Sulev Ingerpuu - Academia.edu (original) (raw)
Papers by Sulev Ingerpuu
Laminins, a large family of abc heterotrimeric proteins mainly found in basement membranes, are s... more Laminins, a large family of abc 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 a (LMa) chains, a5 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 LMa5 chain to further study the biological relevance of a5 laminins, such as laminins 511 (a5b1c1) and 521 (a5b2c1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMa5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited a3b1/a6b1 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...
Arthritis Research & Therapy, 2015
Introduction: Dupuytren's contracture (DC) is a chronic fibroproliferative disease of the hand, w... more 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.
Plos One, 2013
Laminins, a large family of abc heterotrimeric proteins mainly found in basement membranes, are s... more Laminins, a large family of abc 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 a (LMa) chains, a5 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 LMa5 chain to further study the biological relevance of a5 laminins, such as laminins 511 (a5b1c1) and 521 (a5b2c1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMa5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited a3b1/a6b1 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 a3b1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMa5 chain but largely hindered by mAb 4E10 to a LMb1 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 LMa5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.
Journal of Thrombosis and Haemostasis, 2014
Background: Blood platelets secrete upon activation of laminins 411/421 and 511/521, large adhesi... more Background: Blood platelets secrete upon activation of laminins 411/421 and 511/521, large adhesive proteins mainly found in the basement membranes of blood vessels and other tissues. At present, the subcellular localization and secretion mechanisms of platelet laminins are largely unknown. Objectives: Our aim was to compare the subcellular localization of laminins 411/421 and 511/ 521 and specific granule markers in platelets. We also elucidated the role of microvesicles and exosomes in laminin release in platelet activation. Methods: We studied laminin and granule marker protein localization in platelets by using immunofluorescence confocal microscopy and immunoelectron microscopy. Microvesicles and exosomes were separated from material released from platelets on activation by thrombin. The expression of laminins in microvesicles and exosomes was studied by using SDS-PAGE and Western blotting as well as by flow cytometric analysis. The exosomes were immunoprecipitated with magnetic microbeads coated with anti-CD63 antibodies. Results and conclusions: We demonstrate that laminins 411/421 and 511/521 are present in compartments of platelets that do not express a-granule, dense granule, or lysosome marker proteins. Moreover, laminins secreted by activated platelets are mostly found in microvesicles shed from the plasma membrane, while their presence in simultaneously released exosomes is minimum.
Reproductive BioMedicine Online
Research question: How does mucin MUC20 expression change during the menstrual cycle in different... more 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 RNAseq datasets in whole endometrial tissue (n=10) and sorted endometrial epithelial (n=44) or stromal (n=42) cell samples. RNA-Seq results were validated by qRT-PCR in whole tissue (n=10), and sorted epithelial (n=17) and stromal (n=17) cell samples. MUC20 protein localisation 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). Our results indicate to MUC20 as a novel marker of mid-secretory endometrial biology. We propose a model about 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.
Thrombosis and haemostasis, 2006
Following vascular injury, basement membrane (BM) components of the blood vessels are exposed to ... more 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 alph...
Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling ... more Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling functions. They are major components of basement membranes and are found in many organs, including the vasculature and other compartments of bone marrow, thymus, lymph nodes and spleen. However, expression, recognition and use of laminin isoforms by lymphoid cells are poorly understood. In the present study, lymphoid T cells (Jurkat) were found to synthesize laminin α4, β1 and γ1 mRNAs and polypeptides and to assemble the chains into laminin-8. Lymphoblastoid B (NAD-20) cells, lymphoid NK (NKL) cells and blood lymphocytes also contained laminin-8 and, after cell permeabilization, practically all blood lymphocytes reacted with mAbs to laminin β1 and γ1 chains. Following stimulation, blood lymphocytes secreted laminin-8, and this laminin isoform, but not laminin-10/11(α5β1γ1/α5β2γ1), promoted chemokine-induced migration of the cells. In an activation-dependent manner, purified blood CD4 T cells adhered to immobilized laminin-8 and laminin-10/11 by using α6β1 integrin, but minimally to laminin-1 (α1β1γ1). Accordingly, laminin-8 and laminin-10/11, but not laminin-1, strongly costimulated proliferation of the T cells via the same integrin. Thus, lymphoid cells are able to synthesize and secrete complete laminin molecules. In addition, synthesis of laminin-8 and recognition of laminin-8 and-10/11 by lymphocytes indicate relevance of these laminin isoforms in lymphocyte physiology.
