The Experts below are selected from a list of 108 Experts worldwide ranked by ideXlab platform
Alfonso Colombatti - One of the best experts on this subject based on the ideXlab platform.
-
Loss of Multimerin-2 and EMILIN-2 Expression in Gastric Cancer Associate with Altered Angiogenesis
International Journal of Molecular Sciences, 2018Co-Authors: Eva Andreuzzi, Roberto Doliana, Alfonso Colombatti, Alessandra Capuano, Rosanna Pellicani, Evelina Poletto, Stefania Maiero, Mara Fornasarig, Raffaella Magris, Renato CannizzaroAbstract:Gastric cancer is a deadly tumor and a relatively common disease worldwide. Surgical resection and chemotherapy are the main clinical options to treat this type of disease, however the median overall survival rate is limited to one year. Thus, the development of new therapies is a highly necessary clinical need. Angiogenesis is a promising target for this tumor type, however clinical trials with the use of anti-angiogenic drugs have so far not met expectations. Therefore, it is important to better characterize the expression of molecules whose expression levels may impact on the efficacy of the treatments. In this study the characteristics of the gastric tumor associated blood vessels were first assessed by endomicroscopy. Next, we analyzed the expression of Multimerin-2, EMILIN-2 and EMILIN-1, three molecules of the EMI Domain ENdowed (EDEN) protein family. These molecules play important functions in the tumor microenvironment, affecting cancer progression both directly and indirectly impinging on angiogenesis and lymphangiogenesis. All the molecules were highly expressed in the normal mucosa whereas in a number of patients their expression was altered. We consider that better characterizing the gastric tumor microenvironment and the quality of the vasculature may achieve effective patient tailored therapies.
-
Multiple-interactions among EMILIN1 and EMILIN2 N- and C-terminal Domains
Matrix Biology, 2014Co-Authors: Eva Andreuzzi, Alvise Schiavinato, Alfonso Colombatti, Alessandra Capuano, Roberto DolianaAbstract:EMILIN1 and EMILIN2 belong to a family of extracellular matrix glycoproteins characterized by the N-terminal cysteine-rich EMI Domain, a long segment with high probabilty for coiled-coil structure formation and a C-terminal gC1q Domain. To study EMILIN1 and EMILIN2 interaction and assembly we have applied qualitative and quantitative two hybrid systems using constructs corresponding to the gC1q and EMI Domains. The identified interactions were further confirmed in yeast extracts of co-transfected cells followed by co-immunoprecipitation. The data indicated that gC1q Domains are able to self-interact as well as to interact one each other and with the EMI Domains, but no self interactions were detected between the EMI Domains. Furthermore EMILINs interactions were studied in 293-EBNA cells co-transfected with full lenght EMILIN1 and EMILIN2 constructs. Specific antibodies were able to co-immunoprecipitate EMILINs, indicating that also full-lenght proteins can give rise to non-covalent homo- and hetero-multimers even if reduced and alkylated before mixing. Immunofluorescence analysis on mouse cell cultures and tissues sections with specific antibodies showed co-distribution of EMILIN1 and EMILIN2. Thus, we can hypothesize that EMILINs multimers are formed by head-to-tail interaction between C-terminal and N-terminal Domains of EMILIN1 and/or EMILIN2 but also by tail-to-tail interaction between gC1q Domains. These multiple interactions may regulate homo-typic and/or hetero-typic linear and eventually lateral branching assemblies of EMILIN1 and EMILIN2 in tissues.
