BRAFV600E Expression in Thyrocytes Causes Recruitment of Immunosuppressive STABILIN-1 Macrophages.

Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAFV600E genetic alteration is found in 60% of this endocrine cancer. This oncogene is associated with poor prognosis, resistance to radioiodine therapy, and tumor progression. Histological follow-up by anatomo-pathologists revealed that two-thirds of surgically-removed thyroids do not present malignant lesions. Thus, continued fundamental research into the molecular mechanisms of TC downstream of BRAFV600E remains central to better understanding the clinical behavior of these tumors. To study PTC, we used a mouse model in which expression of BRAFV600E was specifically switched on in thyrocytes by doxycycline administration. Upon daily intraperitoneal doxycycline injection, thyroid tissue rapidly acquired histological features mimicking human PTC. Transcriptomic analysis revealed major changes in immune signaling pathways upon BRAFV600E induction. Multiplex immunofluorescence confirmed the abundant recruitment of macrophages, among which a population of LYVE-1+/CD206+/STABILIN-1+ was dramatically increased. By genetically inactivating the gene coding for the scavenger receptor STABILIN-1, we showed an increase of CD8+ T cells in this in situ BRAFV600E-dependent TC. Lastly, we demonstrated the presence of CD206+/STABILIN-1+ macrophages in human thyroid pathologies. Altogether, we revealed the recruitment of immunosuppressive STABILIN-1 macrophages in a PTC mouse model and the interest to further study this macrophage subpopulation in human thyroid tissues.

BRAFV600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment.

Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment.

Sox9 is involved in the thyroid differentiation program and is regulated by crosstalk between TSH, TGFß and thyroid transcription factors.

While the signaling pathways and transcription factors involved in the differentiation of thyroid follicular cells, both in embryonic and adult life, are increasingly well understood, the underlying mechanisms and potential crosstalk between the thyroid transcription factors Nkx2.1, Foxe1 and Pax8 and inductive signals remain unclear. Here, we focused on the transcription factor Sox9, which is expressed in Nkx2.1-positive embryonic thyroid precursor cells and is maintained from embryonic development to adulthood, but its function and control are unknown. We show that two of the main signals regulating thyroid differentiation, TSH and TGFß, modulate Sox9 expression. Specifically, TSH stimulates the cAMP/PKA pathway to transcriptionally upregulate Sox9 mRNA and protein expression, a mechanism that is mediated by the binding of CREB to a CRE site within the Sox9 promoter. Contrastingly, TGFß signals through Smad proteins to inhibit TSH-induced Sox9 transcription. Our data also reveal that Sox9 transcription is regulated by the thyroid transcription factors, particularly Pax8. Interestingly, Sox9 significantly increased the transcriptional activation of Pax8 and Foxe1 promoters and, consequently, their expression, but had no effect on Nkx2.1. Our study establishes the involvement of Sox9 in thyroid follicular cell differentiation and broadens our understanding of transcription factor regulation of thyroid function.

Modelling of Epithelial Growth, Fission and Lumen Formation During Embryonic Thyroid Development: A Combination of Computational and Experimental Approaches.

Organogenesis is the phase of embryonic development leading to the formation of fully functional organs. In the case of the thyroid, organogenesis starts from the endoderm and generates a multitude of closely packed independent spherical follicular units surrounded by a dense network of capillaries. Follicular organisation is unique and essential for thyroid function, i.e. thyroid hormone production. Previous in vivo studies showed that, besides their nutritive function, endothelial cells play a central role during thyroid gland morphogenesis. However, the precise mechanisms and biological parameters controlling the transformation of the multi-layered thyroid epithelial primordium into a multitude of single-layered follicles are mostly unknown. Animal studies used to improve understanding of organogenesis are costly and time-consuming, with recognised limitations. Here, we developed and used a 2-D vertex model of thyroid growth, angiogenesis and folliculogenesis, within the open-source Chaste framework. Our in silico model, based on in vivo images, correctly simulates the differential growth and proliferation of central and peripheral epithelial cells, as well as the morphogen-driven migration of endothelial cells, consistently with our experimental data. Our simulations further showed that reduced epithelial cell adhesion was critical to allow endothelial invasion and fission of the multi-layered epithelial mass. Finally, our model also allowed epithelial cell polarisation and follicular lumen formation by endothelial cell abundance and proximity. Our study illustrates how constant discussion between theoretical and experimental approaches can help us to better understand the roles of cellular movement, adhesion and polarisation during thyroid embryonic development. We anticipate that the use of in silico models like the one we describe can push forward the fields of developmental biology and regenerative medicine.

