A laminin 511 matrix is regulated by TAZ and functions as the ligand for the α6Bß1 integrin to sustain breast cancer stem cells.

Understanding how the extracellular matrix impacts the function of cancer stem cells (CSCs) is a significant but poorly understood problem. We report that breast CSCs produce a laminin (LM) 511 matrix that promotes self-renewal and tumor initiation by engaging the α6Bß1 integrin and activating the Hippo transducer TAZ. Although TAZ is important for the function of breast CSCs, the mechanism is unknown. We observed that TAZ regulates the transcription of the α5 subunit of LM511 and the formation of a LM511 matrix. These data establish a positive feedback loop involving TAZ and LM511 that contributes to stemness in breast cancer.

MUC20 expression marks the receptive phase of the human endometrium.

RESEARCH QUESTION: How does mucin MUC20 expression change during the menstrual cycle in different cell types of human endometrium? DESIGN: Study involved examination of MUC20 expression in two previously published RNA-seq datasets in whole endometrial tissue (n = 10), sorted endometrial epithelial (n = 44) or stromal (n = 42) cell samples. RNA-Seq results were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in whole tissue (n = 10), sorted epithelial (n = 17) and stromal (n = 17) cell samples. MUC20 protein localization and expression were analysed in human endometrium by immunohistochemical analysis of intact endometrial tissue (n = 6) and also Western blot of cultured stromal and epithelial cells (n = 2). RESULTS: MUC20 is differentially expressed in the endometrium between the pre-receptive and receptive phases. We show that MUC20 is predominantly expressed by epithelial cells of the receptive endometrium, both at the mRNA (RNA-Seq, P = 0.005; qRT-PCR, P = 0.039) and protein levels (Western blot; immunohistochemistry, P = 0.029). CONCLUSION: Our results indicate MUC20 as a novel marker of mid-secretory endometrial biology. We propose a model of MUC20 function in the hepatocyte growth factor (HGF)-activated mesenchymal-epithelial transition (MET) receptor signalling specifically in the receptive phase. Further investigations should reveal the precise function of MUC20 in human endometrium and the possible connection between MUC20 and HGF-activated MET receptor signalling. MUC20 could potentially be included in the list of endometrial receptivity markers after further clinical validation.

Optimizing bone morphogenic protein 4-mediated human embryonic stem cell differentiation into trophoblast-like cells using fibroblast growth factor 2 and transforming growth factor-ß/activin/nodal signalling inhibition.

Several studies have demonstrated that human embryonic stem cells (hESC) can be differentiated into trophoblast-like cells if exposed to bone morphogenic protein 4 (BMP4) and/or inhibitors of fibroblast growth factor 2 (FGF2) and the transforming growth factor beta (TGF-ß)/activin/nodal signalling pathways. The goal of this study was to investigate how the inhibitors of these pathways improve the efficiency of hESC differentiation when compared with basic BMP4 treatment. RNA sequencing was used to analyse the effects of all possible inhibitor combinations on the differentiation of hESC into trophoblast-like cells over 12 days. Genes differentially expressed compared with untreated cells were identified at seven time points. Additionally, expression of total human chorionic gonadotrophin (HCG) and its hyperglycosylated form (HCG-H) were determined by immunoassay from cell culture media. We showed that FGF2 inhibition with BMP4 activation up-regulates syncytiotrophoblast-specific genes (CGA, CGB and LGALS16), induces several molecular pathways involved in embryo implantation and triggers HCG-H production. In contrast, inhibition of the TGF-ß/activin/nodal pathway decreases the ability of hESC to form trophoblast-like cells. Information about the conditions needed for hESC differentiation toward trophoblast-like cells helps us to find an optimal model for studying the early development of human trophoblasts in normal and in complicated pregnancy.

The aryl hydrocarbon receptor regulates mouse Fshr promoter activity through an e-box binding site.

The aryl hydrocarbon receptor (AHR) mediates the toxicity of a variety of environmental chemicals. Apart from this, an understanding is emerging that the AHR has a fundamental role in female reproduction. Evidence suggests that AHR participates in regulation of follicle-stimulating hormone receptor (Fshr) transcript level in mouse ovary by binding to the promoter of this gene in vivo. The purpose of this study was to demonstrate the molecular interplay of the Fshr promoter involved in the transactivation by AHR in mouse granulosa cells. We found that AHR activates the Fshr promoter through the region from -209 to -99 bp. In this region, the importance of the E-box motif was revealed by site-directed mutagenesis followed by promoter analysis. By focusing on the DNA/protein interactions, we defined the fact that the intact E-box but not upstream transcription factor 1 (USF1), which is known to bind this motif, is necessary for AHR binding to mouse Fshr promoter. Furthermore, by constructing AHR mutants defective in DNA interaction, we confirmed the importance of DNA binding for AHR's ability to bind to and activate Fshr promoter. Collectively, the present study demonstrates that AHR modulates Fshr transactivation by its direct association through an E-box and not by recruitment via interaction with USFs. These observations suggest that although AHR and USF may respond to different signals, they compete on binding to the same E-box. Our data, together with that from one prior study suggesting involvement of E-box motif in AHR-mediated transcription, provide novel understanding of the way in which AHR may regulate its target genes through E-box sites.

