Thesis (D.Sc.)--Boston University, Henry M. Goldman School of Dental Medicine, 2004 (Oral Biology). / Includes bibliography (leaves 107-151). / Lipopolysaccharide (LPS), a glycolipid found in the outer membrane of Gram negative bacteria, induces the secretion of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-a) and interleukin (IL )-1, by monocytes/macrophages. Excessive and uncontrolled secretion of these compounds leads to multiple pathological conditions, such as septic shock. LPS receptors have been shown to be CD14, TLR4 and MD-2. LPS interaction with these receptors mediates many monocyte/macrophage functions. Even though only CD14 was demonstrated to bind to LPS, and TLR4/MD-2 were capable of transducing signals, data only show that LPS and CD 14 were in close proximity to TLR4 and no direct binding was reported. Quite recently, moesin, a member of the ERM family of proteins, has been also found to function as a receptor for LPS. We have shown that anti-moesin antibody inhibited the release of TNFa by LPS stimulated monocytes. Moesin was also found to be necessary for the detection of LPS, where homozygous knockout mice exhibited 3-fold reduction in neutrophil infiltrates in LPS injected sites when compared to their wild type controls. When moesin gene expression was completely suppressed with antisense oligonucleotides, there was a significant reduction of LPS-induced TNF-a secretion. LPS stimulation of mononuclear phagocytes activates several intracellular signaling pathways including the phosphorylation of IKBa, mitogen-activated protein kinase (MAPK) pathways: extracellular signal-regulated kinases (ERK) 1 / 2 (P44/42), p38. These signaling pathways in tum activate a variety of transcription factors including NF-KB, which coordinates the induction of several genes encoding inflammatory mediators. [TRUNCATED]

Indiana University-Purdue University Indianapolis (IUPUI) / Bone is a common location for breast cancer cell metastasis, and progression of tumor in bone can lead to bone loss and affect human health. Osteocytes have important roles in bone homeostasis and osteogenesis, and their interaction with metastasized cancer cells are known to affect progression of metastasized tumor. However, the potential role of metabolic signaling and actin- cytoskeleton-associated moesin in the interaction of osteocytes and tumor cells remain poorly understood. In this study, we first examined the roles of metabolic signaling, specifically global AMPK modulators and mitochondria-specific AMPK inhibitor (Mito-AIP), as well as mechanical force in beta catenin signaling through interaction between osteocytes and precursor osteoblasts as well as osteocytes and breast cancer cells. We also evaluated the role of metabolic signaling in Rho GTPases including RhoA, Rac1 and Cdc42. We observed that AMPK activator (A769662) and Mito-AMPK stimulated beta catenin translocation to the nucleus, indicating the activation of Wnt signaling, while Mito-AIP did not significantly affect beta catenin activation in osteoblasts. We also observed that osteocyte conditioned medium (CM) treated with Mito-AIP substantially increased beta catenin signaling in osteoblasts, while decreasing beta catenin signaling in breast cancer cells. CM of osteocytes treated with fluid flow increased beta catenin signaling in breast cancer cells. A769662 and Mito-AIP also decreased the activities of RhoA, Rac1, and Cdc42 in cancer cells which are known to regulate cancer cell migration. Additionally, we evaluated the roles of intracellular and extracellular moesin (MSN) protein in well-established oncogenic signaling proteins, such as FAK, Src, and RhoA as well beta catenin signaling. Constitutively active MSN (MSN+) significantly increased FAK and Src activities in cancer cells, but decreased the activity of RhoA. Surprisingly, CM of mesenchymal stem cells treated with MSN+ decreased the activities of FAK, Src, and RhoA, suggesting the inhibitory role of extracellular MSN in tumor-promoting signaling. Our results suggest the distinct role of AMPK signaling, specifically at mitochondria of osteocytes, in the activities of beta-catenin signaling in osteoblasts and breast cancer cells and the distinct role of intracellular and extracellular MSN in these two types of cell.