Vaginal tuberculosis as differential diagnosis of cancer: A case report.

Tuberculosis remains a worrying public health problem. But if pulmonary tuberculosis's symptomatology is well known by the medical profession, this is not the case of genital tuberculosis. We take advantage of a case of vaginal tuberculosis to review the international literature about clinical diagnosis, further tests, and treatment of this extremely rare tuberculosis localization.

Vaginal tuberculosis as differential diagnosis of cancer: A case report.

Tuberculosis remains a worrying public health problem. But if pulmonary tuberculosis's symptomatology is well known by the medical profession, this is not the case of genital tuberculosis. We take advantage of a case of vaginal tuberculosis to review the international literature about clinical diagnosis, further tests, and treatment of this extremely rare tuberculosis localization.

Chronic liver injury promotes hepatocarcinoma cell seeding and growth, associated with infiltration by macrophages.

Ninety percent of hepatocarcinoma (HCC) develops in a chronically damaged liver. Interactions between non-tumor stromal components, especially macrophages, and cancer cells are still incompletely understood. Our aim was to determine whether a chronically injured liver represents a favorable environment for the seeding and growth of HCC cells, and to evaluate the potential roles of macrophages infiltrated within the tumor. HCC cells were injected into the liver in healthy mice (healthy liver group [HL]) and in mice chronically treated with carbon tetrachloride (CCl4 ) for 7 weeks (CCl4 7w group). Livers were examined for the presence of tumor 2 weeks post-injection. Tumor and non-tumor tissues were analyzed for macrophage infiltration, origin (monocytes-derived vs resident macrophages) and polarization state, and MMP production. Fifty-three percent of mice developed neoplastic lesion in the HL group whereas a tumor lesion was found in all livers in the CCl4 7w group. Macrophages infiltrated more deeply the tumors of the CCl4 7w group. Evaluation of factors involved in the recruitment of macrophages and of markers of their polarization state was in favor of prominent infiltration of M2 pro-tumor monocyte-derived macrophages inside the tumors developing in a chronically injured liver. MMP-2 and -9 production, attributed to M2 pro-tumor macrophages, was significantly higher in the tumors of the CCl4 7w group. In our model, chronic liver damage promotes cancer development. Our results suggest that an injured background favors the infiltration of M2 pro-tumor monocyte-derived macrophages. These secrete MMP-2 and MMP-9 that promote tumor progression.

Cellular uptake of proMMP-2:TIMP-2 complexes by the endocytic receptor megalin/LRP-2.

Matrix metalloproteinases (MMPs) are regulated at multiple transcriptional and post-transcriptional levels, among which receptor-mediated endocytic clearance. We previously showed that low-density lipoprotein receptor-related protein-1 (LRP-1) mediates the clearance of a complex between the zymogen form of MMP-2 (proMMP-2) and tissue inhibitor of metalloproteinases, TIMP-2, in HT1080 human fibrosarcoma cells. Here we show that, in BN16 rat yolk sac cells, proMMP-2:TIMP-2 complex is endocytosed through a distinct LRP member, megalin/LRP-2. Addition of receptor-associated protein (RAP), a natural LRP antagonist, caused accumulation of endogenous proMMP-2 and TIMP-2 in conditioned media. Incubation with RAP also inhibited membrane binding and cellular uptake of exogenous iodinated proMMP-2:TIMP-2. Moreover, antibodies against megalin/LRP-2, but not against LRP-1, inhibited binding of proMMP-2:TIMP-2 to BN16 cell surface. BIAcore analysis confirmed direct interaction between the complex and megalin/LRP-2. Conditional renal invalidation of megalin/LRP-2 in mice resulted in accumulation of proMMP-2 and TIMP-2 in their urine, highlighting the physiological relevance of the binding. We conclude that megalin/LRP-2 can efficiently mediate cell-surface binding and endocytosis of proMMP-2:TIMP-2 complex. Therefore megalin/LRP-2 can be considered as a new actor in regulation of MMP-2 activity, an enzyme crucially involved in many pathological processes.

