A link between high serum levels of human chorionic gonadotrophin and chorionic expression of its mature functional receptor (LHCGR) in Down's syndrome pregnancies

Human chorionic gonadotrophin (hCG) is released from placental trophoblasts and is involved in establishing pregnancy by maintaining progesterone secretion from the corpus luteum. Serum hCG is detected in the maternal circulation within the first 2–3 wks of gestation and peaks at the end of the first trimester before declining. In Down's syndrome (DS) pregnancies, serum hCG remains significantly high compared to gestation age-matched uncompromised pregnancies. It has been proposed that increased serum hCG levels could be due to transcriptional hyper-activation of the CGB (hCG beta) gene, or an increased half life of glycosylated hCG hormone, or both. Another possibility is that serum hCG levels remain high due to reduced availability of the hormone's cognate receptor, LHCGR, leading to lack of hormone utilization. We have tested this hypothesis by quantifying the expression of the hCG beta (CGB) RNA, LHCGR RNA and LHCGR proteins in chorionic villous samples. We demonstrate that chorionic expression of hCG beta (CGB) mRNA directly correlates with high serum hCG levels. The steady-state synthesis of LHCGR mRNA (exons 1–5) in DS pregnancies was significantly higher than that of controls, but the expression of full-length LHCGR mRNA (exons 1–11) in DS was comparable to that of uncompromised pregnancies. However, the synthesis of high molecular weight mature LHCGR proteins was significantly reduced in DS compared to uncompromised pregnancies, suggesting a lack of utilization of circulating hCG in DS pregnancies

Mécanisme d'action du récepteur de la progestérone (modulation par les cofacteurs JAB1 et HBO-1)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Impact des isoformes du récepteur de la progestérone sur la progression métastatique du cancer du sein (étude in vitro de la motilité de la lignée MDA-MB-231)

Le récepteur de la progestérone (PR) est un acteur majeur du développement de la glande mammaire. Dans les cellules épithéliales normales, PR est exprimé sous deux isoformes PRA (94 kDa) et PRB (116 kDa) de façon équimolaire. Il est établi que celles-ci ont un impact important sur le développement des cancers du sein, mais leurs rôles dans l évolution métastatique reste très mal connus. Le ratio d expression PRA/PRB étant souvent déséquilibré dans les tumeurs mammaires, nous avons analysé le turnover des deux isoformes. Nous avons démontré que PRA et PRB sont les cibles de modifications post-traductionnelles dirigées par les MAPK qui tendent à stabiliser l une ou l autre isoforme de manière sélective. Ainsi, Erk1/2 (p42/44) inhibe la dégradation de PRB tandis que la p38 stabilise PRA. Il en résulte que le ratio PRA/PRB varie de façon importante en fonction des signalisations extracellulaires impliquant les facteurs de croissance et les cytokines inflammatoires souvent exacerbés dans les cancers. Pour mieux étudier les effets différentiels de PRA et PRB, nous avons établi un modèle cellulaire original exprimant de manière bi-inductible l une ou l autre isoforme de PR, à partir de la lignée cellulaire MDA-MB 231 provenant d une métastase de cancer du sein. En étudiant les variations induites par PRA ou PRB sur le transcriptome de ces cellules, nous avons identifié les gènes cibles spécifiques de ces isoformes. Parmi-eux se trouvent de nombreux gènes impliqués dans les cancers, notamment agissant sur la prolifération, la survie et la motilité cellulaires comme uPA et PAI-1. De plus, en analysant la migration cellulaire, nous avons mis en évidence un effet pro-migratoire de PRB particulièrement important en absence d hormone. En recherchant la cause de ces effets, nous avons découvert que PRB était colocalisé et interagissait avec la kinase d adhésion focale (FAK) qu il active au niveau des points d adhésion focaux. Ces travaux soulignent l incidence du ratio PRA/PRB et du statut du ligand sur les métastases du cancer du sein, aussi bien au niveau de la sélectivité transcriptionnelle que celui des régulations non génomiques impactant la migration cellulaire. Nous suggérons la possibilité de cibler les tumeurs mammaires par des antagonistes sélectifs de PR et des inhibiteurs des voies de signalisation des MAPK ou de FAK.Progesterone receptor (PR) is a major actor of mammary gland development. PR is equally expressed as two main isoforms PRA (94 kDa) and PRB (116kDa) in the mammary gland epithelium. However, breast cancer progression has been associated with abnormalities of their expression ratio through undefined mechanisms. In this study, using a stably transfected cell line, we showed that PRA and PRB stabilizations are differentially regulated by Erk1/2 (p42/44) and p38 MAPKs respectively, leading to strongly influence PRA/PRB ratio. These results highlight the impact of growth factors and inflammatory cytokines on PRA/PRB imbalance in cancer cells. To study the differential effect of PR isoforms, we established an original bi-inducible cell line expressing either PRA and/or PRB. By analyzing variations induced by PRA and PRB on such cell transcriptomes, we identified the isoform-specific targets genes by DNA microarrays. Most of them are implicated in cancers, notably acting on cell proliferation, survival and motility. Furthermore, focusing our studies on cell migration, we showed that PRB acts as a pro-migratory factor particularly powerful in the absence of ligand. We discovered that PRB colocalized and interacted with the focal adhesion kinase (FAK) that was activated in focal adhesion points. Our results highlight the impacts of both PRA/PRB ratio and ligand status on metastatic evolution, in the contexts of transcriptional regulation as well as non-genomic events. We suggest the possibility to target mammary tumors by PR-selective antagonists and/or inhibitors of MAPK and FAK signalings.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Isoformes du récepteur de la progestérone (sélectivité fonctionnelle et ciblage pharmacologique)