Megakaryocytic cells synthesize and platelets secrete alpha5-laminins, and the endothelial laminin isoform laminin 10 (alpha5beta1gamma1) strongly promotes adhesion but not activation of platelets.

Following vascular injury, basement membrane (BM) components of the blood vessels are exposed to circulating cells and may contribute to hemostasis and/or thrombosis. Laminins 8 (LN-8) (alpha4beta1gamma1) and 10 (LN-10) (alpha5beta1gamma1) are major laminin isoforms of the endothelial BM, and LN-8 is also secreted by activated platelets. In the present study, we demonstrate synthesis of alpha5-laminins LN-10 and LN-11 (alpha5beta2gamma1) by megakaryocytic cells, and intracellular expression of these laminin isoforms in blood platelets. In contrast to platelet LN alpha4 chain that had an apparent molecular weight of 180 kDa and associated mostly to LNbeta1 chain, platelet LNalpha5 consisted of 300/350 kDa polypeptides and associated mainly to LNbeta2. Both alpha4- and alpha5-laminins were secreted by platelets following stimulation. When compared to recombinant human (rh) LN-8, rhLN-10 was much more adhesive to platelets, though adhesion to both proteins was largely mediated via alpha6beta1 integrin. In spite of their adhesive properties, rhLN-8 and rhLN-10 induced neither P-selectin expression nor cell aggregation, two signs of platelet activation. This study demonstrates synthesis/expression of heterotrimeric alpha5-laminins in hematopoietic/blood cells, and provides evidence for the adhesive, but not activating, role of endothelial laminin isoforms in platelet biology.

Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells.

Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, ß1, ß2 and γ1 chains, hESC express α2, α3, ß3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain-145 kDa-accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.

Laminin-rich blood vessels display activated growth factor signaling and act as the proliferation centers in Dupuytren's contracture.

INTRODUCTION: Dupuytren's contracture (DC) is a chronic fibroproliferative disease of the hand, which is characterized by uncontrolled proliferation of atypical myofibroblasts at the cellular level. We hypothesized that specific areas of the DC tissue are sustaining the cell proliferation and studied the potential molecular determinants that might contribute to the formation of such niches. METHODS: We studied the expression pattern of cell proliferation marker Ki67, phosphorylated AKT (Ak mouse strain thymoma) kinase, DC-associated growth factors (connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 2 (IGF-2)) and extracellular matrix components (laminins, fibronectin, collagen IV) in DC tissue and normal palmar fascia using immunofluorescence microscopy and quantitative real-time polymerase chain reaction (qPCR). RESULTS: We found that proliferative cells in the DC nodules were concentrated in the immediate vicinity of small blood vessels and localized predominantly in the myofibroblast layer. Correspondingly, the DC-associated blood vessels contained increased levels of phosphorylated AKT, a hallmark of activated growth factor signaling. When studying the expression of potential activators of AKT signaling we found that the expression of bFGF was confined to the endothelium of the small blood vessels, IGF-2 was present uniformly in the DC tissue and CTGF was expressed in the DC-associated sweat gland acini. In addition, the blood vessels in DC nodules contained increased amounts of laminins 511 and 521, which have been previously shown to promote the proliferation and stem cell properties of different cell types. CONCLUSIONS: Based on our findings, we propose that in the DC-associated small blood vessels the presence of growth factors in combination with favorable extracellular matrix composition provide a supportive environment for sustained proliferation of myofibroblasts and thus the blood vessels play an important role in DC pathogenesis.

Transcriptional repression of the Ahr gene by LHCGR signaling in preovulatory granulosa cells is controlled by chromatin accessibility.

Recent advances in establishing the role of the aryl hydrocarbon receptor (Ahr) in normophysiology have discovered its fundamental role, amongst others, in female reproduction. Considering previous studies suggesting the hormonal modulation of Ahr, we aimed to investigate whether in murine granulosa cells (GCs) the gonadotropins regulate Ahr expression and how this is mechanistically implemented. We found that the FSH-like substance--pregnant mare serum gonadotropin--led to stimulation of Ahr expression. More importantly hCG produced relatively rapid reduction of Ahr mRNA in GCs of preovulatory follicles. We show for the first time that LHCGR signaling in regulating the Ahr message involves protein kinase A pathway and is attributable to decreased transcription rate. Finally, we found that Ahr promoter accessibility was decreased by hCG, implicating chromatin remodeling in Ahr gene regulation by LH.

Monoclonal antibodies to human laminin α4 chain globular domain inhibit tumor cell adhesion and migration on laminins 411 and 421, and binding of α6ß1 integrin and MCAM to α4-laminins.

