleen E. SullivanAbstract:The cell autonomous response to viral infection is carefully regulated to induce type I interferons (IFNs), which in turn induce the establishment of an antiviral state. Leucine-rich repeat (in Flightless I) interacting protein-1 (LRRFIP1) and LRRFIP2 are 2 related proteins that have been identified as interacting with MyD88 and Flightless I homolog, a leucine-rich repeat protein. LRRFIP2 positively regulates NFκB and macrophage cytokine production after lipopolysaccharide, but less is known about LRRFIP1. We hypothesized that LRRFIP1 could be more important in antiviral responses, as overexpression led to type I IFN production in a pilot study. The induction of type I IFNs occurred even in the absence of virus, but was enhanced by the presence of virus. Conversely, knockdown of LRRFIP1 compromised IFN expression. We found that LRRFIP1 was rapidly recruited to influenza-containing early endosomes in a p38-dependent fashion. This was specific for virus-containing endosomes as there was almost no colocalization of LRRFIP1 with early endosomes in the absence of virus. Further, LRRFIP1 was recruited to RNA-containing vesicles. Taken together, these data suggest that LRRFIP1 participates in cell responses to virus at early time points and is important for type I IFN induction.
-
Characterization of LRRFIP1.
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2010Co-Authors: Rachel Arakawar. Arakawa, Asen Bagashev, Li Songl. Song, Kelly Maurer, Kathleen E. SullivanAbstract:LRRFIP1 has been identified as a regulator of toll-like receptor (TLR) pathway signaling; however, little is known about its own regulation and function. This study was undertaken to characterize the biochemical properties and its regulation. Over-expression of full length LRRFIP1 led to enhanced responses to lipopolysaccharide (LPS). We examined its expression in monocytic cell lines because they express a broad range of TLRs. We found that its level of expression was not altered by LPS or phorbol myristate acetate (PMA) but that it was up-regulated by nicotine, influenza infection, and serum starvation. Phosphorylation was examined because of the bioinformatically predicted serine phosphorylation sites. Serine phosphorylation was detected and was altered by both poly I:C and nicotine. Finally, we examined the regulation of intracellular localization in response to dsRNA and found that LRRFIP1 colocalized with labeled dsRNA in monocyte lysosomal structures but not with lysosomes lacking dsRNA. These data suggest that LRRFIP1 is phosphorylated in response to immunologic stimuli and it is directed to lysosomal structures.
-
GCF2/LRRFIP1 represses tumor necrosis factor alpha expression.
Molecular and cellular biology, 2005Co-Authors: April R. Suriano, Amy N. Sanford, Nahmah A. Kim, Sarah Kennedy, Mark J. Henderson, Kelly Dietzmann, Kathleen E. SullivanAbstract:Tumor necrosis factor alpha (TNF-α) is an important mediator of inflammation, apoptosis, and the development of secondary lymphoid structures. Multiple polymorphic microsatellites have been identified in and around the gene, and there are also multiple single-base pair biallelic polymorphisms in the introns and promoter. The TNF-α −308 promoter polymorphism is a G-to-A transition which has been statistically associated with various autoimmune disorders. Some studies have found that it may directly mediate the increased transcription of TNF-α in some circumstances. This study characterizes proteins interacting at the polymorphic promoter site. Affinity purification of binding proteins and confirmatory chromatin immunoprecipitation assays were used to identify the proteins. Electrophoretic mobility shift analyses and surface plasmon resonance were used to define binding characteristics. Proteins interacting at this site include GCF2/LRRFIP1 and Ets-1. GCF2/LRRFIP1 appears to act as a repressor and occupies the −308 site in cells that do not make TNF-α. Cells competent to produce TNF-α have Ets-1 bound to the −308 promoter site. Active transcription is accompanied by NF-κB and c-Jun binding to the proximal promoter. Thus, dynamic changes on the TNF-α promoter, particularly at the −308 site, accompany the transition from repressed to active transcription. GCF2/LRRFIP1 is the first TNF-α repressor identified.