Le récepteur de la progestérone (PR) est un cible pharmacologique majeure pour la contraception, et pour le traitement de certaines pertubations endocriniennes ainsi que des cancers hormono-dépendants de l utérus et du sein. Chez la femme, PR est exprimé sous deux isoformes majeures PRA et PRB qui sont des facteurs de transcription fonctionnellement distincts. L expression de PRA vs PRB est souvent altérée dans certaines situations pathologiques selon des mécanismes encore mal identifiés. Dans cette thèse, nous démontrons que des phosphorylations clés regulées par des MAPK distincts contrôlent la stabilité de PRB et PRA. PRA est sélectivement stabilisée par la p38 MAPK tandis que PRB est préférentiellement stabilisé par la p42/44 MAPK. Ces mécanismes différentiels régulent donc le rapport d expression PRA/PRB de façon ligand-dépendente et mettent les fonctions progestatives sous le contrôle de l activité des facteurs de croissance et des cytokines pro-inflammatoires. Or, dans les cellules cancéreuses, la suractivité de certains stimuli extracellulaires provenant de telles signalisations et activant préférentiellement p42/44 et/ou p38 MAPK, pourrait être à l origine des pertubations du rapport PRA/PRB observées dans les tumeurs du sein. Afin d explorer la contribution différentielle des isoformes du PR dans la signalisation cellulaire, nous avons élaboré un modèle cellulaire original permettant de contrôler l expression de PRA et/ou PRB de façon conditionnelle, réversible et dose-dépendante. Par une approche transcriptomique, nous avons identifiés les gènes régulés de façon différentielle par PRA et/ou PRB en absence ou présence de l hormone. Nous montrons que plusieurs aspects de la signalisation de PR comme la sélectivité de la régulation transcriptionnelle, la dialogue-croisée avec des facteurs de croissance ainsi que l efficacité antiproliférative des antiprogestatifs dépendent de l expression differentielle des isoformes du PR. Une nouvelle approche thérapeutique ou préventive possible dans les cancers hormono-dépendants pourrait consister à administrer des antagonistes du PR. Cependant, la plupart des antiprogestatifs disponibles comme la mifépristone présentent des effets agonistes partiels et ne sont pas sélectifs du PR, produisant ainsi des effets indésirables majeurs. Dans un projet collaboratif, et sur la base d études cristallographiques de PR, nous avons synthétisé et caractérisé plusieurs dizaines de molécules antagonistes du PR, nommés APRn. L étude des relations structure-fonctions de ces APRn a permis d identifier les substitutions introduites dans la structure stéroïdienne qui sont responsables des propriétés agonistes/antagonistes de ces molécules. Plusieurs APRn sélectionnés sont dépourvus d effets agonistes partiels, sont spécifiques du PR et inhibent son activité transcriptionnelle par un nouveau mécanisme d action dit passif , en raison de leur capacité particulière à inhiber le recrutement des corégulateurs transcriptionnels. Ces antagonistes sélectifs de PR offrent des perspectives thérapeutiques intéressantes dans les maladies de la reproduction et des cancers hormono-dépendants de l utérus et du sein. L ensemble de nos résultats apportent des informations nouvelles sur les mécanismes impliqués dans la sélectivité fonctionnelle des isoformes du PR en physiopathologie, ainsi que sur la possibilité d un ciblage pharmacologique spécifique par de nouveaux antagonistes utilisables dans le traitement du cancer du sein.Progesterone receptor (PR) is an essential pharmacological target for contraception, female reproductive disorders as well as for hormone-dependent breast and uterine cancers. Human PR is expressed as two major isoforms PRA and PRB which behave as distinct transcriptional factors. PRA vs PRB expression is often altered under pathological conditions notably breast cancer through unknown mechanisms. In this thesis we demonstrate that down-regulations of PRB and PRA proteins are negatively controlled by key phosphorylation events involving distinct MAP kinase signaling. PRA is selectively stabilized by p38 MAPK whereas p42/44 MAPK specifically controls PRB stability leading to unbalanced PRA/PRB ratios in a ligand sensitive manner. In cancer cells, elevated extracellular stimuli such as epidermal growth factors or pro-inflammatory cytokines that preferentially activate p42/44 or p38 MAPK respectively may result in opposite variations in PRA/PRB expression ratio. These results may explain altered PRA/PRB ratios often associated with breast tumors. To get a mechanistic understanding of how varied PRA/PRB ratio contributes in cell signaling, we generated an original bi-inducible PR-isoform cell model allowing selective, reversible and dose-dependent expression of PRA and/or PRB, enabling fine-tune adjustment of PRA/PRB ratio in the same cells. Using this cell-based system, we undertook genome-wide transcriptomic studies to investigate transcriptional regulation driven by unliganded and liganded PR isoforms. We report that several aspects of PR signaling such as target gene selection/transcriptional regulation, cross-talk with growth factors and antiproliferative efficacy of antiprogestin are highly dependent upon variation in PRA/PRB ratio. A new potential therapeutic strategy in PR-dependent pathological conditions may rely on the use of PR antagonists. Most of the currently available antiprogestins such as mifepristone present partial agonist activity and are not selective to PR leading to undesirable side effects. Therefore, in a collaborative project we have synthesized and characterized several new PR antagonist compounds named as APRn. Structure-activity relationship studies allowed identification of the key substitutions in steroidal skeleton responsible for agonist/antagonist character of these molecules. Several selected APRn lack partial agonist effect, are PR specific and inhibit PR transcriptional properties through a new passive mechanism of action i.e. impaired recruitment of transcriptional coregulators. Such PR selective antagonists devoid of partial agonist character might provide important therapeutic perspectives for various reproductive tract abnormalities and hormone-dependent uterine and breast cancers. Altogehter, our results provide mechanistic insights into the functional selectivity of PR isoforms and their pharmacological targeting by the use of PR antagonists.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Anti-Human FSH Receptor Monoclonal Antibodies: Immunochemical and Immunocytochemical Characterization of the Receptor