-
EMILIN2 down‐modulates the Wnt signalling pathway and suppresses breast cancer cell growth and migration
The Journal of Pathology, 2014Co-Authors: Stefano Marastoni, Alfonso Colombatti, Paolo Bonaldo, Alvise Schiavinato, Eva Andreuzzi, Rosanna Pellicani, Alice Paulitti, Roberta Colladel, F. Todaro, Maurizio MongiatAbstract:EMILIN2 is an extracellular matrix (ECM) protein that exerts contradictory effects within the tumour microenvironment: it induces apoptosis in a number of tumour cells, but it also enhances tumour neo-angiogenesis. In this study, we describe a new mechanism by which EMILIN2 attenuates tumour cell viability. Based on sequence homology with the cysteine-rich Domain (CRD) of the Frizzled receptors, we hypothesized that EMILIN2 could affect Wnt signalling activation and demonstrate direct interaction with the Wnt1 ligand. This physical binding leads to decreased LRP6 phosphorylation and to the down-modulation of β-catenin, TAZ and their target genes. As a consequence, EMILIN2 negatively affects the viability, migration and tumourigenic potential of MDA-MB-231 breast cancer cells in a number of two- and three-dimensional in vitro assays. EMILIN2 does not modulate Wnt signalling downstream of the Wnt–Frizzled interaction, since it does not affect the activation of the pathway following treatment with the GSK3 inhibitors LiCl and CHIR99021. The interaction with Wnt1 and the subsequent biological effects require the presence of the EMI Domain, as there is no effect with a deletion mutant lacking this Domain. Moreover, in vivo experiments show that the ectopic expression of EMILIN2, as well as treatment with the recombinant protein, significantly reduce tumour growth and dissEMInation of cancer cells in nude mice. Accordingly, the tumour samples are characterized by a significant down-regulation of the Wnt signalling pathway. Altogether, these findings provide further evidence of the complex regulations governed by EMILIN2 in the tumour microenvironment, and they identify a key extracellular regulator of the Wnt signalling pathway. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
-
The EMILIN/Multimerin Family
Frontiers in Immunology, 2012Co-Authors: Alfonso Colombatti, Roberto Doliana, Giorgio M. Bressan, Maurizio Mongiat, Paola Spessotto, Gennaro EspositoAbstract:EMILINs and Multimerins (EMILIN1, EMILIN2, Multimerin1, and Multimerin2) constitute a four member family that in addition to the shared C-terminus gC1q Domain typical of the gC1q/TNF superfamily members contain a N-terminus unique cysteine-rich EMI Domain. These glycoproteins are homotrimeric and assemble into high molecular weight multimers. They are predominantly expressed in the extracellular matrix and contribute to several cellular functions in part associated with the gC1q Domain and in part not yet assigned nor linked to other specific regions of the sequence. Among the latter is the control of arterial blood pressure, the inhibition of Bacillus anthracis cell cytotoxicity, the promotion of cell death, the proangiogenetic function and a role in platelet hemostasis. The focus of this review is to highlight the multiplicity of functions and Domains of the EMILIN/Multimerin family with a particular emphasis on the regulatory role played by the ligand-receptor interactions of the gC1q Domain.EMILIN1 is the most extensively studied member both from the structural and functional point of view. The structure of the gC1q of EMILIN1 solved by NMR highlights unique characteristics compared to other gC1q Domains: it shows a marked decrease of the contact surface of the trimeric assembly and while conserving the jelly-roll topology with two β-sheets of antiparallel strands it presents a nine-stranded β-sandwich fold instead of the usual ten-stranded fold. This is likely due to the insertion of nine residues that disrupt the ordered strand organization and forma a highly dynamic protruding loop.. In this loop the residue E933 is the site of interaction between gC1q and the α4β1 and α9β1 integrins, and contrary to integrin occupancy that usually upregulates cell growth, when gC1q is ligated by the integrin the cells reduce their proliferative activity.