-
gcf2 LRRFIP1 represses tumor necrosis factor alpha expression
Molecular and Cellular Biology, 2005Co-Authors: April R. Suriano, Amy N. Sanford, Sarah Kennedy, Mark J. Henderson, Kelly Dietzmann, Nahmah Kim, Kathleen E. SullivanAbstract:Tumor necrosis factor alpha (TNF-α) is an important mediator of inflammation, apoptosis, and the development of secondary lymphoid structures. Multiple polymorphic microsatellites have been identified in and around the gene, and there are also multiple single-base pair biallelic polymorphisms in the introns and promoter. The TNF-α −308 promoter polymorphism is a G-to-A transition which has been statistically associated with various autoimmune disorders. Some studies have found that it may directly mediate the increased transcription of TNF-α in some circumstances. This study characterizes proteins interacting at the polymorphic promoter site. Affinity purification of binding proteins and confirmatory chromatin immunoprecipitation assays were used to identify the proteins. Electrophoretic mobility shift analyses and surface plasmon resonance were used to define binding characteristics. Proteins interacting at this site include GCF2/LRRFIP1 and Ets-1. GCF2/LRRFIP1 appears to act as a repressor and occupies the −308 site in cells that do not make TNF-α. Cells competent to produce TNF-α have Ets-1 bound to the −308 promoter site. Active transcription is accompanied by NF-κB and c-Jun binding to the proximal promoter. Thus, dynamic changes on the TNF-α promoter, particularly at the −308 site, accompany the transition from repressed to active transcription. GCF2/LRRFIP1 is the first TNF-α repressor identified.
Takeshi Naitoh - One of the best experts on this subject based on the ideXlab platform.
-
silencing of LRRFIP1 reverses the epithelial mesenchymal transition via inhibition of the wnt β catenin signaling pathway
Cancer Letters, 2015Co-Authors: Daisuke Douchi, Hideo Ohtsuka, Kyohei Ariake, Kunihiro Masuda, Shuhei Kawasaki, Kei Kawaguchi, Koji Fukase, Masaya Oikawa, Fuyuhiko Motoi, Takeshi NaitohAbstract:The canonical Wnt/β-catenin signaling pathway has been shown to promote the epithelial-mesenchymal transition (EMT), which is a crucial process in multiple embryonic developmental processes and the progression of carcinomas. We recently provided evidence that leucine-rich repeat flightless-1-interacting protein 1 (LRRFIP1) promotes cancer metastasis and invasion. In the present study, we identified the signaling elements targeted by LRRFIP1 for promotion of the EMT in pancreatic and lung cancer. LRRFIP1 silencing reversed the EMT, as shown by increased expression of E-cadherin (an epithelial marker) and decreased expression of vimentin (a mesenchymal marker). Silencing of LRRFIP1 up-regulated phosphorylation of β-catenin and decreased its nuclear localization by targeting the β-catenin destruction complex. The expression of β-catenin and E-cadherin in the plasma membrane fraction was increased in LRRFIP1 silenced cancer cells, and the migration and invasion capabilities were strongly inhibited. In addition, this protein was highly expressed at the invasion front of malignant tissue collected from pancreatic cancer patients. Consequently, our data strongly suggested that LRRFIP1 played an important role in the invasion of carcinoma cells. Our data provide experimental evidence that LRRFIP1 is an attractive candidate for targeted therapy in human cancers.
-
Silencing of LRRFIP1 reverses the epithelial-mesenchymal transition via inhibition of the Wnt/β-catenin signaling pathway.
Cancer letters, 2015Co-Authors: Daisuke Douchi, Hideo Ohtsuka, Kyohei Ariake, Kunihiro Masuda, Shuhei Kawasaki, Kei Kawaguchi, Koji Fukase, Masaya Oikawa, Fuyuhiko Motoi, Takeshi NaitohAbstract:Abstract The canonical Wnt/β-catenin signaling pathway has been shown to promote the epithelial–mesenchymal transition (EMT), which is a crucial process in multiple embryonic developmental processes and the progression of carcinomas. We recently provided evidence that leucine-rich repeat flightless-1-interacting protein 1 (LRRFIP1) promotes cancer metastasis and invasion. In the present study, we identified the signaling elements targeted by LRRFIP1 for promotion of the EMT in pancreatic and lung cancer. LRRFIP1 silencing reversed the EMT, as shown by increased expression of E-cadherin (an epithelial marker) and decreased expression of vimentin (a mesenchymal marker). Silencing of LRRFIP1 up-regulated phosphorylation of β-catenin and decreased its nuclear localization by targeting the β-catenin destruction complex. The expression of β-catenin and E-cadherin in the plasma membrane fraction was increased in LRRFIP1 silenced cancer cells, and the migration and invasion capabilities were strongly inhibited. In addition, this protein was highly expressed at the invasion front of malignant tissue collected from pancreatic cancer patients. Consequently, our data strongly suggested that LRRFIP1 played an important role in the invasion of carcinoma cells. Our data provide experimental evidence that LRRFIP1 is an attractive candidate for targeted therapy in human cancers.
