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J Francisco Humberto R Nociti - One of the best experts on this subject based on the ideXlab platform.
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leucine rich amelogenin peptide lrap uptake by cementoblast requires Flotillin 1 mediated endocytosis
Journal of Cellular Physiology, 2017Co-Authors: Luciane Martins, Adriana Franco Paes Leme, Kamila Rosamilia Kantovitz, Em Nome De Luciane Martins, Enilson Antonio Sallum, Marcio Zaffalon Casati, J Francisco Humberto R NocitiAbstract:Basic, pre-clinical, and clinical studies have documented the potential of amelogenin, and its variants, to affect cell response and tissue regeneration. However, the mechanisms are unclear. Thus, the aim of the present study was to identify, in cementoblasts, novel binding partners for an alternatively spliced amelogenin form (Leucine-Rich Amelogenin Peptide-LRAP), which is supposed to act as a signaling molecule in epithelial-mesenchymal interactions. LRAP-binding protein complexes from immortalized murine cementoblasts (OCCM-30) were achieved by capture affinity assay (GST pull down) and proteins present in these complexes were identified by mass spectrometry and immunoblotting. Flotillin-1, which functions as a platform for signal transduction, vesicle trafficking, endocytosis, and exocytosis, was identified and confirmed by co-precipitation and co-localization assays as a protein-binding partner for LRAP in OCCM-30 cells. In addition, we found that exogenously added GST-LRAP recombinant protein was internalized by OCCM-30 cells, predominantly localized in the perinuclear region and, that inhibition of Flotillin1-dependent functions by small interference RNA (siRNA) methodology significantly affected LRAP uptake and its biological properties on OCCM-30 cells, including LRAP effect on the expression of genes encoding osteocalcin (Ocn), bone sialoprotein (Bsp), and runt-related transcription factor 2 (RunX2). In conclusion, LRAP uptake by cementoblast involves Flotillin-assisted endocytosis, which suggests an involvement of LRAP in lipid-raft-dependent signaling pathways which are mediated by Flotillin-1. J. Cell. Physiol. 232: 556-565, 2017. © 2016 Wiley Periodicals, Inc.
Ritva Tikkanen - One of the best experts on this subject based on the ideXlab platform.
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cholinergic transactivation of the egfr in hacat keratinocytes stimulates a Flotillin 1 dependent mapk mediated transcriptional response
International Journal of Molecular Sciences, 2015Co-Authors: Sina Kuhne, Antje Banning, Wymke Ockenga, Ritva TikkanenAbstract:Acetylcholine and its receptors regulate numerous cellular processes in keratinocytes and other non-neuronal cells. Muscarinic acetylcholine receptors are capable of transactivating the epidermal growth factor receptor (EGFR) and, downstream thereof, the mitogen-activated protein kinase (MAPK) cascade, which in turn regulates transcription of genes involved in cell proliferation and migration. We here show that cholinergic stimulation of human HaCaT keratinocytes results in increased transcription of matrix metalloproteinase MMP-3 as well as several ligands of the epidermal growth factor family. Since both metalloproteinases and the said ligands are involved in the transactivation of the EGFR, this transcriptional upregulation may provide a positive feed-forward loop for EGFR/MAPK activation. We here also show that the cholinergic EGFR and MAPK activation and the upregulation of MMP-3 and EGF-like ligands are dependent on the expression of Flotillin-1 which we have previously shown to be a regulator of MAPK signaling.
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Flotillin-1 facilitates toll-like receptor 3 signaling in human endothelial cells
Basic Research in Cardiology, 2014Co-Authors: Christian Fork, Benjamin J Nichols, Ritva Tikkanen, Juliane Hitzel, Ralf P. BrandesAbstract:Endothelial cells are important elements in the vascular response to danger-associated molecules signaling through toll-like receptors (TLRs). Flotillin-1 and -2 are markers of membrane rafts but their true endothelial function is unknown. We hypothesized that Flotillins are required for TLR signaling in human umbilical vein endothelial cells (HUVECs). Knockdown of Flotillin-1 by shRNA decreased the TLR3-mediated poly-I:C-induced but not the TLR4-mediated LPS-induced inflammatory activation of HUVEC. As TLR3 but not TLR4 signals through the endosomal compartment, Flotillin-1 might be involved in the transport of poly-I:C to its receptor. Consistently, uptake of poly-I:C was attenuated by Flotillin-1 knockdown and probably involved the scavenger receptor SCARA4 as revealed by knockdown of this receptor. To determine the underlying mechanism, SILAC proteomics was performed. Down-regulation of Flotillin-1 led to a reduction of the structural caveolae proteins caveolin-1, cavin-1 and -2, suggesting a role of Flotillin-1 in caveolae formation. Flotillin-1 and caveolin-1 colocalized within the cell, and knockdown of Flotillin-1 decreased caveolin-1 expression in an endoplasmic reticulum stress-dependent manner. Importantly, downregulation of caveolin-1 also attenuated TLR3-induced signaling. To demonstrate the importance of this finding, cell adhesion was studied. Flotillin-1 shRNA attenuated the poly-I:C-mediated induction of the adhesion molecules VCAM-1 and ICAM-1. As a consequence, the poly-I:C-induced adhesion of peripheral blood mononuclear cells onto HUVECs was significantly attenuated by Flotillin-1 shRNA. Collectively, these data suggest that interaction between Flotillin-1 and caveolin-1 may facilitate the transport of TLR3-ligands to its intracellular receptor and enables inflammatory TLR3 signaling.