-
EMIlin1 deficiency causes structural and functional defects of lymphatic vasculature
Molecular and Cellular Biology, 2008Co-Authors: Carla Danussi, Roberto Doliana, Giorgio M. Bressan, Paola Spessotto, Alessandra Petrucco, Bruna Wassermann, Patrizia Sabatelli, Monica Montesi, Alfonso ColombattiAbstract:Lymphatic and blood vascular systems have distinct structural characteristics that reflect their specific and complementary functions. The lymphatic vasculature represents a second circulatory system and maintains tissue fluid homeostasis; it plays a major role in the absorption of dietary fat and in immune response, transporting lymphocytes and antigen-presenting cells to regional lymph nodes; finally, it provides routes for tumor metastasis (8). The lymphatic system consists of a complex network of lymphatic capillaries, which are uniquely adapted for the uptake of protein-rich lymph from tissue interstitium, and collecting lymphatic vessels that transport lymph back to the blood vascular system. The latter are surrounded by a basement membrane and smooth muscle cells, which are less organized than in blood vessels and, in addition, have intraluminal valves, which prevent lymph backflow (26). By contrast, lymphatic capillaries are blind-end vessels, lined by a single thin layer of overlapping lymphatic endothelial cells (LECs) directly connected to the surrounding extracellular matrix (ECM) by means of anchoring elastic filaments (18). These structures play a fundamental role in lymphatic-vessel function and represent one of the main distinguishing features between lymphatic and blood capillaries. When interstitial fluid pressure increases, anchoring filaments exert tension on LECs, thereby widening the capillary lumen and opening the overlapping cell junctions, which enable fluid and macromolecule uptake and cell entry. It is thought that abnormalities of anchoring filaments may reduce adsorption from the interstitium and propulsion of lymph and cells and promote pathological conditions, such as lymphedema or diseases related to impaired immune responses (9, 18). Furthermore, the perivascular ECM plays an integral role in lymphatic-vessel function, as the fluid equilibrium is controlled by the cooperation of both lymphatic function and the ECM (40). The elasticity and hydration of a tissue is determined by the composition and organization of the ECM. Extensive and chronic degradation of the ECM eventually renders lymphatic vessels nonresponsive to the changes in the interstitium and therefore causes dysfunction (28, 30). Lymphatic and blood endothelial cells express different lineage-specific molecules involved in the regulation of their biological functions that are frequently used as distinguishing markers, such as vascular endothelial growth factor receptor 3 (VEGFR-3) (22), podoplanin (3), Prox-1 (44), LYVE-1 (1, 34), neuropilin 2 (46), CCL21 (24), and desmoplakin (12). Recently, a comparative microarray analysis of gene expression profiles of lymphatic and blood endothelial cells identified previously unknown lymphatic lineage genes, including macrophage mannose receptor 1, plakoglobin, the chemokine CCL20, the integrin α9β1 (19, 32), and EMILIN1 (33). EMILIN1 is an ECM glycoprotein associated with elastic fibers (4, 6) and composed of an N-terminal cysteine-rich Domain and the EMI Domain (11), followed by a coiled-coil structure, a short collagenous stalk, and a C-terminal gC1q Domain (7). EMILIN1 is particularly abundant in the walls of large blood vessels, such as the aorta (10), and has been implicated in multiple functions. EMILIN1 is involved in elastogenesis and in the maintenance of blood vascular cell morphology (48). It interacts with the α4β1 integrin through the gC1q1 Domain (36) and has strong adhesive and migratory properties for different cell types (10, 36, 37). EMILIN1, via the EMI Domain, regulates pro-transforming growth factor beta (TGF-β) maturation and is involved in blood pressure homeostasis (47). To directly investigate the physiological function of EMILIN1 in lymphatic vessels, we studied the effects of its absence in mice that had targeted deletions in the EMIlin1 gene (48). Here, we report that EMILIN1 is highly expressed by LECs in vitro and that it colocalizes with lymphatic vessels in several mouse tissues. Importantly, EMIlin1 deficiency results in hyperplasia and enlargement of lymphatic vessels and in a significant reduction of anchoring filaments compared to those of wild-type (WT) mice. The lymphatic vessels of EMIlin1−/− mice are functionally altered. We found that lack of EMILIN1 leads to a mild lymphedema associated with inefficient lymph drainage and increased leakage. In addition, EMIlin1−/− mice develop larger lymphangiomas than their WT littermates. Altogether, these findings demonstrate an important role of EMILIN1 in the structure-function relationship of lymphatic vessels and identify EMILIN1 as a lymphangiogenesis modulator.
Roberto Doliana - One of the best experts on this subject based on the ideXlab platform.