Daisuke Douchi - One of the best experts on this subject based on the ideXlab platform.
-
silencing of LRRFIP1 reverses the epithelial mesenchymal transition via inhibition of the wnt β catenin signaling pathway
Cancer Letters, 2015Co-Authors: Daisuke Douchi, Hideo Ohtsuka, Kyohei Ariake, Kunihiro Masuda, Shuhei Kawasaki, Kei Kawaguchi, Koji Fukase, Masaya Oikawa, Fuyuhiko Motoi, Takeshi NaitohAbstract:The canonical Wnt/β-catenin signaling pathway has been shown to promote the epithelial-mesenchymal transition (EMT), which is a crucial process in multiple embryonic developmental processes and the progression of carcinomas. We recently provided evidence that leucine-rich repeat flightless-1-interacting protein 1 (LRRFIP1) promotes cancer metastasis and invasion. In the present study, we identified the signaling elements targeted by LRRFIP1 for promotion of the EMT in pancreatic and lung cancer. LRRFIP1 silencing reversed the EMT, as shown by increased expression of E-cadherin (an epithelial marker) and decreased expression of vimentin (a mesenchymal marker). Silencing of LRRFIP1 up-regulated phosphorylation of β-catenin and decreased its nuclear localization by targeting the β-catenin destruction complex. The expression of β-catenin and E-cadherin in the plasma membrane fraction was increased in LRRFIP1 silenced cancer cells, and the migration and invasion capabilities were strongly inhibited. In addition, this protein was highly expressed at the invasion front of malignant tissue collected from pancreatic cancer patients. Consequently, our data strongly suggested that LRRFIP1 played an important role in the invasion of carcinoma cells. Our data provide experimental evidence that LRRFIP1 is an attractive candidate for targeted therapy in human cancers.
-
Silencing of LRRFIP1 reverses the epithelial-mesenchymal transition via inhibition of the Wnt/β-catenin signaling pathway.
Cancer letters, 2015Co-Authors: Daisuke Douchi, Hideo Ohtsuka, Kyohei Ariake, Kunihiro Masuda, Shuhei Kawasaki, Kei Kawaguchi, Koji Fukase, Masaya Oikawa, Fuyuhiko Motoi, Takeshi NaitohAbstract:Abstract The canonical Wnt/β-catenin signaling pathway has been shown to promote the epithelial–mesenchymal transition (EMT), which is a crucial process in multiple embryonic developmental processes and the progression of carcinomas. We recently provided evidence that leucine-rich repeat flightless-1-interacting protein 1 (LRRFIP1) promotes cancer metastasis and invasion. In the present study, we identified the signaling elements targeted by LRRFIP1 for promotion of the EMT in pancreatic and lung cancer. LRRFIP1 silencing reversed the EMT, as shown by increased expression of E-cadherin (an epithelial marker) and decreased expression of vimentin (a mesenchymal marker). Silencing of LRRFIP1 up-regulated phosphorylation of β-catenin and decreased its nuclear localization by targeting the β-catenin destruction complex. The expression of β-catenin and E-cadherin in the plasma membrane fraction was increased in LRRFIP1 silenced cancer cells, and the migration and invasion capabilities were strongly inhibited. In addition, this protein was highly expressed at the invasion front of malignant tissue collected from pancreatic cancer patients. Consequently, our data strongly suggested that LRRFIP1 played an important role in the invasion of carcinoma cells. Our data provide experimental evidence that LRRFIP1 is an attractive candidate for targeted therapy in human cancers.
