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Matrix metalloproteinases (MMPs) are enzymes that are able to cleave all the components of the extracellular matrix but also an increasing number of non-matrix proteins such as growth factors and cytokines. They play a key role in many processes such as morphogenesis, wound healing and menstrual shedding. They involve cell proliferation, survival and migration, angiogenesis and bioactivity of other proteases. An abnormal expression of these MMPs could be involved in the development of cancers, rheumatoid arthritis or endometriosis.
Human endometrium is an outstanding model for studying the mechanisms of matrix remodelling. The menstrual shedding of the superficial layer, adjacent to the uterine cavity, is triggered by a set of MMPs mainly produced by stromal fibroblasts in response to the drop of the plasmatic levels of ovarian steroids. Recently, a characterization of the menstrual endometrium transcriptome within distinct tissue compartments showed that a largely unknown MMP, MMP-27, was overexpressed in the areas of stromal lysis. The aim of the study was to investigate the expression profile of the MMP-27 in the cycling endometrium, its cellular origin, its control by the ovarian steroids and its possible association with endometriosis.
Levels of MMP-27 mRNA, detected by real-time PCR, steadily increased during the secretory phase to culminate at the menstrual phase and decreased during the proliferative phase of the menstrual cycle. Cultured endometrial explants confirmed that the ovarian steroids repress MMP-27 mRNA expression. However, variations were noticed between patients within each phase probably owing to MMP- 27 cellular origin. Indeed, MMP-27 was stained in large cells scattered throughout the stroma and around blood vessels. These cells also expressed CD163, a specific marker of macrophages. MMP-27 was also abundant in endometriotic lesions of the ovary but not in deep endometriosis lesion of the recto-vaginal wall.
Western blotting assay, performed with 4 different antibodies, suggested that many MMP-27 isoforms are expressed in endometrium. This hypothesis was supported by menstrual endometrium cDNA sequencing which showed alternative splicing of exon 4, exon 5 or both with maintenance of the readingframe. In the absence of the amino acids coded by exon 4 or exon 5, the protease is respectively devoid of the structural zinc binding motif and an odd cystein residue of unknown function, or of the catalytic site. Consequences of these mutations on the MMP-27 structure and activity will be investigated later with recombinant isoforms whose expression is underway.
In conclusion, MMP-27 expression culminates in the stroma of menstrual endometrium, like many other MMPs. Nevertheless, its particular cellular origin and the presence of many isoforms likely determine particular activities of this protease. Its presence in specific endometriotic lesions also suggests its participation in the development of this pathology Les métalloprotéinases matricielles (MMPs) sont des protéases capables de cliver l’ensemble des composants protéiques de la matrice extracellulaire mais également un nombre croissant de protéines non-matricielles telles que des facteurs de croissance et des cytokines. Elles participent activement à de nombreux processus comme la morphogenèse, la cicatrisation et la menstruation. Elles influencent la prolifération, la survie et la migration cellulaires, la vascularisation et la bioactivité d’autres protéases. Une expression anormale de MMPs interviendrait dans le développement de cancers, d’arthrite rhumatoïde ou encore de l’endométriose.
L’endomètre humain constitue un modèle remarquable pour l’étude des mécanismes d’un remodelage matriciel. La destruction menstruelle de sa couche superficielle, qui jouxte la cavité utérine, est réalisée par un ensemble de MMPs principalement produites par les fibroblastes du stroma en réponse à la chute de concentration des stéroïdes ovariens. Une caractérisation récente du transcriptome des différents compartiments tissulaires de l’endomètre menstruel a indiqué qu’une MMP encore largement inconnue, la MMP-27, était surexprimée dans le stroma des zones de lyse. L’objectif de ce mémoire était de préciser le profil de l’expression endométriale de la MMP-27 au cours du cycle menstruel, son origine cellulaire, son contrôle par les stéroïdes ovariens et son association éventuelle avec l’endométriose.
Les quantités d’ARNm de la MMP-27, mesurées par PCR en temps réel, augmentent progressivement en fin de cycle menstruel, culminent pendant la phase menstruelle puis diminuent au début du cycle suivant. Toutefois, ces évolutions globales sont superposées à des variations inter-patientes importantes qui s’expliquent vraisemblablement par l’origine cellulaire de la MMP-27. En effet, par immunomarquage, la MMP-27 est localisée dans de larges cellules dispersées dans le stroma et autour des vaisseaux sanguins, qui expriment également le marqueur CD163 spécifique des macrophages. La MMP-27 est également abondante dans les lésions d’endométriose ovarienne mais pas dans les lésions d’endométriose profonde de la lame recto-vaginale.
Des expériences de Western blotting, réalisées avec 4 anticorps différents, suggèrent que plusieurs isoformes de MMP-27 sont exprimées dans l’endomètre. Cette hypothèse est soutenue par le séquençage d’ADNc issus d’endomètres menstruels qui montre un épissage alternatif de l’exon 4 et/ou de l’exon 5 conservant la phase de lecture. En l’absence des acides aminés codés par les séquences de l’exon 4 ou de l’exon 5, la protéase produite est dépourvue respectivement du motif de fixation d’un atome de zinc structural, et du domaine catalytique. Les conséquences de cette mutation sur la structure et l’activité de la MMP-27 pourront être investiguées ultérieurement grâce à des formes recombinantes dont l’expression est en cours.
En conclusion, l’expression de la MMP-27 culmine dans le stroma de l’endomètre menstruel, comme celle d’autres MMPs. Toutefois, son origine cellulaire particulière et plus restreinte, par les macrophages, ainsi que l’existence d’isoformes différentes déterminent vraisemblablement des activités particulière de cette protéase. Sa présence dans certaines lésions d’endométriose suggère par ailleurs sa participation au développement de cette pathologie