Dysregulation of protein expression, in particular expression of proto-oncogenes and tumor-suppressor genes whose function play key roles in cell growth, adhesion and migration, are hallmarks of human malignancies. Transmembrane and immunoglobulin-containing domain 1 (TMIGD1) was recently discovered as a cell adhesion molecule (CAM) that plays an important role in epithelial cell function by regulating epithelial cell polarity and adhesion. The extracellular domain of TMIGD1 contains two Ig domains that are involved in cell-cell interaction, followed by a transmembrane region and short cytoplasmic domain with potential to relay signal transduction. Our further investigation demonstrated TMIGD1 is downregulated in human colon cancer, suggesting a potentially important role for TMIGD1 in the regulation colorectal cancer. However, the molecular mechanisms of TMIGD1-mediated signal transduction, which could relay its function in epithelial cells, are not known. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we have identified moesin as a possible TMIGD1 binding protein. Moesin, a member of the Ezrin/Radixin/Moesin (ERM) family of proteins, is upregulated in human tumors. Moesin stimulates cell migration, tumor invasion, adherence and modulates cytoskeletal actin assembly. Similar to other ERM family proteins, moesin contains an N-terminal FERM domain, which binds to transmembrane proteins, and a C-terminal C-ERMAD domain, which binds F-actin. The overall goal of this study was to determine the binding of moesin with TMIGD1 and the specific domain involved in mediating the binding of moesin with TMIGD1. Our study in vitro and in vivo binding assays demonstrate that moesin interacts with the cytoplasmic domain of TMIGD1 via its FERM domain. Moreover, we demonstrate TMIGD1 interaction with moesin inhibits phosphorylation of moesin, indicating that perhaps TMIGD1 inhibits tumor cell migration through inhibition of phosphorylation of moesin. Additionally, TMIGD1 alters cellular localization of moesin, suggesting that altered cellular localization by TMIGD1 could account for inhibition of phosphorylation of moesin. We propose that TMIGD1 sequesters moesin near the cell membrane, preventing its interaction with PIP2, which is required for its phosphorylation and hence inhibits moesin activation. Altogether, the data presented in this work identifies moesin as a key signaling component of TMIGD1. Moesin directly interacts with TMIGD1 via its FERM domain. Recruitment of moesin to TMIGD1 blocks phosphorylation of moesin, suggesting that TMIGD1 exerts its effect in tumor cells in part by inhibition of moesin activation. / 2018-07-11T00:00:00Z

No description available.

Willin is a novel member of the Four-point-one Ezrin Radixin Moesin (FERM) protein superfamily, containing an N-terminal FERM domain most like the Ezrin-Radixin-Moesin (ERM) family but also the closely related protein Merlin. Willin was initially discovered as a yeast two-hybrid binding partner of neurofascin155, and this interaction has now been confirmed by both co-localisation studies and the use of two different biochemical methods. Like neurofascin155, Willin also localises to detergent resistant membranes, and like the ERM family, it is able to bind to phospholipids. The expression of Willin appears to be toxic as the production of cell-lines stably expressing Willin proved to be not possible and this appears to be because it induces apoptosis in cultured cells. This is a proliferation control function consistent with the suggestion that Willin is the human homologue of the Drosophila tumour suppressor ‘Expanded’. Three antibodies to Willin were also characterised and a novel splice variant, Willin2, subcloned into a GFP-tagged plasmid for comparison with the original form.