-
gcf2 LRRFIP1 promotes colorectal cancer metastasis and liver invasion through integrin dependent rhoa activation
Cancer Letters, 2012Co-Authors: Kyohei Ariake, Daisuke Douchi, Hideo Ohtsuka, Koji Fukase, Masaya Oikawa, Fuyuhiko Motoi, Toshiki Rikiyama, Yu Katayose, Shinichi Egawa, Michiaki UnnoAbstract:The precise relationship between GCF2 expression and carcinogenesis has not yet been established. To clarify the metastatic potential of GCF2 in colorectal cancer, HT-29 cells stably suppressing GCF2 expression were injected into the spleens of severe combined immunodeficient (SCID) mice. GCF2 suppression reduced the number of metastatic foci in the liver and reduced fibronectin-induced cell adhesion, migration, and invasion. Downstream from the integrin signaling pathways, GCF2 regulates RhoA interaction with the RGS domain of Leukemia associated RhoGEF (LARG). Altogether, our results suggest that GCF2 plays an important role in colorectal cancer metastasis by regulating RhoA-induced cell adhesion, migration, and invasion.
-
GCF2/LRRFIP1 promotes colorectal cancer metastasis and liver invasion through integrin-dependent RhoA activation.
Cancer letters, 2012Co-Authors: Kyohei Ariake, Daisuke Douchi, Hideo Ohtsuka, Koji Fukase, Masaya Oikawa, Fuyuhiko Motoi, Toshiki Rikiyama, Yu Katayose, Shinichi Egawa, Michiaki UnnoAbstract:The precise relationship between GCF2 expression and carcinogenesis has not yet been established. To clarify the metastatic potential of GCF2 in colorectal cancer, HT-29 cells stably suppressing GCF2 expression were injected into the spleens of severe combined immunodeficient (SCID) mice. GCF2 suppression reduced the number of metastatic foci in the liver and reduced fibronectin-induced cell adhesion, migration, and invasion. Downstream from the integrin signaling pathways, GCF2 regulates RhoA interaction with the RGS domain of Leukemia associated RhoGEF (LARG). Altogether, our results suggest that GCF2 plays an important role in colorectal cancer metastasis by regulating RhoA-induced cell adhesion, migration, and invasion.
Pauline Labbe - One of the best experts on this subject based on the ideXlab platform.
-
The alternatively spliced LRRFIP1 Isoform-1 is a key regulator of the Wnt/β-catenin transcription pathway
BBA - Biochimica et Biophysica Acta, 2017Co-Authors: Pauline Labbe, Emilie Faure, Simon Lecointe, Florence Kyndt, Cecile Duplaa, Solena Le Scouarnec, Marie Marrec, Thierry Le Tourneau, Bernard Offmann, Stephane ZaffranAbstract:The GC-rich Binding Factor 2/Leucine Rich Repeat in the Flightless 1 Interaction Protein 1 gene (GCF2/LRRFIP1) is predicted to be alternatively spliced in five different isoforms. Although important peptide sequence differences are expected to result from this alternative splicing, to date, only the gene transcription regulator properties of LRRFIP1-Iso5 were unveiled. Based on molecular, cellular and biochemical data, we show here that the five isoforms define two molecular entities with different expression profiles in human tissues, subcellular localizations, oligomerization properties and transcription enhancer properties of the canonical Wnt pathway. We demonstrated that LRRFIP1-Iso3, -4 and -5, which share over 80% sequence identity, are primarily located in the cell cytoplasm and form homo and hetero-multimers between each other. In contrast, LRRFIP1-Iso1 and -2 are primarily located in the cell nucleus in part thanks to their shared C-terminal domain. Furthermore, we showed that LRRFIP1-Iso1 is preferentially expressed in the myocardium and skeletal muscle. Using the in vitro Topflash reporter assay we revealed that among LRRFIP1 isoforms, LRRFIP1-Iso1 is the strongest enhancer of the β-catenin Wnt canonical transcription pathway thanks to a specific N-terminal domain harboring two critical tryptophan residues (W76, 82). In addition, we showed that the Wnt enhancer properties of LRRFIP1-Iso1 depend on its homo-dimerisation which is governed by its specific coiled coil domain. Together our study identified LRRFIP1-Iso1 as a critical regulator of the Wnt canonical pathway with a potential role in myocyte differentiation and myogenesis.