Matrix Metalloproteinases --- Matrix Metalloproteinases, Secreted --- Endometrium

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Matrix Metalloproteinases --- Matrix Metalloproteinases --- metabolism. --- pharmacology.

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The host laboratory has previously found that matrix metalloproteinase (MMP)-27 is present in CD206- positive macrophages located in the functional layer of the menstrual endometrium and in superficial endometriotic lesions. Preliminary examinations carried outside the lab have suggested that mice invalidated for MMP-27 feature bone marrow hyperplasia and enlarged spleen. Since these are two characteristics of leukemia, the overall aim of my master thesis was to determine whether there is a link between MMP-27 and hematologic diseases. This issue was addressed through two complementary experimental strategies. First, I studied the expression of human MMP-27 in tissue or cell samples from cancer patients. On the other hand, I compared the organs of the hematopoietic system in MMP-27 KO mice to those in wild-type mice. I demonstrated an intense immunostaining of MMP-27 in macrophages in sections of lymph nodes of patients suffering from non-Hodgkin lymphoma, and to a lesser extent, in unidentified cells in lymph nodes or spleen of patients with other forms of hematological cancers. The expression of MMP-27 in these tumors was also measured by quantitative PCR and compared to non-pathological tissue, but the results obtained cannot be interpreted. Moreover, I showed by Western blotting that MMP-27 is expressed by peripheral blood mononuclear cells (PBMCs) of patients with hematological cancers. Migration profiles were variable, emphasizing the diversity of the isoforms of MMP-27.Comparison of MMP-27 KO, heterozygous or wild-type mice focused on the analysis of blood counts as well as the detailed histology (hemalun-eosin staining and May-Grünwald Giemsa), and immunostaining of proliferating cells (Ki-67) and apoptosis (caspase) on sections olymph nodes and spleens.Although my work confirms the presence of MMP-27 in the organs of the hematopoietic system, my two complementary approaches have not established a clear link between the expression (or reduced expression) and hematological cancers. Le laboratoire d’accueil a précédemment découvert que la métallo protéinase matricielle (MMP¨)-27 est présente dans les macrophages CD206 positifs localisés dans la couche fonctionnelle de l’endomètre menstruel ainsi que dans des lésions superficielles d’endométriose. Des examens préliminaires réalisés en dehors du laboratoire ont suggéré que des souris invalidées pour la MMP-27 présentent une splénomégalie et une hyperplasie médullaire. Puisqu’il s’agit de deux caractéristiques des leucémies, l’objectif global de mon mémoire a été de déterminer s’il existe un lien entre la MMP-27 et des pathologies hématologiques. Cette question a été abordée grâce à deux stratégies expérimentales complémentaires. D’une part, j’ai étudié l’expression de la MMP-27 humaine dans des échantillons tissulaires ou cellulaires prélevés sur des patients cancéreux. D’autre part, j’ai comparé les organes du système hématopoïétique de souris KO pour la MMP-27 à ceux de souris wild-type. J’ai mis en évidence un immunomarquage intense de la MMP-27 dans des macrophages présents dans des coupes de ganglions de patients atteints d’un lymphome non Hodgkinien et dans des proportions moindres, dans des cellules non identifiées de ganglions ou de la rate de patients atteints d’autres formes de cancers hématologiques. L’expression de MMP-27 dans ces tumeurs a également été mesurée par PCR quantitative et comparée à des tissus non pathologiques, mais les résultats obtenus ne peuvent être interprétés. Par ailleurs, j’ai montré, par Western Blotting, que la MMP-27 est exprimée par des cellules mononuclées du sang périphérique (PBMCs) de patients atteints de cancers hématologiques. Les profils de migration étaient variables, soulignant la diversité des isoformes de la MMP-27. La comparaison de souris KO, hérérozygotes ou wild-type pour la MMP27 a porté sur l’analyse d’hémogrammes ainsi que sur l’examen histologique approfondi (coloration hémalunéosine et May-Grünwald Giemsa) et l’immunomarquage de cellules prolifératives ( Ki-67) ou apoptotiques (caspase) sur des coupes de ganglions et de rates. Bien que mon travail confirme la présence de MMP-27 dans les organes du système hématopoïétique, mes deux approches complémentaires n’ont pas permis d’établir un lien clair entre son expression (ou la réduction de son expression) et les cancers hématologiques.