-
Loss of Multimerin-2 and EMILIN-2 Expression in Gastric Cancer Associate with Altered Angiogenesis
International Journal of Molecular Sciences, 2018Co-Authors: Eva Andreuzzi, Roberto Doliana, Alfonso Colombatti, Alessandra Capuano, Rosanna Pellicani, Evelina Poletto, Stefania Maiero, Mara Fornasarig, Raffaella Magris, Renato CannizzaroAbstract:Gastric cancer is a deadly tumor and a relatively common disease worldwide. Surgical resection and chemotherapy are the main clinical options to treat this type of disease, however the median overall survival rate is limited to one year. Thus, the development of new therapies is a highly necessary clinical need. Angiogenesis is a promising target for this tumor type, however clinical trials with the use of anti-angiogenic drugs have so far not met expectations. Therefore, it is important to better characterize the expression of molecules whose expression levels may impact on the efficacy of the treatments. In this study the characteristics of the gastric tumor associated blood vessels were first assessed by endomicroscopy. Next, we analyzed the expression of Multimerin-2, EMILIN-2 and EMILIN-1, three molecules of the EMI Domain ENdowed (EDEN) protein family. These molecules play important functions in the tumor microenvironment, affecting cancer progression both directly and indirectly impinging on angiogenesis and lymphangiogenesis. All the molecules were highly expressed in the normal mucosa whereas in a number of patients their expression was altered. We consider that better characterizing the gastric tumor microenvironment and the quality of the vasculature may achieve effective patient tailored therapies.
-
Multiple-interactions among EMILIN1 and EMILIN2 N- and C-terminal Domains
Matrix Biology, 2014Co-Authors: Eva Andreuzzi, Alvise Schiavinato, Alfonso Colombatti, Alessandra Capuano, Roberto DolianaAbstract:EMILIN1 and EMILIN2 belong to a family of extracellular matrix glycoproteins characterized by the N-terminal cysteine-rich EMI Domain, a long segment with high probabilty for coiled-coil structure formation and a C-terminal gC1q Domain. To study EMILIN1 and EMILIN2 interaction and assembly we have applied qualitative and quantitative two hybrid systems using constructs corresponding to the gC1q and EMI Domains. The identified interactions were further confirmed in yeast extracts of co-transfected cells followed by co-immunoprecipitation. The data indicated that gC1q Domains are able to self-interact as well as to interact one each other and with the EMI Domains, but no self interactions were detected between the EMI Domains. Furthermore EMILINs interactions were studied in 293-EBNA cells co-transfected with full lenght EMILIN1 and EMILIN2 constructs. Specific antibodies were able to co-immunoprecipitate EMILINs, indicating that also full-lenght proteins can give rise to non-covalent homo- and hetero-multimers even if reduced and alkylated before mixing. Immunofluorescence analysis on mouse cell cultures and tissues sections with specific antibodies showed co-distribution of EMILIN1 and EMILIN2. Thus, we can hypothesize that EMILINs multimers are formed by head-to-tail interaction between C-terminal and N-terminal Domains of EMILIN1 and/or EMILIN2 but also by tail-to-tail interaction between gC1q Domains. These multiple interactions may regulate homo-typic and/or hetero-typic linear and eventually lateral branching assemblies of EMILIN1 and EMILIN2 in tissues.
-
The EMILIN/Multimerin Family
Frontiers in Immunology, 2012Co-Authors: Alfonso Colombatti, Roberto Doliana, Giorgio M. Bressan, Maurizio Mongiat, Paola Spessotto, Gennaro EspositoAbstract:EMILINs and Multimerins (EMILIN1, EMILIN2, Multimerin1, and Multimerin2) constitute a four member family that in addition to the shared C-terminus gC1q Domain typical of the gC1q/TNF superfamily members contain a N-terminus unique cysteine-rich EMI Domain. These glycoproteins are homotrimeric and assemble into high molecular weight multimers. They are predominantly expressed in the extracellular matrix and contribute to several cellular functions in part associated with the gC1q Domain and in part not yet assigned nor linked to other specific regions of the sequence. Among the latter is the control of arterial blood pressure, the inhibition of Bacillus anthracis cell cytotoxicity, the promotion of cell death, the proangiogenetic function and a role in platelet hemostasis. The focus of this review is to highlight the multiplicity of functions and Domains of the EMILIN/Multimerin family with a particular emphasis on the regulatory role played by the ligand-receptor interactions of the gC1q Domain.EMILIN1 is the most extensively studied member both from the structural and functional point of view. The structure of the gC1q of EMILIN1 solved by NMR highlights unique characteristics compared to other gC1q Domains: it shows a marked decrease of the contact surface of the trimeric assembly and while conserving the jelly-roll topology with two β-sheets of antiparallel strands it presents a nine-stranded β-sandwich fold instead of the usual ten-stranded fold. This is likely due to the insertion of nine residues that disrupt the ordered strand organization and forma a highly dynamic protruding loop.. In this loop the residue E933 is the site of interaction between gC1q and the α4β1 and α9β1 integrins, and contrary to integrin occupancy that usually upregulates cell growth, when gC1q is ligated by the integrin the cells reduce their proliferative activity.