-
the alternatively spliced LRRFIP1 isoform 1 is a key regulator of the wnt β catenin transcription pathway
Biochimica et Biophysica Acta, 2017Co-Authors: Pauline Labbe, Emilie Faure, Simon Lecointe, Florence Kyndt, Thierry Le Tourneau, Cecile Duplaa, Solena Le Scouarnec, Marie Marrec, Bernard Offmann, Stephane ZaffranAbstract:The GC-rich Binding Factor 2/Leucine Rich Repeat in the Flightless 1 Interaction Protein 1 gene (GCF2/LRRFIP1) is predicted to be alternatively spliced in five different isoforms. Although important peptide sequence differences are expected to result from this alternative splicing, to date, only the gene transcription regulator properties of LRRFIP1-Iso5 were unveiled. Based on molecular, cellular and biochemical data, we show here that the five isoforms define two molecular entities with different expression profiles in human tissues, subcellular localizations, oligomerization properties and transcription enhancer properties of the canonical Wnt pathway. We demonstrated that LRRFIP1-Iso3, -4 and -5, which share over 80% sequence identity, are primarily located in the cell cytoplasm and form homo and hetero-multimers between each other. In contrast, LRRFIP1-Iso1 and -2 are primarily located in the cell nucleus in part thanks to their shared C-terminal domain. Furthermore, we showed that LRRFIP1-Iso1 is preferentially expressed in the myocardium and skeletal muscle. Using the in vitro Topflash reporter assay we revealed that among LRRFIP1 isoforms, LRRFIP1-Iso1 is the strongest enhancer of the β-catenin Wnt canonical transcription pathway thanks to a specific N-terminal domain harboring two critical tryptophan residues (W76, 82). In addition, we showed that the Wnt enhancer properties of LRRFIP1-Iso1 depend on its homo-dimerisation which is governed by its specific coiled coil domain. Together our study identified LRRFIP1-Iso1 as a critical regulator of the Wnt canonical pathway with a potential role in myocyte differentiation and myogenesis.
-
mecanismes moleculaires et cellulaires impliques dans le prolapsus valvulaire mitral roles des genes LRRFIP1 et ptprf
2016Co-Authors: Pauline LabbeAbstract:Le Prolapsus Valvulaire Mitral (PVM) est une pathologie cardiaque frequente (2.5% de la population generale) dont le seul traitement est la chirurgie valvulaire. Il existe des formes non syndromiques pour lesquelles l'implication de facteurs genetiques a ete demontree. A partir d'analyses genetiques sur des familles atteintes de PVM, LRRFIP et PTPRF ont ete identifies comme de nouveaux genes potentiellement responsables de PVM. Les mutations (respectivement R94G et I328M) ciblent des isoformes de LRRFIP1 (LRRFIP1-iso1) et PTPRF (short-PTPRF) dont les roles physiologiques ne sont pas encore connus. Mon travail a consiste a etudier le role de ces deux proteines ainsi que l'effet des mutations dans des modeles de reexpression cellulaires. Nous avons identifie LRRFIP1-iso1 comme un (co-)facteur de transcription de la voie de signalisation Wnt canonique de part 1) sa localisation nucleaire 2) sa forte activation des promoteurs TCF/LEF 3) son interaction avec la β- catenine 4) son activation/repression de plusieurs cibles transcriptionnelles importantes pour le developpement du coeur et des valves. L'effet physiopathologique de la mutation R94G n'a neanmoins pas ete mis en evidence dans nos modeles cellulaires. Dans un second projet, j’ai caracterise l'expression et la localisation subcellulaire d'une nouvelle isoforme de PTPRF qui est exprimee dans les valves humaines et interagit avec les Heparane Sulfate Proteoglycans (HSPGs) dont le role majeur a ete montre dans le developpement des valves. Ce travail met en evidence un role potentiel de LRRFIP1 et PTPRF dans le developpement et/ou l'homeostasie des valves cardiaques.