Thyroid follicle development requires Smad1/5- and endothelial cell-dependent basement membrane assembly.

Thyroid follicles, the functional units of the thyroid gland, are delineated by a monolayer of thyrocytes resting on a continuous basement membrane. The developmental mechanisms of folliculogenesis, whereby follicles are formed by the reorganization of a non-structured mass of non-polarized epithelial cells, are largely unknown. Here we show that assembly of the epithelial basement membrane is crucial for folliculogenesis and is controlled by endothelial cell invasion and by BMP-Smad signaling in thyrocytes. Thyroid-specific Smad1 and Smad5 double-knockout (Smad1/5(dKO)) mice displayed growth retardation, hypothyroidism and defective follicular architecture. In Smad1/5(dKO) embryonic thyroids, epithelial cells remained associated in large clusters and formed small follicles. Although similar follicular defects are found in Vegfa knockout (Vegfa(KO)) thyroids, Smad1/5(dKO) thyroids had normal endothelial cell density yet impaired endothelial differentiation. Interestingly, both Vegfa(KO) and Smad1/5(dKO) thyroids displayed impaired basement membrane assembly. Furthermore, conditioned medium (CM) from embryonic endothelial progenitor cells (eEPCs) rescued the folliculogenesis defects of both Smad1/5(dKO) and Vegfa(KO) thyroids. Laminin α1, ß1 and γ1, abundantly released by eEPCs into CM, were crucial for folliculogenesis. Thus, epithelial Smad signaling and endothelial cell invasion promote folliculogenesis via assembly of the basement membrane.

Triterpenes from the exudate of Gardenia urvillei.

A cycloartane gardurvilleic acid, three 3,4-seco-cycloartanes securvienol, secodienurvilleic acid, securvitriol, a 3,4;9,10-seco-cycloartane gardheptlactone, two dammaranes urvilone, urvilol, along with eight known cycloartanes and 3,4-seco-cycloartanes and four known dammaranes have been isolated from the bud exudate of Gardenia urvillei, an endemic tree to the New Caledonian dry forest. Two other dammarane derivatives have been obtained by semisynthesis. The structures of the original compounds were determined by spectroscopic methods and chemical correlations. In association with previously published data, the description of oxidized side-chains in position 17 are now available for two couples of diastereoisomers. Evaluation of anti-parasite activity and cytotoxicity has shown noticeable results for some of the isolated triterpenes.

Induction of post-menstrual regeneration by ovarian steroid withdrawal in the functionalis of xenografted human endometrium.