Reproductive biomedicine online, Jan 12, 2017
Several studies have demonstrated that human embryonic stem cells (hESC) can be differentiated in... more 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-β)/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 gene...
This information is current as) 1 γ 1 β 4 α Migrate on Laminin-8 (Monocytic Cells Synthesize, Adh... more This information is current as) 1 γ 1 β 4 α Migrate on Laminin-8 (Monocytic Cells Synthesize, Adhere to, and
Exp Cell Res, 1999
Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are charac... more Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin gamma1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to gamma1 and beta1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin beta1 antibody-Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to beta1 and gamma1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin alpha4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20-35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to beta1 and alpha6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (alpha4beta1gamma1) and that the cells adhere to the protein by using alpha6beta1 integrin.
Eksperimentalʹnai͡a onkologii͡a, 1990
Four monoclonal antibodies (MAbs) from series IGR to human peripheral blood neutrophilic granuloc... more Four monoclonal antibodies (MAbs) from series IGR to human peripheral blood neutrophilic granulocyte cell surface antigens were obtained by the conventional hybridoma technique. Specificity of MAbs AGR was determined to various leukemic cell lines and human peripheral blood cells. Overlapping in characteristics of antigens (molecular weight, localization, expression on induced leukemic cell line HL-60) to MAbs IGR-1 4C7, IGR-1 5B6 and IGR-2 IA6 suggests their identity. These, apparently, cannot be analogous to the well known granulocyte cell surface glycoproteins LFA-1, CR-3, p150, 95 or GP 130. The characteristics of MAbs IGR-1 and IGR-2 permit concluding that the antibodies should be useful in normal and leukemic myelomonocytic cell linear differentiation studies.
Cell Biology International Reports, 1990
Biology of Reproduction, 2012
The aryl hydrocarbon receptor (AHR) mediates the toxicity of a variety of environmental chemicals... more 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 À209 to À99 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.
Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling ... more Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling functions. They are major components of basement membranes and are found in many organs, including the vasculature and other compartments of bone marrow, thymus, lymph nodes and spleen. However, expression, recognition and use of laminin isoforms by lymphoid cells are poorly understood. In the present study, lymphoid T cells (Jurkat) were found to synthesize laminin α4, β1 and γ1 mRNAs and polypeptides and to assemble the chains into laminin-8. Lymphoblastoid B (NAD-20) cells, lymphoid NK (NKL) cells and blood lymphocytes also contained laminin-8 and, after cell permeabilization, practically all blood lymphocytes reacted with mAbs to laminin β1 and γ1 chains. Following stimulation, blood lymphocytes secreted laminin-8, and this laminin isoform, but not laminin-10/11(α5β1γ1/α5β2γ1), promoted chemokine-induced migration of the cells. In an activation-dependent manner, purified blood CD4 T cells adhered to immobilized laminin-8 and laminin-10/11 by using α6β1 integrin, but minimally to laminin-1 (α1β1γ1). Accordingly, laminin-8 and laminin-10/11, but not laminin-1, strongly costimulated proliferation of the T cells via the same integrin. Thus, lymphoid cells are able to synthesize and secrete complete laminin molecules. In addition, synthesis of laminin-8 and recognition of laminin-8 and-10/11 by lymphocytes indicate relevance of these laminin isoforms in lymphocyte physiology.
Eksperimentalʹnai͡a onkologii͡a, 1990
Four monoclonal antibodies (Mabs) of series IGR-1 and IGR-2 to nuclear antigens of neutrophilic g... more Four monoclonal antibodies (Mabs) of series IGR-1 and IGR-2 to nuclear antigens of neutrophilic granulocytes of human peripheral blood were obtained. Mabs IGR-1 2B8 and IGR-1 6B5 are bound to their specific antigens in the nuclei of all the investigated human cell lines. These Mabs were also specific for metaphase chromosomes of cell lines HL-60 and U-937. Investigations on the ultrastructural level showed that Mabs IGR-1 6B5 reacted with the HL-60 nuclear heterochromatin region. Mabs IGR-1 3D3 and IGR-2 2F1 manifested high specificity only for the nuclei of mature neutrophils and of plasma cells.
Plos One, 2013
Laminins, a large family of abc heterotrimeric proteins mainly found in basement membranes, are s... more Laminins, a large family of abc 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 a (LMa) chains, a5 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 LMa5 chain to further study the biological relevance of a5 laminins, such as laminins 511 (a5b1c1) and 521 (a5b2c1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMa5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited a3b1/a6b1 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 a3b1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMa5 chain but largely hindered by mAb 4E10 to a LMb1 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 LMa5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.