-
0268 involvement of LRRFIP1 gene and canonical wnt pathway in mitral valve prolapse mvp
Archives of Cardiovascular Diseases Supplements, 2015Co-Authors: Pauline Labbe, Emilie Faure, Simon Lecointe, Florence Kyndt, Thierry Le Tourneau, Jose Luis De La Pompa, Cecile Duplaa, Stephane Zaffran, Jeanjacques Schott, Jean MerotAbstract:Heart valves diseases affect 3% of world population, and surgery is often the only therapeutic mean. A genetic study performed on a family in which several members exhibited a MVP identified a R94G mutation on LRRFIP1 gene. LRRFIP1 alternative transcription splicing gives rise to five isoforms in humans, three of which are targeted by the mutation (Iso1, 3 and 4). Previous studies only focused on LRRFIP1-iso5 that was first described as a transcription factor interacting with positive (Dishevelled) and negative (Flightless-1) regulators of the canonical Wnt β-catenin dependant pathway. As it may participate and regulate crucial events of cardiac valve development and homeostasis involving Wnt pathway, we hypothesised that LRRFIP1 could be involved in MVP pathology. We first analysed the expression of LRRFIP1 in valves by RNA sequencing and quantitative PCR and showed that LRRFIP1- iso1 is expressed in human valves. In mouse, it prevails during embryonic development and then levels down to that other isoforms expression. We thus focused on LRRFip-iso1. Using cell fractionation, we showed a nuclear localization of LRRFIP1-iso1 while other isoforms are strictly cytoplasmic. Using luciferase-based Wnt reporter assays and co-IP, we further demonstrated that out of the five isoforms, LRRFIP1-iso1 is the strongest interactor of Dvl-1 and Fli-1, and the strongest activator of the canonical Wnt pathway. Although activation requires beta-catenin, it does not involve beta-catenin stabilization nor activation. Using site directed mutagenesis, we mapped the domain responsible for Wnt pathway activation to the 25 amino-acids region surrounding arginine 94 and showed that R94G mutation also decreases Wnt activation. This work demonstrates the involvement of LRRFIP1-iso1 in canonical Wnt pathway activation. Taken together, our results suggest a potential role for LRRFIP1 in valvulogenesis and/or valve homeostasis regulation that may be impeded by the R94G mutation.
-
0174: LRRFIP1 and Wnt pathway involvement in mitral valve prolapse
Archives of Cardiovascular Diseases Supplements, 2014Co-Authors: Pauline Labbe, Florence Kyndt, Thierry Le Tourneau, Cecile Duplaa, Stephane Zaffran, Jeanjacques Schott, Jean MerotAbstract:Heart valve diseases affect 3% of world population, and surgery is often the only therapeutic mean. A genetic study performed on a family in which several members exhibited a mitral valve prolapse (MVP) identified a mutation on LRRFIP1 gene. LRRFIP1 undergoes extensive alternative transcription splicing giving rise to five isoforms in humans and the mutation we identified results in R94G substitution in three (Iso1, 3 and 4) out of the five isoforms. Previous studies essentially focused on LRRFIP1-Iso5 that was first described as a transcription factor interacting with positive (Dishevelled) and negative (Flightess-1) regulators of the canonical Wnt b-catenin dependent pathway. LRRFIP1 thus appeared as an interesting gene in MVP as it may participate and regulate two crucial events of cardiac valve development and homeostasis involving Wnt pathway: epithelial to mesenchymal transition and cell proliferation. Interestingly, we showed using RNA sequencing analysis and RTPCR experiments that LRRFIP1-Iso1 is the most expressed isoform in human valves. Furthermore, LRRFIP1-Iso1 has poor homology with the other isoforms and nothing is known about its function. We thus focused on LRRFIP1-Iso1 and analyzed its cellular localization, its role in Wnt pathway and the impact of R94G mutation in HEK293 cells. Cell fractionation experiments revealed a nuclear localization of LRRFIP1-Iso1 while other isoforms are strictly cytoplasmic. We then showed by Luciferase assays and co-immunoprecipitations that out of the five isoforms, LRRFIP1-Iso1 activates the canonical Wnt pathway at the highest levels and interacts mainly with Fli-1. Furthermore, R94G mutation decreases this interaction. Thus, our studies suggest that LRRFIP1-Iso1 may activate the canonical Wnt pathway by opposing Fli-1 inhibitory effects. R94G mutation may alter this key regulatory loop, deregulate gene transcription and consequently alter valvulogenesis and/or valve homeostasy.
Stephane Zaffran - One of the best experts on this subject based on the ideXlab platform.