<p>In this study, we first examined the roles of metabolic signaling, specifically global AMPK modulators and mitochondria-specific AMPK inhibitor (Mito-AIP), as well as mechanical force in beta catenin signaling through interaction between osteocytes and precursor osteoblasts as well as osteocytes and breast cancer cells. We also evaluated the role of metabolic signaling in Rho GTPases including RhoA, Rac1 and Cdc42. We observed that AMPK activator (A769662) and Mito-AMPK stimulated beta catenin translocation to the nucleus, indicating the activation of Wnt signaling, while Mito-AIP did not significantly affect beta catenin activation in osteoblasts. We also observed that osteocyte conditioned medium (CM) treated with Mito-AIP substantially increased beta catenin signaling in osteoblasts, while decreasing beta catenin signaling in breast cancer cells. CM of osteocytes treated with fluid flow increased beta catenin signaling in breast cancer cells. A769662 and Mito-AIP also decreased the activities of RhoA, Rac1, and Cdc42 in cancer cells which are known to regulate cancer cell migration.</p><p>Additionally, we evaluated the roles of intracellular and extracellular moesin (MSN) protein in well-established oncogenic signaling proteins, such as FAK, Src, and RhoA as well beta catenin signaling. Constitutively active MSN (MSN+) significantly increased FAK and Src activities in cancer cells, but decreased the activity of RhoA. Surprisingly, CM of mesenchymal stem cells treated with MSN+ decreased the activities of FAK, Src, and RhoA, suggesting the inhibitory role of extracellular MSN in tumor-promoting signaling. Our results suggest the distinct role of AMPK signaling, specifically at mitochondria of osteocytes, in the activities of beta-catenin signaling in osteoblasts and breast cancer cells and the distinct role of intracellular and extracellular MSN in these two types of cell.</p>

No description available.

Les kinases constituent une famille majeure de protéines qui régulent divers processus par la phosphorylation de leurs substrats, mais aussi par leur activité non- catalytique. Ce rôle indépendant de l’activité kinase a été observé chez quelques protéines dont des membres de la famille Sterile-20. La kinase Ste20 Slik de Drosophila aide au maintien de l’intégrité des tissus épithéliaux en phosphorylant l’ERM Moesin et peut aussi induire une prolifération cellulaire non-autonome indépendamment de son activité catalytique. La méthode de régulation de ces deux rôles était jusqu’ici inconnue. Nous avons identifié 19 sites de phosphorylation chez Slik par spectrométrie de masse. À l’aide de mutants, nous démontrons que les deux fonctions de Slik sont régulées par la phosphorylation d’au moins 2 résidus conservés de son segment d’activation par un mécanisme d’auto- et/ou trans-phosphorylation. Cette étude amène une meilleure compréhension de la régulation de l’intégrité épithéliale et de la croissance, deux processus clés qui sont souvent déréglés dans le cancer et certaines maladies génétiques. / Kinases constitute a major protein family which regulate diverse pathways through the phosphorylation of their substrates, but also through their non-catalytic activity. This kinase-independent role has been observed in a few proteins such as certain members of the Sterile-20 family. The Drosophila Ste20 kinase Slik helps to maintain epithelial integrity by phosphorylating the ERM Moesin and it can also drive non- autonomous cellular proliferation in a kinase-independent fashion. The mechanism of regulation of these two roles was unknown up until now. We have identified 19 phosphorylation sites in Slik by mass spectrometry. Using Slik mutants, we show that its two functions are regulated by the phosphorylation of at least 2 conserved residues of its activation segment by an auto- and/or trans-phosphorylation mechanism. This research brings a better understanding of the regulation of epithelial maintenance and growth, two key processes which are deregulated in cancer and certain genetic diseases.