-
EMIlin1 deficiency causes structural and functional defects of lymphatic vasculature
Molecular and Cellular Biology, 2008Co-Authors: Carla Danussi, Roberto Doliana, Giorgio M. Bressan, Paola Spessotto, Alessandra Petrucco, Bruna Wassermann, Patrizia Sabatelli, Monica Montesi, Alfonso ColombattiAbstract:Lymphatic and blood vascular systems have distinct structural characteristics that reflect their specific and complementary functions. The lymphatic vasculature represents a second circulatory system and maintains tissue fluid homeostasis; it plays a major role in the absorption of dietary fat and in immune response, transporting lymphocytes and antigen-presenting cells to regional lymph nodes; finally, it provides routes for tumor metastasis (8). The lymphatic system consists of a complex network of lymphatic capillaries, which are uniquely adapted for the uptake of protein-rich lymph from tissue interstitium, and collecting lymphatic vessels that transport lymph back to the blood vascular system. The latter are surrounded by a basement membrane and smooth muscle cells, which are less organized than in blood vessels and, in addition, have intraluminal valves, which prevent lymph backflow (26). By contrast, lymphatic capillaries are blind-end vessels, lined by a single thin layer of overlapping lymphatic endothelial cells (LECs) directly connected to the surrounding extracellular matrix (ECM) by means of anchoring elastic filaments (18). These structures play a fundamental role in lymphatic-vessel function and represent one of the main distinguishing features between lymphatic and blood capillaries. When interstitial fluid pressure increases, anchoring filaments exert tension on LECs, thereby widening the capillary lumen and opening the overlapping cell junctions, which enable fluid and macromolecule uptake and cell entry. It is thought that abnormalities of anchoring filaments may reduce adsorption from the interstitium and propulsion of lymph and cells and promote pathological conditions, such as lymphedema or diseases related to impaired immune responses (9, 18). Furthermore, the perivascular ECM plays an integral role in lymphatic-vessel function, as the fluid equilibrium is controlled by the cooperation of both lymphatic function and the ECM (40). The elasticity and hydration of a tissue is determined by the composition and organization of the ECM. Extensive and chronic degradation of the ECM eventually renders lymphatic vessels nonresponsive to the changes in the interstitium and therefore causes dysfunction (28, 30). Lymphatic and blood endothelial cells express different lineage-specific molecules involved in the regulation of their biological functions that are frequently used as distinguishing markers, such as vascular endothelial growth factor receptor 3 (VEGFR-3) (22), podoplanin (3), Prox-1 (44), LYVE-1 (1, 34), neuropilin 2 (46), CCL21 (24), and desmoplakin (12). Recently, a comparative microarray analysis of gene expression profiles of lymphatic and blood endothelial cells identified previously unknown lymphatic lineage genes, including macrophage mannose receptor 1, plakoglobin, the chemokine CCL20, the integrin α9β1 (19, 32), and EMILIN1 (33). EMILIN1 is an ECM glycoprotein associated with elastic fibers (4, 6) and composed of an N-terminal cysteine-rich Domain and the EMI Domain (11), followed by a coiled-coil structure, a short collagenous stalk, and a C-terminal gC1q Domain (7). EMILIN1 is particularly abundant in the walls of large blood vessels, such as the aorta (10), and has been implicated in multiple functions. EMILIN1 is involved in elastogenesis and in the maintenance of blood vascular cell morphology (48). It interacts with the α4β1 integrin through the gC1q1 Domain (36) and has strong adhesive and migratory properties for different cell types (10, 36, 37). EMILIN1, via the EMI Domain, regulates pro-transforming growth factor beta (TGF-β) maturation and is involved in blood pressure homeostasis (47). To directly investigate the physiological function of EMILIN1 in lymphatic vessels, we studied the effects of its absence in mice that had targeted deletions in the EMIlin1 gene (48). Here, we report that EMILIN1 is highly expressed by LECs in vitro and that it colocalizes with lymphatic vessels in several mouse tissues. Importantly, EMIlin1 deficiency results in hyperplasia and enlargement of lymphatic vessels and in a significant reduction of anchoring filaments compared to those of wild-type (WT) mice. The lymphatic vessels of EMIlin1−/− mice are functionally altered. We found that lack of EMILIN1 leads to a mild lymphedema associated with inefficient lymph drainage and increased leakage. In addition, EMIlin1−/− mice develop larger lymphangiomas than their WT littermates. Altogether, these findings demonstrate an important role of EMILIN1 in the structure-function relationship of lymphatic vessels and identify EMILIN1 as a lymphangiogenesis modulator.