-
The alternatively spliced LRRFIP1 Isoform-1 is a key regulator of the Wnt/β-catenin transcription pathway
BBA - Biochimica et Biophysica Acta, 2017Co-Authors: Pauline Labbe, Emilie Faure, Simon Lecointe, Florence Kyndt, Cecile Duplaa, Solena Le Scouarnec, Marie Marrec, Thierry Le Tourneau, Bernard Offmann, Stephane ZaffranAbstract:The GC-rich Binding Factor 2/Leucine Rich Repeat in the Flightless 1 Interaction Protein 1 gene (GCF2/LRRFIP1) is predicted to be alternatively spliced in five different isoforms. Although important peptide sequence differences are expected to result from this alternative splicing, to date, only the gene transcription regulator properties of LRRFIP1-Iso5 were unveiled. Based on molecular, cellular and biochemical data, we show here that the five isoforms define two molecular entities with different expression profiles in human tissues, subcellular localizations, oligomerization properties and transcription enhancer properties of the canonical Wnt pathway. We demonstrated that LRRFIP1-Iso3, -4 and -5, which share over 80% sequence identity, are primarily located in the cell cytoplasm and form homo and hetero-multimers between each other. In contrast, LRRFIP1-Iso1 and -2 are primarily located in the cell nucleus in part thanks to their shared C-terminal domain. Furthermore, we showed that LRRFIP1-Iso1 is preferentially expressed in the myocardium and skeletal muscle. Using the in vitro Topflash reporter assay we revealed that among LRRFIP1 isoforms, LRRFIP1-Iso1 is the strongest enhancer of the β-catenin Wnt canonical transcription pathway thanks to a specific N-terminal domain harboring two critical tryptophan residues (W76, 82). In addition, we showed that the Wnt enhancer properties of LRRFIP1-Iso1 depend on its homo-dimerisation which is governed by its specific coiled coil domain. Together our study identified LRRFIP1-Iso1 as a critical regulator of the Wnt canonical pathway with a potential role in myocyte differentiation and myogenesis.
-
the alternatively spliced LRRFIP1 isoform 1 is a key regulator of the wnt β catenin transcription pathway
Biochimica et Biophysica Acta, 2017Co-Authors: Pauline Labbe, Emilie Faure, Simon Lecointe, Florence Kyndt, Thierry Le Tourneau, Cecile Duplaa, Solena Le Scouarnec, Marie Marrec, Bernard Offmann, Stephane ZaffranAbstract:The GC-rich Binding Factor 2/Leucine Rich Repeat in the Flightless 1 Interaction Protein 1 gene (GCF2/LRRFIP1) is predicted to be alternatively spliced in five different isoforms. Although important peptide sequence differences are expected to result from this alternative splicing, to date, only the gene transcription regulator properties of LRRFIP1-Iso5 were unveiled. Based on molecular, cellular and biochemical data, we show here that the five isoforms define two molecular entities with different expression profiles in human tissues, subcellular localizations, oligomerization properties and transcription enhancer properties of the canonical Wnt pathway. We demonstrated that LRRFIP1-Iso3, -4 and -5, which share over 80% sequence identity, are primarily located in the cell cytoplasm and form homo and hetero-multimers between each other. In contrast, LRRFIP1-Iso1 and -2 are primarily located in the cell nucleus in part thanks to their shared C-terminal domain. Furthermore, we showed that LRRFIP1-Iso1 is preferentially expressed in the myocardium and skeletal muscle. Using the in vitro Topflash reporter assay we revealed that among LRRFIP1 isoforms, LRRFIP1-Iso1 is the strongest enhancer of the β-catenin Wnt canonical transcription pathway thanks to a specific N-terminal domain harboring two critical tryptophan residues (W76, 82). In addition, we showed that the Wnt enhancer properties of LRRFIP1-Iso1 depend on its homo-dimerisation which is governed by its specific coiled coil domain. Together our study identified LRRFIP1-Iso1 as a critical regulator of the Wnt canonical pathway with a potential role in myocyte differentiation and myogenesis.