STUDY QUESTION: Does the endometrial functionalis have the potential to undergo self-renewal after menstruation and how is this process controlled by ovarian steroids? SUMMARY ANSWER: Endometrial xenografts subjected to withdrawal of estradiol and progesterone shrink but also show signs of proliferation and tissue repair; new estradiol supply prevents atrophy but is not sufficient to increase graft volume. WHAT IS KNOWN ALREADY: Menstruation, i.e. cyclic proteolysis of the extracellular matrix of endometrial functionalis, is induced by a fall in estrogen and progesterone concentration and is followed by tissue regeneration. However, there is debate about whether regenerating cells must originate from the basalis or from stem cells and whether new estrogen supply is required for the early repair concomitant with menstruation. STUDY DESIGN, SIZE, DURATION: Fragments from human endometrial functionalis (from 24 hysterectomy specimens) were xenografted in ovariectomized SCID mice and submitted to a 4-day estradiol and progesterone withdrawal (to mimic menstruation) followed by re-exposure to estradiol (to mimic the proliferative phase). We measured signs of proliferation and changes in graft volume. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrium was collected from spontaneously cycling women. Cell proliferation was examined by immunolabeling Ki-67, cyclin D1 and phosphorylated-histone H3. Xenograft volume was measured by magnetic resonance imaging. Xenograft histomorphometry was performed to determine how the different tissue compartments contributed to volume change. MAIN RESULTS AND THE ROLE OF CHANCE: Hormone withdrawal induced a rapid decrease in graft volume mainly attributable to stroma condensation and breakdown, concomitant with an increase of proliferation markers. Reinsertion of estradiol pellets after induced menstruation blocked volume decrease and stimulated epithelial and stromal growth, but, surprisingly, did not induce graft enlargement. Reinsertion of both estradiol and progesterone pellets blocked apoptosis. LIMITATIONS, REASONS FOR CAUTION: Mechanisms of endometrial remodeling are different in women and mice and the contribution of circulating inflammatory cells in both species remains to be clarified. Moreover, during human menstruation, endometrial fragments resulting from tissue proteolysis can be expelled by the menstrual flow, unlike in this model. WIDER IMPLICATIONS OF THE FINDINGS: Menstruation is a multifocal event within the functionalis. This is the first evidence that endometrial fragments that are not shed after menstrual tissue breakdown can support endometrial regeneration. Endometriosis is commonly thought to result from the retrograde migration of menstrual fragments of the degraded functionalis into the peritoneal cavity. Our study supports their potential to regenerate as ectopic endometrium. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Fonds de la Recherche Scientifique Médicale, Concerted Research Actions, Communauté Française de Belgique, Région wallonne, Région bruxelloise and Loterie nationale. P.H. and B.F.J. are research associates of the Belgian Fonds de la Recherche Scientifique (F.R.S.-F.N.R.S.). E.M. is Associate Editor at Human Reproduction. There is no conflict of interest to declare.

Matrix metalloproteinase-27 is expressed in CD163+/CD206+ M2 macrophages in the cycling human endometrium and in superficial endometriotic lesions.

Matrix metalloproteinases (MMPs) are key enzymes involved in extracellular matrix remodelling. In the human endometrium, the expression and activity of several MMPs are maximal during the menstrual phase. Moreover, MMPs are thought to be involved in the pathogenesis of endometriosis and cancers, in particular with invasion and metastasis. We recently reported that MMP-27 is a unique MMP with an intracellular retention motif. We investigated the expression and cellular localization of MMP-27 in the cycling human endometrium and in endometriotic lesions. MMP-27 mRNA was detected throughout the menstrual cycle. Despite large interpatient variations, mRNA levels increased from the proliferative to the secretory phase, to peak during the menstrual phase. MMP-27 was immunolocalized in large isolated cells scattered throughout the stroma and around blood vessels: these cells were most abundant at menstruation and were identified by immunofluorescence as CD45(+), CD163(+) and CD206(+) macrophages. CD163(+) macrophages were also abundant in endometriotic lesions, but showed different patterns in ovarian or peritoneal endometriotic lesions (co-labelling for CD206 and MMP-27) and rectovaginal lesions (no co-labelling). In conclusion, MMP-27 is expressed in a subset of endometrial macrophages related to menstruation and in ovarian and peritoneal endometriotic lesions.

Hypoxia is not required for human endometrial breakdown or repair in a xenograft model of menstruation.

Menstrual endometrial breakdown induced by estradiol and progesterone withdrawal is regularly attributed to vasospasm of spiral arteries causing ischemia and hypoxia. We investigated whether hypoxia actually occurred in an in vivo model of menstruation. Three complementary approaches were used to look for signs of hypoxia in fragments of human functionalis xenografted to ovariectomized immunodeficient mice bearing pellets-releasing estradiol and progesterone, and then deprived of ovarian steroids. Hormone withdrawal 21 d after grafting induced menstrual breakdown and MMP expression within 4 d. Local partial oxygen pressure (pO2) was measured by electron paramagnetic resonance using implanted lithium phtalocyanine crystals. In mice with hormone maintenance until sacrifice, pO2 was low one week after grafting (14.8±3.4 mmHg) but increased twofold from the second week when tissue was largely revascularized. After 3 wk, pO2 was not modified by hormone withdrawal but was slightly increased on hormone reimpregnation 4 d after removal (34.7±6.1 mmHg) by comparison with hormone maintenance (27.1±8.6 mmHg). These results were confirmed using fluorescence quenching-based OxyLite measurements. In a further search for signs of hypoxia, we did not find significant HIF1-α immunostaining, nor pimonidazole adducts after hormone withdrawal. We conclude that hypoxia is not needed to trigger menstrual-like tissue breakdown or repair in human endometrial xenograft.