-
Regulation of the Extrinsic Apoptotic Pathway by the Extracellular Matrix Glycoprotein EMILIN2
Molecular and Cellular Biology, 2007Co-Authors: Maurizio Mongiat, Roberto Doliana, Giovanni Ligresti, Stefano Marastoni, E. Lorenzon, Alfonso ColombattiAbstract:Over the last few decades, increasing evidence points to a key role exerted by the extracellular environment in determining cell behavior in terms of gene expression patterns, differentiation, proliferation, and cell death (27). The apoptotic process is finely regulated, and in mammals, it is triggered by two major pathways: the “intrinsic” pathway, orchestrated mainly by the mitochondrion, and the receptor-mediated extrinsic pathway (1, 16). Effector caspase-3, -6, and -7 in turn cleave a specific set of cellular substrates including poly(ADP-ribose) polymerase (PARP) (9). These events ultimately result in the typical morphological changes observed in the course of apoptosis. The extrinsic pathway is triggered by specific receptors of the tumor necrosis factor receptor (TNFR) superfamily: Fas (CD95), DR4 (TRAIL-R1), and DR5 (TRAIL-R2) (1, 21, 23, 28, 38). Upon the binding of their respective ligands, death receptors cluster and redistribute in lipid rafts (10, 17, 34). This is followed by a common intracellular signaling pathway that includes the formation of the death-inducing signaling complex (DISC) and the activation of the initiator caspase-8 (3, 35) and caspase-10 (39). Active caspase-8 and caspase-10 in turn activate effector caspase-3, -6, and -7. This pathway is specifically inhibited by the FLICE-inhibitory protein FLIP (18). Selective interactions of the cell with components of the extracellular matrix (ECM) play an important role in regulating cell death and survival during organogenesis and tissue remodeling. In fact, it has been shown that specific components of the ECM may act as tuning factors for apoptosis. For instance, the ECM proteins TSP1 (19), endostatin (33), SPARC (30), and CCN1 (36) are reported to induce apoptosis in several cell types. Elastin microfibril interface-located proteins (EMILINs) are a family of ECM glycoproteins containing the EMI Domain (11). EMILIN2 was identified based on a two-hybrid screening using the gC1q-like Domain of the prototype of the family, EMILIN1, as bait. Similarly to EMILIN1, EMILIN2 contains an EMI Domain, a cysteine-rich region of about 80 amino acids at the N terminus of the molecule, an alpha-helical large Domain with high probability for coiled-coil structure formation, a collagenous stalk, and a C-terminal gC1q Domain. A proline-rich Domain following the coiled-coil region is a distinctive feature of EMILIN2 (12). EMILINs show the highest level of similarity at the EMI and gC1q Domains. EMILIN1 is expressed around the blood vessels and in a variety of organs (6), and it is detected at the interface between the amorphous core of the elastic fibers and the surrounding microfibrils (5, 7), hence the acronym EMILIN. EMILIN1 null mice displayed defects in the endothelial cell layer, interruptions of the elastic lamellae of large vessels (41), and chronic hypertension (40). On the contrary, the biological function of EMILIN2 is unclear, but expression analyses using mouse suggest an important role for EMILIN2 in organogenesis (4). In this study, we unveil a previously unknown function for EMILINs. We find that EMILIN2, through direct binding and subsequent activation of the death receptors DR4 and DR5, induces apoptosis in a number of tumor cells. Our results add further support to the role of ECM proteins in the regulation of cell survival and tissue homeostasis and disclose a novel mechanism for ECM protein-regulated cell death.