-
0268 involvement of LRRFIP1 gene and canonical wnt pathway in mitral valve prolapse mvp
Archives of Cardiovascular Diseases Supplements, 2015Co-Authors: Pauline Labbe, Emilie Faure, Simon Lecointe, Florence Kyndt, Thierry Le Tourneau, Jose Luis De La Pompa, Cecile Duplaa, Stephane Zaffran, Jeanjacques Schott, Jean MerotAbstract:Heart valves diseases affect 3% of world population, and surgery is often the only therapeutic mean. A genetic study performed on a family in which several members exhibited a MVP identified a R94G mutation on LRRFIP1 gene. LRRFIP1 alternative transcription splicing gives rise to five isoforms in humans, three of which are targeted by the mutation (Iso1, 3 and 4). Previous studies only focused on LRRFIP1-iso5 that was first described as a transcription factor interacting with positive (Dishevelled) and negative (Flightless-1) regulators of the canonical Wnt β-catenin dependant pathway. As it may participate and regulate crucial events of cardiac valve development and homeostasis involving Wnt pathway, we hypothesised that LRRFIP1 could be involved in MVP pathology. We first analysed the expression of LRRFIP1 in valves by RNA sequencing and quantitative PCR and showed that LRRFIP1- iso1 is expressed in human valves. In mouse, it prevails during embryonic development and then levels down to that other isoforms expression. We thus focused on LRRFip-iso1. Using cell fractionation, we showed a nuclear localization of LRRFIP1-iso1 while other isoforms are strictly cytoplasmic. Using luciferase-based Wnt reporter assays and co-IP, we further demonstrated that out of the five isoforms, LRRFIP1-iso1 is the strongest interactor of Dvl-1 and Fli-1, and the strongest activator of the canonical Wnt pathway. Although activation requires beta-catenin, it does not involve beta-catenin stabilization nor activation. Using site directed mutagenesis, we mapped the domain responsible for Wnt pathway activation to the 25 amino-acids region surrounding arginine 94 and showed that R94G mutation also decreases Wnt activation. This work demonstrates the involvement of LRRFIP1-iso1 in canonical Wnt pathway activation. Taken together, our results suggest a potential role for LRRFIP1 in valvulogenesis and/or valve homeostasis regulation that may be impeded by the R94G mutation.
-
0174: LRRFIP1 and Wnt pathway involvement in mitral valve prolapse
Archives of Cardiovascular Diseases Supplements, 2014Co-Authors: Pauline Labbe, Florence Kyndt, Thierry Le Tourneau, Cecile Duplaa, Stephane Zaffran, Jeanjacques Schott, Jean MerotAbstract:Heart valve diseases affect 3% of world population, and surgery is often the only therapeutic mean. A genetic study performed on a family in which several members exhibited a mitral valve prolapse (MVP) identified a mutation on LRRFIP1 gene. LRRFIP1 undergoes extensive alternative transcription splicing giving rise to five isoforms in humans and the mutation we identified results in R94G substitution in three (Iso1, 3 and 4) out of the five isoforms. Previous studies essentially focused on LRRFIP1-Iso5 that was first described as a transcription factor interacting with positive (Dishevelled) and negative (Flightess-1) regulators of the canonical Wnt b-catenin dependent pathway. LRRFIP1 thus appeared as an interesting gene in MVP as it may participate and regulate two crucial events of cardiac valve development and homeostasis involving Wnt pathway: epithelial to mesenchymal transition and cell proliferation. Interestingly, we showed using RNA sequencing analysis and RTPCR experiments that LRRFIP1-Iso1 is the most expressed isoform in human valves. Furthermore, LRRFIP1-Iso1 has poor homology with the other isoforms and nothing is known about its function. We thus focused on LRRFIP1-Iso1 and analyzed its cellular localization, its role in Wnt pathway and the impact of R94G mutation in HEK293 cells. Cell fractionation experiments revealed a nuclear localization of LRRFIP1-Iso1 while other isoforms are strictly cytoplasmic. We then showed by Luciferase assays and co-immunoprecipitations that out of the five isoforms, LRRFIP1-Iso1 activates the canonical Wnt pathway at the highest levels and interacts mainly with Fli-1. Furthermore, R94G mutation decreases this interaction. Thus, our studies suggest that LRRFIP1-Iso1 may activate the canonical Wnt pathway by opposing Fli-1 inhibitory effects. R94G mutation may alter this key regulatory loop, deregulate gene transcription and consequently alter valvulogenesis and/or valve homeostasy.