Dinuclear mixed-valence Co(III)Co(II) complexes derived from a macrocyclic ligand: unique example of a Co(III)Co(II) complex showing catecholase activity.

The work in this paper presents the syntheses, characterization, catecholase activity, and electrospray ionization mass spectroscopic (ESI-MS positive) study of three mixed-valence dinuclear Co(III)Co(II) complexes of composition [Co(III)Co(II)L(N(3))(3)]·CH(3)CN (1), [Co(III)Co(II)L(OCN)(3)]·CH(3)CN (2), and [Co(III)Co(II)L(µ-CH(3)COO)(2)](ClO(4)) (3), derived from a tetraimino diphenolate macrocyclic ligand H(2)L, obtained on [2 + 2] condensation of 4-ethyl-2,6-diformylphenol and 2,2'-dimethyl-1,3-diaminopropane. While 1 and 2 are diphenoxo-bridged, 3 is a heterobridged bis(µ-phenoxo)bis(µ-acetate) system. Utilizing 3,5-di-tert-butyl catechol (3,5-DTBCH(2)) as the substrate, the catecholase activity of all the three complexes has been checked in methanol/acetonitrile/N,N-dimethyl formamide. While 2 and 3 are inactive, complex 1 shows catecholase activity with turnover numbers of 482.16 h(-1) and 45.38 h(-1) in acetonitrile and methanol, respectively. Electrospray ionization mass (ESI-MS positive) spectra of complexes 1-3 have been recorded in acetonitrile solutions and the positive ions have been well characterized. The ESI-MS positive spectrum of complex 1 in the presence of 3,5-DTBCH(2) has also been recorded and, interestingly, two positive ions [Co(III)Co(II)L(N(3))(2)(3,5-DTBCH(-))H](+) and [Co(II)Co(II)L(µ-3,5-DTBCH(2-))Na](+) have been identified.

Cell cholesterol modulates metalloproteinase-dependent shedding of low-density lipoprotein receptor-related protein-1 (LRP-1) and clearance function.

Low-density lipoprotein receptor-related protein-1 (LRP-1) is a plasma membrane scavenger and signaling receptor, composed of a large ligand-binding subunit (515-kDa α-chain) linked to a shorter transmembrane subunit (85-kDa ß-chain). LRP-1 cell-surface level and function are controlled by proteolytic shedding of its ectodomain. Here, we identified ectodomain sheddases in human HT1080 cells and demonstrated regulation of the cleavage by cholesterol by comparing the classical fibroblastoid type with a spontaneous epithelioid variant, enriched ∼ 2-fold in cholesterol. Two membrane-associated metalloproteinases were involved in LRP-1 shedding: a disintegrin and metalloproteinase-12 (ADAM-12) and membrane-type 1 matrix metalloproteinase (MT1-MMP). Although both variants expressed similar levels of LRP-1, ADAM-12, MT1-MMP, and specific tissue inhibitor of metalloproteinases-2 (TIMP-2), LRP-1 shedding from epithelioid cells was ∼4-fold lower than from fibroblastoid cells. Release of the ectodomain was triggered by cholesterol depletion in epithelioid cells and impaired by cholesterol overload in fibroblastoid cells. Modulation of LRP-1 shedding on clearance was reflected by accumulation of gelatinases (MMP-2 and MMP-9) in the medium. We conclude that cholesterol exerts an important control on LRP-1 levels and function at the plasma membrane by modulating shedding of its ectodomain, and therefore represents a novel regulator of extracellular proteolytic activities.