Paolo Bonaldo - One of the best experts on this subject based on the ideXlab platform.
-
Overlapping, complementary and site-specific expression pattern of genes of the EMILIN/Multimerin family
Matrix Biology, 2020Co-Authors: Paola Braghetta, Paolo Bonaldo, Miriam Zanetti, Alessandra Ferrari, Paola De Gemmis, Dino Volpin, Giorgio M. BressanAbstract:The EDEN gene superfamily comprises genes that contain the EMI Domain, a structural motif recently identified in proteins of the extracellular matrix. We report here the detailed expression pattern of genes of the EMILIN/Multimerin family, the most numerous group of EDEN superfamily, during mouse development. In situ hybridization has revealed that the EMILIN/Multimerin genes are particularly expressed in the cardio-vascular system and in mesenchymal cells. In general, the territories of expression of each gene are partially overlapping or complementary with that of other members of the family and, usually, more than one gene of the family is active in different tissues, consistent with the possibility of functional compensation. The analysis is particularly relevant in the interpretation of gene targeting experiments.
-
EMILIN2 down‐modulates the Wnt signalling pathway and suppresses breast cancer cell growth and migration
The Journal of Pathology, 2014Co-Authors: Stefano Marastoni, Alfonso Colombatti, Paolo Bonaldo, Alvise Schiavinato, Eva Andreuzzi, Rosanna Pellicani, Alice Paulitti, Roberta Colladel, F. Todaro, Maurizio MongiatAbstract:EMILIN2 is an extracellular matrix (ECM) protein that exerts contradictory effects within the tumour microenvironment: it induces apoptosis in a number of tumour cells, but it also enhances tumour neo-angiogenesis. In this study, we describe a new mechanism by which EMILIN2 attenuates tumour cell viability. Based on sequence homology with the cysteine-rich Domain (CRD) of the Frizzled receptors, we hypothesized that EMILIN2 could affect Wnt signalling activation and demonstrate direct interaction with the Wnt1 ligand. This physical binding leads to decreased LRP6 phosphorylation and to the down-modulation of β-catenin, TAZ and their target genes. As a consequence, EMILIN2 negatively affects the viability, migration and tumourigenic potential of MDA-MB-231 breast cancer cells in a number of two- and three-dimensional in vitro assays. EMILIN2 does not modulate Wnt signalling downstream of the Wnt–Frizzled interaction, since it does not affect the activation of the pathway following treatment with the GSK3 inhibitors LiCl and CHIR99021. The interaction with Wnt1 and the subsequent biological effects require the presence of the EMI Domain, as there is no effect with a deletion mutant lacking this Domain. Moreover, in vivo experiments show that the ectopic expression of EMILIN2, as well as treatment with the recombinant protein, significantly reduce tumour growth and dissEMInation of cancer cells in nude mice. Accordingly, the tumour samples are characterized by a significant down-regulation of the Wnt signalling pathway. Altogether, these findings provide further evidence of the complex regulations governed by EMILIN2 in the tumour microenvironment, and they identify a key extracellular regulator of the Wnt signalling pathway. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
-
EMI a novel cysteine rich Domain of EMIlins and other extracellular proteins interacts with the gc1q Domains and participates in multimerization
FEBS Letters, 2000Co-Authors: Roberto Doliana, Paolo Bonaldo, Alfonso ColombattiAbstract:The N-terminal cysteine-rich Domain (EMI Domain) of EMILIN-1 is a new protein Domain that is shared with two proteins (multimerin and EMILIN-2) and with four additional database entries. The EMI Domains are always located at the N-terminus, have a common gene organization, and belong to proteins that are forming or are compatible with multimer formation. The potential role of the EMI Domain in the assembly of EMILIN-1 was investigated by the two-hybrid system. No reporter gene activity was detected when EMI-1 was co-transformed with the C-terminal gC1q-1 Domain excluding a head-to-tail multimerization; conversely, a strong interaction was detected when the EMI-1 Domain was co-transformed with the gC1q-2 Domain of EMILIN-2.