Matrix Metalloproteinases --- Hematologic Neoplasms

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The matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases and play key roles in a number of physiological and pathological functions. One of these endopeptidases is the poorly characterized MMP-27 which is investigated in the laboratory. Preliminary data indicate that MMP-27 is an intracellular enzyme unlike other MMPs that can be secreted or localized at the cell surface. MMP-27 is retained in the endoplasmic reticulum in part due to a unique C-terminal extension (CTE). Although limited secretion is obtained with a recombinant form of MMP-27 lacking the CTE (MMP27), this protein is still retained in the endoplasmic reticulum when compared with MMP-10 used as a secretion control. In western blot, several bands appear at molecular weights both higher and lower than the value deduced from the primary sequence.The goal of my Master thesis was to identify motifs in MMP27.The matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases and play key roles in a number of physiological and pathological functions. One of these endopeptidases is the poorly characterized MMP-27 which is investigated in the laboratory. Preliminary data indicate that MMP-27 is an intracellular enzyme unlike other MMPs that can be secreted or localized at the cell surface. MMP-27 is retained in the endoplasmic reticulum in part due to a unique C-terminal extension (CTE). Although limited secretion is obtained with a recombinant form of MMP-27 lacking the CTE (MMP27), this protein is still retained in the endoplasmic reticulum when compared with MMP-10 used as a secretion control. In western blot, several bands appear at molecular weights both higher and lower than the value deduced from the primary sequence.The goal of my Master thesis was to identify motifs in MMP27 that influence its migration in western blots and its subcellular localization. For this purpose I have generated different variants of MMP27 with structural modifications obtained by point mutations or domain exchange with homologous sequences from MMP10. The effects of these modifications were assessed by western blot and immunofluorescence. The results indicate that the N-terminal moiety of MMP27(induding the inactivation domain of the zymogen and the catalytic domain) contains motifs that influence MMP-27 progression along the secretory pathway. 1 also found that three asparagines are N-glycosylated in the pro-, catalytic and hemopexin-like domains of MMP27. These modifications explain the two highest bands in western blots. On the opposite, the lowest band corresponds to a cytosolic form of MMP27. The matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases and play key roles in a number of physiological and pathological functions. One of these endopeptidases is the poorly characterized MMP-27 which is investigated in the laboratory. Preliminary data indicate that MMP-27 is an intracellular enzyme unlike other MMPs that can be secreted or localized at the cell surface. MMP-27 is retained in the endoplasmic reticulum in part due to a unique C-terminal extension (CTE). Although limited secretion is obtained with a recombinant form of MMP-27 lacking the CTE (MMP27, this protein is still retained in the endoplasmic reticulum when compared with MMP-10 used as a secretion control. In western blot, several bands appear at molecular weights both higher and lower than the value deduced from the primary sequence. The goal of my Master thesis was to identify motifs in MMP27 that influence its migration in western blots and its subcellular localization. For this purpose I have generated different variants of MMP27 with structural modifications obtained by point mutations or domain exchange with homologous sequences from MMP10. The effects of these modifications were assessed by western blot and immunofluorescence.The results indicate that the N-terminal moiety of MMP27 (induding the inactivation domain of the zymogen and the catalytic domain) contains motifs that influence MMP-27 progression along the secretory pathway. I also found that three asparagines are N-glycosylated in the pro-, catalytic and hemopexin-like domains of MMP27. These modifications explain the two highest bands in western blots. On the opposite, the lowest band corresponds to a cytosolic form of MMP2 7ôCTE deprived of signal