Ovarian steroids, mitogen-activated protein kinases, and/or aspartic proteinases cooperate to control endometrial remodeling by regulating gene expression in the stroma and glands.

Explants from nonmenstrual endometria cultured in the absence of ovarian hormones undergo tissue breakdown. Addition of estradiol and progesterone (EP) prevents proteolysis. Explants include stromal and epithelial compartments which play different but complementary roles in endometrial physiology, including tissue remodeling and hormonal response. In order to characterize the cell type-specific contribution to regulation of tissue breakdown, we characterized the transcriptomes of microdissected stromal and glandular areas from endometrial explants cultured with or without EP. The datasets were also compared to other published endometrial transcriptomes. Finally, the contribution of proteolysis, hypoxia, and MAPKs to the regulation of selected genes was further investigated in explant culture. This analysis identified distinct gene expression profiles in stroma and glands, with differential response to EP, but functional clustering underlined convergence in biological processes, further indicating that endometrial remodeling requires cooperation between the two compartments through expression of cell type-specific genes. Only partial overlaps were observed between lists of genes involved in different occurrences of endometrial breakdown, pointing to a limited number of potentially crucial regulators but also to the requirement for additional mechanisms controlling tissue remodeling. We identified a group of genes differentially regulated by EP in stroma and glands among which some were sensitive to MAPKs and/or aspartic proteinases and were not induced by hypoxia. In conclusion, MAPKs and/or aspartic proteinases likely act in concert with EP to locally and specifically control differential expression of genes between degrading and preserved areas of the human endometrium.

Bis(2,2'-bipyridyl-κN,N')bis-(2-hydroxy-benzoato)-κO;κO,O-cadmium(II) methanol solvate.

The title compound, [Cd(C(7)H(5)O(3))(2)(C(10)H(8)N(2))(2)]·CH(3)OH, contains one monomeric seven-coordinate cadmium complex and one methanol solvate mol-ecule. The Cd(II) atom is coordinated to two 2,2'-bipyridyl ligands via the N atoms and to two salicylate anions (Hsal(-)) via the carboxyl-ate O atoms, which act as monodentate ligand for the one and bidentate ligand for the second. The Cd(II) atom exhibits a {6 + 1} environment, approximately described as a distorted capped octa-hedron with the apical positions occupied by one of the two N atoms belonging to one bipyridyl ligand and one of the two carboxyl-ate O atoms from the monodentate Hsal(-) ligand. Two intra-molecular six-membered hydrogen-bonded rings are present, generated from inter-actions between the carboxyl-ate and hydr-oxy groups of the salicylate ligands. There is one inter-molecular hydrogen-bonding inter-action involving the methanol solvent mol-ecule and the carboxyl-ate group from the monodentate Hsal(-) ligand. The crystal packing is governed by π-π stacking inter-actions [centroid-centroid distance = 3.783 (4) Å] which occur between bipyridyl ligands, by C-H⋯O and C-H⋯π inter-actions and by numerous van der Waals contacts.

Spatiotemporal coupling of focal extracellular matrix degradation and reconstruction in the menstrual human endometrium.

Coupling of focal degradation and renewal of the functional layer of menstrual endometrium is a key event of the female reproductive biology. The precise mechanisms by which the various endometrial cell populations control extracellular matrix (ECM) degradation in the functionalis while preserving the basalis and the respective contribution of basalis and functionalis in endometrium regeneration are still unclear. We therefore compared the transcriptome of stromal and glandular cells isolated by laser capture microdissection from the basalis as well as degraded and preserved areas of the functionalis in menstrual endometria. Data were validated by in situ hybridization. Expression profile of selected genes was further analyzed throughout the menstrual cycle, and their response to ovarian steroids withdrawal was studied in a mouse xenograft model. Immunohistochemistry confirmed the results at the protein level. Algorithms for sample clustering segregated biological samples according to cell type and tissue depth, indicating distinct gene expression profiles. Pairwise comparisons identified the greatest numbers of differentially expressed genes in the lysed functionalis when compared with the basalis. Strikingly, in addition to genes products associated with tissue degradation (matrix metalloproteinase and plasmin systems) and apoptosis, superficial lysed stroma was enriched in gene products associated with ECM biosynthesis (collagens and their processing enzymes). These results support the hypothesis that fragments of the functionalis participate in endometrial regeneration during late menstruation. Moreover, menstrual reflux of lysed fragments overexpressing ECM components and adhesion molecules could easily facilitate implantation of endometriotic lesions.