Le précurseur de la protéine amyloïde (APP) est une protéine transmembranaire qui, après coupure séquentielle par les sécrétases β et γ, produit des peptides Aβ trouvés dans les plaques séniles de patientsatteints d’Alzheimer. Par contre, la forme soluble de l’APP (sAPPα), produite après coupure par une sécrétase α, augmente la survie cellulaire, la croissance des neurites et la synaptogénèse. L’APP est coupéeau site α par 3 métalloprotéases : ADAM9, ADAM10 et ADAM17.Notre laboratoire a montré que la stimulation du récepteur purinergique P2X7 (P2X7R) provoque la coupure protéolytique du précurseur de la protéine amyloïde (APP). Le Dr Delarasse a établi que la voie non amyloïdogénique est mise en jeu et que c'est le fragment sAPPα, neuroprotecteur, qui est produit. Deplus, le laboratoire a précédemment démontré que ce ne sont pas les alpha-sécrétases ADAM9, 10 et 17 qui sont responsables du clivage protéolytique de l'APP après stimulation du P2X7R dans les cellules de neuroblastome Neuro2a.Durant mes travaux de thèse, nous avons étudié la voie biochimique menant à la libération du fragments APPα. L’activation du P2X7R stimule la phosphorylation et la translocation rapide à la membrane plasmique de protéines, appelées ezrine, radixine et moesine (ERM) qui ont la capacité d’établir un lien entre la région cytosolique du P2X7R et la F-actine. Les ERM jouent un rôle crucial dans la coupure protéolytique de l’APP par les métalloprotéases ADAM. En effet, l’inhibition de l’expression des ERM par RNA interférence aboutit à une absence de coupure de l’APP. Par ailleurs, nous avons observé que les MAPKERK1/2 et JNK et la ROCKinase sont nécessaires à la phosphorylation activatrice des ERM et jouent donc un rôle en amont des ERM. Enfin, nous avons mis en évidence le rôle de la PI3K en aval des ERM.Par ailleurs, nous avons démontré que l’activation du récepteur purinergique P2X7 entraînait la coupure protéolytique de la molécule NrCAM par ADAM17 aboutissant à la libération du fragment soluble del’ectodomaine de NrCAM. Les résultats obtenus indiquent que la coupure de NrCAM est dépendante de l’activation et de la fixation des ERM à NrCAM. Ces résultats suggèrent fortement que les ERM sont indispensables à la coupure protéolytique de différents substrats après stimulation du P2X7R.Les données obtenues mettent en évidence un mécanisme moléculaire original et important qui fait jouer aux ERM un rôle central de « liens moléculaires » dans le clivage protéolytique des protéines transmembranaires. A ce stade de notre étude, nous émettons l’hypothèse que les ERM agissent en aval du récepteur P2X7, en liant les substrats et/ou les protéases qu’ils regroupent à la membrane plasmique favorisant ainsi le clivage des substrats. / The amyloid protein precursor (APP) can be cleaved in neural cells by α-secretases to produce the soluble APP ectodomain (sAPPα), which is neuroprotective. We have shown previously that activation of the purinergic receptor P2X7 (P2X7R), a member of the P2X receptor family of ATP-gated cation channels, triggers sAPPα shedding from neural cells. Here, we demonstrate that theactivation of Ezrin/Radixin/Moesin proteins (ERM) is required for the P2X7R-dependent proteolyticprocessing of APP leading to sAPPα release. Indeed, the down regulation of ERM by siRNA blocksthe P2X7R-dependent shedding of sAPPα. We also show that P2X7R stimulation triggers thephosphorylation of ERM. Thus, ezrin translocates to the plasma membrane to interact with P2X7R.Using specific pharmacological inhibitors, we have established the order in which several enzymestrigger the P2X7R-dependent release of sAPPα. Thus, a Rho-kinase and the MAPK modules ERK1/2and JNK act upstream of ERM while a PI3Kinase activity is triggered downstream. This work for the first time identifies ERM as major partners in the regulated non-amyloidogenic processing of APP. Inaddition, we have recently established that the stimulation of P2X7R leads to the proteolytic cleavage of NrCAM by ADAM17 and the shedding of the soluble extracellular domain of NrCAM. Our results clearly show that the proteolytic cleavage of NrCAM is dependant of ERM activation and fixation tothe intracellular region of NrCAM. Thus, our results strongly suggest that ERM are required for the proteolytic cleavage of numerous substrates after P2X7R stimulation. Our findings suggest that ERM play a central role in the proteolytic cleavage of transmembrane proteins and act as molecular linkswhich aggregate ADAMs and substrates at the plasma membrane promoting the cleavage of substrates.