α4-Laminins, such as laminins 411 and 421, are mesenchymal laminins expressed by vascular and lymphatic endothelial cells, leukocytes and other normal cell types. These laminins are recognized by α6ß1 and α6ß4 integrins and MCAM (CD146), and promote adhesion and migration of the cells. α4-Laminins are also expressed and secreted by some tumor cells and strongly promote tumor cell migration. Moreover, the abluminal side of blood and/or lymphatic vessels and the nerve perineurium, common tracks of tumor cell dissemination, express α4-laminins, and these laminin isoforms, when expressed in the stroma, may contribute to tumor invasion. In the present study, we examined ten mAbs to human laminin α4 chain for their reactivity with the isolated laminin α4 globular domain, their ability to inhibit tumor cell adhesion and migration on laminins 411 and 421, and their effect on the binding of α6ß1 integrin and MCAM to both α4-laminins. Most of the mAbs reacted with the laminin α4 globular domain, but only two, mAbs FC10 and 084, significantly inhibited tumor cell adhesion and migration on laminin-411. When used in combination, these antibodies practically abolished the cell adhesion and migration on laminin-411 and significantly reduced the cellular responses on laminin-421. Accordingly, mAbs FC10 and 084 significantly inhibited the binding of purified α6ß1 integrin and MCAM to laminins 411 and 421. These results indicate that mAbs to the laminin α4 globular domain are able to inhibit tumor cell adhesion and migration on laminins 411 and 421, and that α6ß1 integrin and MCAM bind α4-laminins at very close sites on the globular domain. These reagents contribute to a better understanding of the biology of α4-laminins and may have a therapeutic potential in malignant and inflammatory diseases.

Laminin production and basement membrane deposition by mesenchymal stem cells upon adipogenic differentiation.

The aim was to study laminin (LM) synthesis, integration, and deposition into the basement membrane (BM) during adipogenesis. Human bone marrow-derived mesenchymal stromal cells (MSCs) were induced along the adipogenic lineage. LM chain mRNA and protein levels were followed using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining, transmission electron microscopy (TEM), and immunoprecipitation. MSCs produced low levels of LM mRNAs but were not surrounded by BM in IF and TEM imaging. LM-α4, LM-ß1, and LM-γ1 mRNAs increased during adipogenesis 3.9-, 5.8-, and 2.8-fold by day 28. LM-411 was immunoprecipitated from the ECM of the differentiated cells. Immunostaining suggested deposition of LM-411 and some LM-421. BM build-up was probably organized in part by integrin (Int) α6ß1. At day 28, TEM images revealed BM-like structures around fat droplet-containing cells. The first signs of BM formation and Int α6ß1 were seen using IF imaging at day 14. Laminin-411 and Int α6ß1 were expressed in vivo in mature human subcutaneous fat tissue. Undifferentiated human MSCs did not organize LM subunits into BM, whereas LM-411 and some LM-421 are precipitated in the BM around adipocytes. This is the first demonstration of LM-411 precipitation during hMSC adipogenesis around adipocytes as a structural scaffold and Int-regulated signaling element.

A novel monoclonal antibody to human laminin α5 chain strongly inhibits integrin-mediated cell adhesion and migration on laminins 511 and 521.

Laminins, a large family of αßγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα) chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs) to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5ß1γ1) and 521 (α5ß2γ1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3ß1/α6ß1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3ß1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMß1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.

An endothelial laminin isoform, laminin 8 (alpha4beta1gamma1), is secreted by blood neutrophils, promotes neutrophil migration and extravasation, and protects neutrophils from apoptosis.

During extravasation, neutrophils migrate through the perivascular basement membrane (BM), a specialized extracellular matrix rich in laminins. Laminins 8 (LN-8) (alpha4beta1gamma1) and 10 (LN-10) (alpha5beta1gamma1) are major components of the endothelial BM, but expression, recognition, and use of these laminin isoforms by neutrophils are poorly understood. In the present study, we provide evidence, using a panel of novel monoclonal antibodies against human laminin alpha4 (LNalpha4) chain, that neutrophils contain and secrete LN-8, and that this endogenous laminin contributes to chemoattractant-induced, alphaMbeta2-integrin-dependent neutrophil migration through albumin-coated filters. Phorbol ester-stimulated neutrophils adhered to recombinant human (rh) LN-8, rhLN-10, and mouse LN-1 (mLN-1) (alpha1beta1gamma1) via alphaMbeta2-integrin, and these laminin isoforms strongly promoted chemoattractant-induced neutrophil migration via the same integrin. However, only rhLN-8 enhanced the spontaneous migration. In addition, recruitment of neutrophils into the peritoneum following an inflammatory stimulus was impaired in LNalpha4-deficient mice. rhLN-8 also protected isolated neutrophils from spontaneous apoptosis. This study is the first to identify a specific laminin isoform in neutrophils and provides evidence for the role of LN-8 in the adhesion, migration, extravasation, and survival of these cells.