International audienceThe extracellular domain of the human FSH receptor was expressed in Escherichia coli as a fusion protein with ubiquitin. It was tagged with a poly-His tract which was used for its purification. Immunization of mice allowed the preparation of high affinity antireceptor monoclonal antibodies. The latter fell into two categories:  some of them were inhibited hormone binding and adenylate cyclase activation whereas others were devoid of these properties. None of the antibodies had agonistic activity (i.e., stimulated adenylate cyclase). Immunoaffinity chromatography allowed us to purify the native receptor in a single step either from a permanently transfected L cell line (75% recovery) or from human ovaries (33% recovery). Immunoblotting of the receptor in human ovaries showed the presence of a major band of 87 kDa and of a minor band of 81 kDa. Endoglycosidase digestion and pulse−chase experiments showed the former to be the mature receptor and the latter the precursor containing mannose-rich carbohydrates. Thus, as in the case for the LH receptor, there was an accumulation (albeit to a lower degree) of the precursor in target cells. We did not detect variant forms of the protein corresponding to the alternative mRNA transcripts previously described. Additive binding to the receptor of several antibodies, but not of the same antibody, allowed us to establish a sandwich-type ELISA for the receptor (sensitivity ∼1 fmol) and to obtain evidence against the existence of previously described oligomeric forms of the protein. All monoclonal antibodies were able to label the receptor immunocytochemically in transfected cells, and two of them were also able to detect it at the markedly lower physiological concentrations, i.e., in human Sertoli and granulosa cells