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phosphatidylinositol 3 kinase dependent upregulation of the epidermal growth factor receptor upon Flotillin 1 depletion in breast cancer cells
BMC Cancer, 2013Co-Authors: Nina Kurrle, Antje Banning, Melanie Meister, Wymke Ockenga, Frauke Vollner, Sina Kuhne, Bincy A John, Ritva TikkanenAbstract:Background: Flotillin-1 and Flotillin-2 are two homologous and ubiquitously expressed proteins that are involved in signal transduction and membrane trafficking. Recent studies have reported that Flotillins promote breast cancer progression, thus making them interesting targets for breast cancer treatment. In the present study, we have investigated the underlying molecular mechanisms of Flotillins in breast cancer. Methods: Human adenocarcinoma MCF7 breast cancer cells were stably depleted of Flotillins by means of lentivirus mediated short hairpin RNAs. Western blotting, immunofluorescence and quantitative real-time PCR were used to analyze the expression of proteins of the epidermal growth factor receptor (EGFR) family. Western blotting was used to investigate the effect of EGFR stimulation or inhibition as well as phosphatidylinositol 3-kinase (PI3K) inhibition on mitogen activated protein kinase (MAPK) signaling. Rescue experiments were performed by stable transfection of RNA intereference resistant Flotillin proteins. Results: We here show that stable knockdown of Flotillin-1 in MCF7 cells resulted in upregulation of EGFR mRNA and protein expression and hyperactivation of MAPK signaling, whereas ErbB2 and ErbB3 expression were not affected. Treatment of the Flotillin knockdown cells with an EGFR inhibitor reduced the MAPK signaling, demonstrating that the increased EGFR expression and activity is the cause of the increased signaling. Stable ectopic expression of Flotillins in the knockdown cells reduced the increased EGFR expression, demonstrating a direct causal relationship between Flotillin-1 expression and EGFR amount. Furthermore, the upregulation of EGFR was dependent on the PI3K signaling pathway which is constitutively active in MCF7 cells, and PI3K inhibition resulted in reduced EGFR expression. Conclusions: This study demonstrates that Flotillins may not be suitable as cancer therapy targets in cells that carry certain other oncogenic mutations such as PI3K activating mutations, as unexpected effects are prone to emerge upon Flotillin knockdown which may even facilitate cancer cell growth and proliferation.
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Flotillin 1 reggie 2 protein plays dual role in activation of receptor tyrosine kinase mitogen activated protein kinase signaling
Journal of Biological Chemistry, 2012Co-Authors: Monia Amaddii, Antje Banning, Melanie Meister, Ana Tomasovic, Juliane Mooz, Krishnaraj Rajalingam, Ritva TikkanenAbstract:Our previous work has shown that the membrane microdomain-associated Flotillin proteins are potentially involved in epidermal growth factor (EGF) receptor signaling. Here we show that knockdown of Flotillin-1/reggie-2 results in reduced EGF-induced phosphorylation of specific tyrosines in the EGF receptor (EGFR) and in inefficient activation of the downstream mitogen-activated protein (MAP) kinase and Akt signaling. Although Flotillin-1 has been implicated in endocytosis, its depletion affects neither the endocytosis nor the ubiquitination of the EGFR. However, EGF-induced clustering of EGFR at the cell surface is altered in cells lacking Flotillin-1. Furthermore, we show that Flotillins form molecular complexes with EGFR in an EGF/EGFR kinase-independent manner. However, knockdown of Flotillin-1 appears to affect the activation of the downstream MAP kinase signaling more directly. We here show that Flotillin-1 forms a complex with CRAF, MEK1, ERK, and KSR1 (kinase suppressor of RAS) and that Flotillin-1 knockdown leads to a direct inactivation of ERK1/2. Thus, Flotillin-1 plays a direct role during both the early phase (activation of the receptor) and late (activation of MAP kinases) phase of growth factor signaling. Our results here unveil a novel role for Flotillin-1 as a scaffolding factor in the regulation of classical MAP kinase signaling. Furthermore, our results imply that other receptor-tyrosine kinases may also rely on Flotillin-1 upon activation, thus suggesting a general role for Flotillin-1 as a novel factor in receptor-tyrosine kinase/MAP kinase signaling.