La migration cellulaire est un mécanisme important pour divers processus cellulaires tels que l’embryogenèse et la cicatrisation. De même, elle participe à des processus pathologiques notamment l’invasion des cellules malignes et la formation des métastases cancéreux. La dissémination métastatique est un processus très compliqué. L’acquisition du pouvoir migratoire invasif par la cellule maligne ainsi que son potentiel métastatique est gérée par le cytosquelette qui est dynamiquement modifié et contrôlé par des voies de signalisation intracellulaires. Cependant, la physiologie des cellules métastatiques et les cascades de signalisation qui les poussent à métastaser ne sont toujours pas comprises. Les protéines Ezrine, Radixine et Moésine (ERMs) jouent un rôle important dans l’organisation du cytosquelette au cortex cellulaire et elles sont des déterminantes clés de la migration cellulaire. Ainsi, une dérégulation à ce niveau peut conduire à une migration cellulaire aberrante. D’où l’implication des ERMs dans différents cancers agressifs et invasifs. Les ERMs sont régulées en aval de plusieurs acteurs cellulaires notamment les récepteurs membranaires. Plusieurs études ont rapporté que le récepteur au thromboxane A2 (RTXA2), un récepteur couplé à la protéine G (RCPG) favorise les métastases. Il a été décrit surtout dans le cadre de cancer du sein triple négatif (CSTN), l’un des cancers les plus mortels chez la femme. Les RCPG possèdent un rôle central dans presque toutes les fonctions physiologiques et constituent la plus grande famille des cibles médicamenteuses. D’une manière intéressante, les deux laboratoires de Dr Sébastien Carréno et Dr Michel Bouvier, ont découvert que le RTXA2 active les protéines ERMs à travers la GTPase RhoA. Dans ce projet de recherche on a identifié une nouvelle voie de signalisation liant le RTXA2 aux ERMs à travers la GTPase RhoA et la kinase SLK. Cette voie est impliquée dans la migration des cellules de cancer du sein triple négatif. Ainsi, on a pu démontrer que la moésine et la kinase SLK agissaient en aval du récepteur étudié pour favoriser la vitesse et la directionnalité de la migration des cellules de CSTN. 6 On a montré que la migration cellulaire dirigée en aval du RTXA2 est due à une polarité de la moésine au front de la migration. On a constaté aussi que la moésine est responsable d’une polarité des filaments d’actine au front de la migration suite à une activation du récepteur. Ce travail a mis en évidence une nouvelle cascade de signalisation importante pour la migration des cellules cancéreuses agressives triples négatives du sein ce que pourrait être une nouvelle cible des thérapies anti-métastatiques. / Cell migration is an important mechanism for various cellular processes such as embryogenesis and cicatrization. Likewise, it controls pathological processes including the invasion of malignant cells and the formation of metastases. Metastasis is a very complicated process. The acquisition of invasive migratory power by a malignant cell as well as its metastatic potential is regulated by the cytoskeleton which is dynamically modified and controlled by intracellular signaling pathways. However, metastatic cells physiology and the cascades causing their metastases are not clear yet. Ezrin, Radixin and Moesin (ERMs) proteins have an important role in organizing the cytoskeleton at the cell cortex and they are key determinants of cell motility. Thus, a deregulation at this point may lead to an aberrant cell migration. Hence, the involvement of ERMs in various aggressive and invasive cancers. ERMs are regulated downstream of several cellular actors in particular membrane receptors. Several studies have reported that the thromboxane A2 receptor (TXA2R), a G protein coupled receptor (GPCR) promotes metastasis. It has been described especially in the context of triple negative breast cancer (TNBC), one of the deadliest cancers in women. GPCR have a central role in almost all physiological functions and constitute the largest family of drug targets. Interestingly, the two laboratories of Dr Sébastien Carréno and Dr Michel Bouvier, have discovered that the TXA2R activates ERM proteins through the GTPase RhoA. In this research project, we have identified a new signaling pathway linking the TXA2 receptor to ERMs via RhoA and the kinase SLK. This pathway is involved in the migration of triple negative breast cancer cells. Thus, we demonstrated that moesin and SLK acted downstream of the receptor to promote the speed and directionality of TNBC cells migration. We discovered that the directed cell migration downstream of TXA2R is due to a polarization of moesin at the leading edge. We also observed that moesin is responsible for actin filaments polarity at the leading edge following an activation of the receptor. So, this work has revealed a new signaling cascade important for the migration of aggressive triple negative breast cancer cells which could be a new target for anti-metastatic therapies.