JAB1 Interacts with Both the Progesterone Receptor and SRC-1

International audienceJAB1 (Jun activation domain-binding protein-1) has previously been described as a coactivator of AP1 transcription factor. We show here, by yeast and mammalian two-hybrid analyses and by pull-down experiments, that JAB1 also interacts with both the progesterone receptor (PR) and the steroid receptor coactivator 1 (SRC-1) and that it stabilizes PR-SRC-1 complexes. We also show that JAB1 potentiates the activity of a variety of transcription factors known to associate with SRC-1 (nuclear receptors, activator protein-1, and nuclear factor κB). This occurs without any modification of PR or SRC-1 concentration. JAB1 is a subunit of a large multiprotein complex that has been called the COP9 signalosome. The latter is present in plant and animal cells and has been shown to be involved in a variety of cellular mechanisms including transcription regulation, cell cycle control, and phosphorylation cascades. We now show that it is also involved in the mechanisms of action of nuclear receptors and of their coactivators

: PR-dependent breast cancer cell motility

International audienceProgesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(-) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl-amino)-phenyl-17β-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes

p38 and p42/44 MAPKs Differentially Regulate Progesterone Receptor A and B Isoform Stabilization.: Distinct MAPKs regulate PRA and PRB stability

International audienceProgesterone receptor (PR) isoforms (PRA and PRB) are implicated in the progression of breast cancers frequently associated with imbalanced PRA/PRB expression ratio. Antiprogestins represent potential antitumorigenic agents for such hormone-dependent cancers. To investigate the mechanism(s) controlling PR isoforms degradation/stability in the context of agonist and antagonist ligands, we used endometrial and mammary cancer cells stably expressing PRA and/or PRB. We found that the antiprogestin RU486 inhibited the agonist-induced turnover of PR isoforms through active mechanism(s) involving distinct MAPK-dependent phosphorylations. p42/44 MAPK activity inhibited proteasome-mediated degradation of RU486-bound PRB but not PRA in both cell lines. Ligand-induced PRB turnover required neosynthesis of a mandatory down-regulating partner whose interaction/function is negatively controlled by p42/44 MAPK. Such regulation strongly influenced expression of various endogenous PRB target genes in a selective manner, supporting functional relevance of the mechanism. Interestingly, in contrast to PRB, PRA stability was specifically increased by MAPK kinase kinase 1-induced p38 MAPK activation. Selective inhibition of p42/p44 or p38 activity resulted in opposite variations of the PRA/PRB expression ratio. Moreover, MAPK-dependent PR isoforms stability was independent of PR serine-294 phosphorylation previously proposed as a major sensor of PR down-regulation. In sum, we demonstrate that MAPK-mediated cell signaling differentially controls PRA/PRB expression ratio at posttranslational level through ligand-sensitive processes. Imbalance in PRA/PRB ratio frequently associated with carcinogenesis might be a direct consequence of disorders in MAPK signaling that might switch cellular responses to hormonal stimuli and contribute towards pathogenesis