Laminins, a large family of abc heterotrimeric proteins mainly found in basement membranes, are s... more Laminins, a large family of abc 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 a (LMa) chains, a5 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 LMa5 chain to further study the biological relevance of a5 laminins, such as laminins 511 (a5b1c1) and 521 (a5b2c1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMa5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited a3b1/a6b1 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...
Arthritis Research & Therapy, 2015
Introduction: Dupuytren's contracture (DC) is a chronic fibroproliferative disease of the hand, w... more 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.
Plos One, 2013
Laminins, a large family of abc heterotrimeric proteins mainly found in basement membranes, are s... more Laminins, a large family of abc 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 a (LMa) chains, a5 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 LMa5 chain to further study the biological relevance of a5 laminins, such as laminins 511 (a5b1c1) and 521 (a5b2c1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMa5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited a3b1/a6b1 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 a3b1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMa5 chain but largely hindered by mAb 4E10 to a LMb1 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 LMa5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.
Journal of Thrombosis and Haemostasis, 2014
Background: Blood platelets secrete upon activation of laminins 411/421 and 511/521, large adhesi... more Background: Blood platelets secrete upon activation of laminins 411/421 and 511/521, large adhesive proteins mainly found in the basement membranes of blood vessels and other tissues. At present, the subcellular localization and secretion mechanisms of platelet laminins are largely unknown. Objectives: Our aim was to compare the subcellular localization of laminins 411/421 and 511/ 521 and specific granule markers in platelets. We also elucidated the role of microvesicles and exosomes in laminin release in platelet activation. Methods: We studied laminin and granule marker protein localization in platelets by using immunofluorescence confocal microscopy and immunoelectron microscopy. Microvesicles and exosomes were separated from material released from platelets on activation by thrombin. The expression of laminins in microvesicles and exosomes was studied by using SDS-PAGE and Western blotting as well as by flow cytometric analysis. The exosomes were immunoprecipitated with magnetic microbeads coated with anti-CD63 antibodies. Results and conclusions: We demonstrate that laminins 411/421 and 511/521 are present in compartments of platelets that do not express a-granule, dense granule, or lysosome marker proteins. Moreover, laminins secreted by activated platelets are mostly found in microvesicles shed from the plasma membrane, while their presence in simultaneously released exosomes is minimum.
Reproductive BioMedicine Online
Research question: How does mucin MUC20 expression change during the menstrual cycle in different... more 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 RNAseq datasets in whole endometrial tissue (n=10) and sorted endometrial epithelial (n=44) or stromal (n=42) cell samples. RNA-Seq results were validated by qRT-PCR in whole tissue (n=10), and sorted epithelial (n=17) and stromal (n=17) cell samples. MUC20 protein localisation 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). Our results indicate to MUC20 as a novel marker of mid-secretory endometrial biology. We propose a model about 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.
Thrombosis and haemostasis, 2006
Following vascular injury, basement membrane (BM) components of the blood vessels are exposed to ... more 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 alph...
Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling ... more Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling functions. They are major components of basement membranes and are found in many organs, including the vasculature and other compartments of bone marrow, thymus, lymph nodes and spleen. However, expression, recognition and use of laminin isoforms by lymphoid cells are poorly understood. In the present study, lymphoid T cells (Jurkat) were found to synthesize laminin α4, β1 and γ1 mRNAs and polypeptides and to assemble the chains into laminin-8. Lymphoblastoid B (NAD-20) cells, lymphoid NK (NKL) cells and blood lymphocytes also contained laminin-8 and, after cell permeabilization, practically all blood lymphocytes reacted with mAbs to laminin β1 and γ1 chains. Following stimulation, blood lymphocytes secreted laminin-8, and this laminin isoform, but not laminin-10/11(α5β1γ1/α5β2γ1), promoted chemokine-induced migration of the cells. In an activation-dependent manner, purified blood CD4 T cells adhered to immobilized laminin-8 and laminin-10/11 by using α6β1 integrin, but minimally to laminin-1 (α1β1γ1). Accordingly, laminin-8 and laminin-10/11, but not laminin-1, strongly costimulated proliferation of the T cells via the same integrin. Thus, lymphoid cells are able to synthesize and secrete complete laminin molecules. In addition, synthesis of laminin-8 and recognition of laminin-8 and-10/11 by lymphocytes indicate relevance of these laminin isoforms in lymphocyte physiology.