Metalloproteinase-dependent shedding of low-density lipoprotein receptor-related protein-1 ectodomain decreases endocytic clearance of endometrial matrix metalloproteinase-2 and -9 at menstruation.

Cyclic elimination of the endometrium functional layer through menstrual bleeding results from intense tissue breakdown by proteolytic enzymes, mainly members of the matrix metalloproteinase (MMP) family. In contrast to menstrual-restricted MMPs, e.g. interstitial collagenase (MMP-1), gelatinases A (MMP-2) and B (MMP-9) mRNAs are abundant throughout the cycle without detectable tissue degradation at proliferative and secretory phases, implying a tight posttranslational control of both gelatinases. This paper addresses the role of low-density lipoprotein receptor-related protein (LRP)-1 in the endocytic clearance of endometrial gelatinases. LRP-1 mRNA and protein were studied using RT-PCR, Western blotting, and immunolabeling. Posttranslational control of LRP-1 was analyzed in explant culture. The receptor-associated protein (RAP), used as LRP antagonist, strongly increased (pro)gelatinase accumulation in medium conditioned by endometrial explants, suggesting a role for LRP-1 in their clearance. Although LRP-1 mRNA remained constant throughout the cycle, the protein ectodomain vanished at menses. LRP-1 immunolabeling selectively disappeared in areas of extracellular matrix breakdown in menstrual samples. It also disappeared from explants cultured without estrogen and progesterone (EP) due to ectodomain shedding in the medium. The shedding was inhibited by metalloproteinase inhibitors, including a disintegrin and metalloproteinase (ADAM) inhibitor, and by tissue inhibitors of MMPs (TIMP)-3 and -2, but barely by TIMP-1, pointing to ADAM-12 as the putative sheddase. In good agreement, ADAM-12 mRNA expression was repressed by EP. In conclusion, the efficient LRP-1-mediated clearance of gelatinase activity in nonbleeding endometrium is abrogated upon EP withdrawal, due to shedding of LRP-1 ectodomain by a metalloproteinase, presumably ADAM-12, itself regulated by EP.

Opposite regulation of transforming growth factors-beta2 and -beta3 expression in the human endometrium.

TGF-betas have been reported to mediate the repression by progesterone of several matrix metalloproteinases in the human endometrium, thereby preventing menstrual breakdown. Because of conflicting reports on the expression profiles, source, and regulation of the TGF-beta system in this tissue, we investigated by real-time RT-PCR and ELISA the expression of the three TGF-betas (total and mature forms) and their two receptors throughout the menstrual cycle, and their regulation by ovarian steroids in cultured explants including their microdissected epithelial and stromal compartments. Regulation by cAMP and MAPK was further investigated. This comprehensive study on a large collection of endometrial samples evidenced a differential regulation of TGF-beta isoforms expression, both in vivo and in explant culture. In vivo, TGF-beta2 increased by about 5-fold at the mid-late secretory phase then declined after menstruation; TGF-beta3 increased at menstruation and remained high during the proliferative phase; TGF-beta1 was maximal at menstruation. In explants cultured without ovarian steroids both TGF-beta2 and -beta3 were preferentially expressed in the stroma. Ovarian steroids strongly repressed both TGF-beta2 and -beta3 in stroma but only TGF-beta2 in glands. cAMP prevented inhibition by ovarian steroids of TGF-beta2 but not -beta3. In presence of ovarian steroids, MAPK inhibitors (p38 and ERK pathways) stimulated TGF-beta3 but inhibited TGF-beta2 expression. In conclusion, TGF-beta2 and -beta3 are differentially expressed during the menstrual cycle and regulated by progesterone in epithelial vs stromal cells. The opposite regulation of TGF-beta2 and -beta3 by cAMP and MAPK could account for their distinct expression in vivo.