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Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2
PloS one, 2012Co-Authors: Ana Tomasovic, Stephanie Traub, Ritva TikkanenAbstract:Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified Flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of Flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with Flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and Flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and Flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway.
Luciane Martins - One of the best experts on this subject based on the ideXlab platform.
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leucine rich amelogenin peptide lrap uptake by cementoblast requires Flotillin 1 mediated endocytosis
Journal of Cellular Physiology, 2017Co-Authors: Luciane Martins, Adriana Franco Paes Leme, Kamila Rosamilia Kantovitz, Em Nome De Luciane Martins, Enilson Antonio Sallum, Marcio Zaffalon Casati, J Francisco Humberto R NocitiAbstract:Basic, pre-clinical, and clinical studies have documented the potential of amelogenin, and its variants, to affect cell response and tissue regeneration. However, the mechanisms are unclear. Thus, the aim of the present study was to identify, in cementoblasts, novel binding partners for an alternatively spliced amelogenin form (Leucine-Rich Amelogenin Peptide-LRAP), which is supposed to act as a signaling molecule in epithelial-mesenchymal interactions. LRAP-binding protein complexes from immortalized murine cementoblasts (OCCM-30) were achieved by capture affinity assay (GST pull down) and proteins present in these complexes were identified by mass spectrometry and immunoblotting. Flotillin-1, which functions as a platform for signal transduction, vesicle trafficking, endocytosis, and exocytosis, was identified and confirmed by co-precipitation and co-localization assays as a protein-binding partner for LRAP in OCCM-30 cells. In addition, we found that exogenously added GST-LRAP recombinant protein was internalized by OCCM-30 cells, predominantly localized in the perinuclear region and, that inhibition of Flotillin1-dependent functions by small interference RNA (siRNA) methodology significantly affected LRAP uptake and its biological properties on OCCM-30 cells, including LRAP effect on the expression of genes encoding osteocalcin (Ocn), bone sialoprotein (Bsp), and runt-related transcription factor 2 (RunX2). In conclusion, LRAP uptake by cementoblast involves Flotillin-assisted endocytosis, which suggests an involvement of LRAP in lipid-raft-dependent signaling pathways which are mediated by Flotillin-1. J. Cell. Physiol. 232: 556-565, 2017. © 2016 Wiley Periodicals, Inc.
Valenti Gomez Martinez - One of the best experts on this subject based on the ideXlab platform.
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noves funcions de Flotillin 1 en la regulacio del proces de mitosi i la via de senyalitzacio del receptor notch1
TDX (Tesis Doctorals en Xarxa), 2009Co-Authors: Valenti Gomez MartinezAbstract:Flotillin-1 es una proteina associada a membrana plasmatica implicada en processos de transit de vesicules, reordenacio del citoesquelet i transduccio de senyals. Estudis previs en el laboratori han demostrat que Flotillin-1 es capac de translocar-se a nucli en resposta a un estimul mitogenic i afavorir la proliferacio de diverses linies cel·lulars. Els mecanismes mitjancant els quals provoca aquests efectes son desconeguts i objecte del present estudi. D'una banda demostrem que Flotillin-1 es un factor regulador de la cinasa Aurora B, una proteina que interve en el control de la mitosi i mes concretament en el anaphase checkpoint. El knock-down de Flotillin-1 provoca events mitotics aberrants, acompanyats del descens tant en l'expressio d'Aurora B com de la seva activitat mesurada com els nivells de fosforilacio de la histona H3. Flotillin-1 interacciona amb Aurora B i evita la seva degradacio per la via del proteasoma. D'altra banda, Flotillin-1 interacciona amb el receptor transmembrana Notch1, implicat en nombrosos processos de regulacio de proliferacio, diferenciacio, apoptosi, etc. Flotillin-1 regula la localitzacio subcel·lular de Notch1 aixi com la seva capacitat com activador transcripcional. La deplecio o mutacio de Flotillin-1 dificulta l'entrada de Notch1 a nucli i l'expressio dels gens diana de les families Hes/Hrt. En conjunt, es presenta a Flotillin-1 com una proteina capac d'actuar a diferents nivells i regular processos i vies de senyalitzacio cel·lular que li confereixen un paper com a regulador de la proliferacio cel·lular.