Reproductive biomedicine online, Jan 12, 2017
Several studies have demonstrated that human embryonic stem cells (hESC) can be differentiated in... more 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-β)/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 gene...
This information is current as) 1 γ 1 β 4 α Migrate on Laminin-8 (Monocytic Cells Synthesize, Adh... more This information is current as) 1 γ 1 β 4 α Migrate on Laminin-8 (Monocytic Cells Synthesize, Adhere to, and
Exp Cell Res, 1999
Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are charac... more Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin gamma1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to gamma1 and beta1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin beta1 antibody-Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to beta1 and gamma1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin alpha4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20-35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to beta1 and alpha6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (alpha4beta1gamma1) and that the cells adhere to the protein by using alpha6beta1 integrin.
Eksperimentalʹnai͡a onkologii͡a, 1990
Four monoclonal antibodies (MAbs) from series IGR to human peripheral blood neutrophilic granuloc... more Four monoclonal antibodies (MAbs) from series IGR to human peripheral blood neutrophilic granulocyte cell surface antigens were obtained by the conventional hybridoma technique. Specificity of MAbs AGR was determined to various leukemic cell lines and human peripheral blood cells. Overlapping in characteristics of antigens (molecular weight, localization, expression on induced leukemic cell line HL-60) to MAbs IGR-1 4C7, IGR-1 5B6 and IGR-2 IA6 suggests their identity. These, apparently, cannot be analogous to the well known granulocyte cell surface glycoproteins LFA-1, CR-3, p150, 95 or GP 130. The characteristics of MAbs IGR-1 and IGR-2 permit concluding that the antibodies should be useful in normal and leukemic myelomonocytic cell linear differentiation studies.
Cell Biology International Reports, 1990
Biology of Reproduction, 2012
The aryl hydrocarbon receptor (AHR) mediates the toxicity of a variety of environmental chemicals... more 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 À209 to À99 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.
Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling ... more Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling functions. They are major components of basement membranes and are found in many organs, including the vasculature and other compartments of bone marrow, thymus, lymph nodes and spleen. However, expression, recognition and use of laminin isoforms by lymphoid cells are poorly understood. In the present study, lymphoid T cells (Jurkat) were found to synthesize laminin α4, β1 and γ1 mRNAs and polypeptides and to assemble the chains into laminin-8. Lymphoblastoid B (NAD-20) cells, lymphoid NK (NKL) cells and blood lymphocytes also contained laminin-8 and, after cell permeabilization, practically all blood lymphocytes reacted with mAbs to laminin β1 and γ1 chains. Following stimulation, blood lymphocytes secreted laminin-8, and this laminin isoform, but not laminin-10/11(α5β1γ1/α5β2γ1), promoted chemokine-induced migration of the cells. In an activation-dependent manner, purified blood CD4 T cells adhered to immobilized laminin-8 and laminin-10/11 by using α6β1 integrin, but minimally to laminin-1 (α1β1γ1). Accordingly, laminin-8 and laminin-10/11, but not laminin-1, strongly costimulated proliferation of the T cells via the same integrin. Thus, lymphoid cells are able to synthesize and secrete complete laminin molecules. In addition, synthesis of laminin-8 and recognition of laminin-8 and-10/11 by lymphocytes indicate relevance of these laminin isoforms in lymphocyte physiology.
Eksperimentalʹnai͡a onkologii͡a, 1990
Four monoclonal antibodies (Mabs) of series IGR-1 and IGR-2 to nuclear antigens of neutrophilic g... more Four monoclonal antibodies (Mabs) of series IGR-1 and IGR-2 to nuclear antigens of neutrophilic granulocytes of human peripheral blood were obtained. Mabs IGR-1 2B8 and IGR-1 6B5 are bound to their specific antigens in the nuclei of all the investigated human cell lines. These Mabs were also specific for metaphase chromosomes of cell lines HL-60 and U-937. Investigations on the ultrastructural level showed that Mabs IGR-1 6B5 reacted with the HL-60 nuclear heterochromatin region. Mabs IGR-1 3D3 and IGR-2 2F1 manifested high specificity only for the nuclei of mature neutrophils and of plasma cells.
Plos One, 2013
Laminins, a large family of abc heterotrimeric proteins mainly found in basement membranes, are s... more Laminins, a large family of abc 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 a (LMa) chains, a5 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 LMa5 chain to further study the biological relevance of a5 laminins, such as laminins 511 (a5b1c1) and 521 (a5b2c1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMa5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited a3b1/a6b1 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 a3b1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMa5 chain but largely hindered by mAb 4E10 to a LMb1 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 LMa5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.