peptide and resulting from translation from an alternative start codon. The contribution of this glycosylation to inhibition of progression in the secretory pathway requires further investigation. CTE deprived of signal peptide and resulting from translation from an alternative start codon. The contribution of this glycosylation to inhibition of progression in the secretory pathway requires further investigation. CTE that influence its migration in western blots and its subcellular localization. For this purpose 1 have generated different variants of MMP27 with structural modifications obtained by point mutations or domain exchange with homologous sequences from MMP10 . The effects of these modifications were assessed by western blot and immunofluorescence.The results indicate that the N-terminal moiety of MMP27 (induding the inactivation domain of the zymogen and the catalytic domain) contains motifs that influence MMP-27 progression along the secretory pathway. I also found that three asparagines are N-glycosylated in the pro-, catalytic and hemopexin-like domains of MMP27. These modifications explain the two highest bands in western blots. On the opposite, the lowest band corresponds to a cytosolic form of MMP27. The matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases and play key roles in a number of physiological and pathological functions. One of these endopeptidases is the poorly characterized MMP-27 which is investigated in the laboratory. Preliminary data indicate that MMP-27 is an intracellular enzyme unlike other MMPs that can be secreted or localized at the cell surface. MMP-27 is retained in the endoplasmic reticulum in part due to a unique C-terminal extension (CTE). Although limited secretion is obtained with a recombinant form of MMP-27 lacking the CTE (MMP27ôCTE), this protein is still retained in the endoplasmic reticulum when compared with MMP-10 used as a secretion control. In western blot, several bands appear at molecular weights both higher and lower than the value deduced from the primary sequence.The goal of my Master thesis was to identify motifs in MMP2 7ôCTE that influence its migration in western blots and its subcellular localization. For this purpose I have generated different variants of MMP27 with structural modifications obtained by point mutations or domain exchange with homologous sequences from MMP10. The effects of these modifications were assessed by western blot and immunofluorescence. The results indicate that the N-terminal moiety of MMP27 (induding the inactivation domain of the zymogen and the catalytic domain) contains motifs that influence MMP-27 progression along the secretory pathway. I also found that three asparagines are N-glycosylated in the pro-, catalytic and hemopexin-like domains of MMP27. These modifications explain the two highest bands in western blots. On the opposite, the lowest band corresponds to a cytosolic form of MMP27deprived of signal peptide and resulting from translation from an alternative start codon. The contribution of this glycosylation to inhibition of progression in the secretory pathway requires further investigation. CTE deprived of signal peptide and resulting from translation from an alternative start codon. The contribution of this glycosylation to inhibition of progression in the secretory pathway requires further investigation. Les métalloprotéinases matricielles (MMPs) sont une famille d'endopeptidases dépendantes du zinc qui interviennent dans plusieurs fonctions physiologiques et pathologiques. La MMP-27 est, à ce jour, une protéine très peu connue qui fait l'objet d'une étude approfondie dans le laboratoire d'accueil. Des expériences pilotes indiquent que, contrairement aux autres MMPs qui sont sécrétées ou présentées à la membrane plasmique,la MMP-27 reste intracellulaire, principalement dans le réticulum endoplasmique (R.E.). La structure en domaines de la MMP-27 est très semblable à celle des autres MMPs mais une extension C-terminale (CTE) unique contribue partiellement à sa rétention intracellulaire. Toutefois, bien qu'une forme recombinante de MMP-27 dépourvue de CTE (appelée MMP- 27l

Matrix Metalloproteinases --- MMP27 protein, rat --- Endoplasmic Reticulum

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Matrix Metalloproteinases --- Arthritis, Rheumatoid --- Synovial Fluid --- blood. --- enzymology. --- Theses --- blood --- enzymology