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noves funcions de la proteina Flotillin 1 en la regulacio del proces de mitosi i de la via de senyalitzacio de notch1
2009Co-Authors: Valenti Gomez MartinezAbstract:Flotillin-1 es una proteina asociada a membrana plasmatica implicada en procesos de trafico de vesiculas, reordenacion del citoesqueleto y trasnduccion de senales. Estudios previos en el laboratorio han demostrado que Flotillin-1 es capaz de translocarse a nucleo en respuesta a un estimulo mitogenico i favorecer la proliferacion de diversas lineas celulares. Los mecanismos mediante los cuales provoca estos efectos son desconocidos i objeto del presente estudio. En una primera parte demostramos que Flotillin-1 interacciona con el receptor transmembrana Notch1, implicado en numerosos procesos de proliferacion, diferenciacion, apoptosis, etc. Flotillin-1 regula la localizacion subcelular de Notch1 asi como su capacidad como activador transcripcional. La inhibicion o mutacion de Flotillin-1 dificulta la entrada de Notch1 a nucleo i la expression de sus genes diana de las familias Hes/Hrt. En un segundo bloque se muestra el papel regulador de Flotillin-1 sobre la quinasa Aurora B, una proteina que interviene en el control del proceso de mitosis, concretamente en el anaphase checkpoint. El knock-down de Flotillin-1 provoca la aparicion de fenotipos mitoticos aberrantes, acompanados del descenso tanto en la expresion de Aurora B como de su actividad medida como los niveles de fosforilacion de su substrato histona H3. Flotillin-1 interacciona con Aurora B i evita su degradacion por la via del proteasoma. En conjunto, se presenta Flotillin-1 como una proteina capaz de actuar a diferentes niveles i regular procesos i vias de senalizacion celular que le confieren un papel como regulador de la proliferacion celular.
Rosanna Paciucci - One of the best experts on this subject based on the ideXlab platform.
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regulation of aurora b kinase by the lipid raft protein Flotillin 1
Journal of Biological Chemistry, 2010Co-Authors: Valenti Gomez, Marta Sese, Anna Santamaria, Juan D Martinez, Elisabeth Castellanos, Marta Soler, Timothy M Thomson, Rosanna PaciucciAbstract:The lipid raft protein Flotillin-1 was previously shown to be required for cell proliferation. Here we show that it is critical for the maintenance of the levels of the mitotic regulator Aurora B. Knockdown of Flotillin-1 induced aberrant mitotic events similar to those produced by Aurora B depletion and led to a marked decline in Aurora B levels and activity. Transfection of wild-type full-length Flotillin-1 or forms directed to the nucleus increased Aurora B levels and activity. Flotillin-1 interacted with Aurora B directly through its SPFH domain in a complex distinct from the chromosomal passenger protein complex, and the two proteins co-purified in nuclear, non-raft fractions. These observations are the first evidence for a function of Flotillin-1 outside of lipid rafts and suggest its critical role in the maintenance of a pool of active Aurora B.
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ptov1 enables the nuclear translocation and mitogenic activity of Flotillin 1 a major protein of lipid rafts
Molecular and Cellular Biology, 2005Co-Authors: Anna Santamaria, Valenti Gomez, Elisabeth Castellanos, Timothy M Thomson, Patricia Benedit, Jaime Renaupiqueras, Juan Morote, Jaume Reventos, Rosanna PaciucciAbstract:PTOV1 is a mitogenic protein that shuttles between the nucleus and the cytoplasm in a cell cycle-dependent manner. It consists of two homologous domains arranged in tandem that constitute a new class of protein modules. We show here that PTOV1 interacts with the lipid raft protein Flotillin-1, with which it copurifies in detergent-insoluble floating fractions. Flotillin-1 colocalized with PTOV1 not only at the plasma membrane but, unexpectedly, also in the nucleus, as demonstrated by immunocytochemistry and subcellular fractionation of endogenous and exogenous Flotillin-1. Flotillin-1 entered the nucleus concomitant with PTOV1, shortly before the initiation of the S phase. Protein levels of PTOV1 and Flotillin-1 oscillated during the cell cycle, with a peak in S. Depletion of PTOV1 significantly inhibited nuclear localization of Flotillin-1, whereas depletion of Flotillin-1 did not affect nuclear localization of PTOV1. Depletion of either protein markedly inhibited cell proliferation under basal conditions. Overexpression of PTOV1 or Flotillin-1 strongly induced proliferation, which required their localization to the nucleus, and was dependent on the reciprocal protein. These observations suggest that PTOV1 assists Flotillin-1 in its translocation to the nucleus and that both proteins are required for cell proliferation.