-
EMI, a novel cysteine‐rich Domain of EMILINs and other extracellular proteins, interacts with the gC1q Domains and participates in multimerization
FEBS Letters, 2000Co-Authors: Roberto Doliana, Paolo Bonaldo, Alfonso ColombattiAbstract:The N-terminal cysteine-rich Domain (EMI Domain) of EMILIN-1 is a new protein Domain that is shared with two proteins (multimerin and EMILIN-2) and with four additional database entries. The EMI Domains are always located at the N-terminus, have a common gene organization, and belong to proteins that are forming or are compatible with multimer formation. The potential role of the EMI Domain in the assembly of EMILIN-1 was investigated by the two-hybrid system. No reporter gene activity was detected when EMI-1 was co-transformed with the C-terminal gC1q-1 Domain excluding a head-to-tail multimerization; conversely, a strong interaction was detected when the EMI-1 Domain was co-transformed with the gC1q-2 Domain of EMILIN-2.
Akira Kudo - One of the best experts on this subject based on the ideXlab platform.
-
Periostin function in communication with extracellular matrices
Journal of Cell Communication and Signaling, 2018Co-Authors: Akira KudoAbstract:Periostin is a secretory protein with a multi-Domain structure, comprising an amino-terminal cysteine-rich EMI Domain, four internal FAS 1 Domains, and a carboxyl-terminal hydrophilic Domain. These adjacent Domains bind to extracellular matrix proteins (type I collagen, fibronectin, tenascin-C, and laminin γ2), and BMP-1 that catalyzes crosslinking of type I collagen, and proteoglycans, which play a role in cell adhesion. The binding sites on periostin have been demonstrated to contribute to the mechanical strength of connective tissues, enhancing intermolecular interactions in close proximity and their assembly into extracellular matrix architectures, where periostin plays further essential roles in physiological maintenance and pathological progression. Furthermore, periostin also binds to Notch 1 and CCN3, which have functions in maintenance of stemness, thus opening up a new field of periostin action.
-
Periostin promotes secretion of fibronectin from the endoplasmic reticulum.
Biochemical and Biophysical Research Communications, 2016Co-Authors: Takashi Nishiyama, Akira KudoAbstract:Extracellular matrix (ECM) proteins are synthesized in the endoplasmic reticulum (ER), transported to the extracellular milieu through the secretory pathway, and assembled into an extracellular architecture. A previous study of ours showed that periostin, a secretory protein, interacts with fibronectin and is involved in ECM remodeling. Here we show that periostin played a role in fibronectin secretion from the ER. Co-immunoprecipitation and in situ proximity ligation assays revealed an interaction between periostin and fibronectin in the ER. Although accumulation of fibronectin was detected in the ER of fibroblastic C3H10T1/2 cells, forced expression of periostin in those cells decreased the accumulation of fibronectin in the ER, suggesting that periostin promoted the secretion of fibronectin. A substitution mutant of tryptophan at the position 65 to alanine in the EMI Domain of periostin, which caused periostin to lose its ability to interact with fibronectin, did not decrease the accumulation. Furthermore, targeted disruption of periostin in mice caused the non-fibrillar and ectopic deposition of fibronectin in the periodontal ligament. Thus, these results demonstrate a subcellular role of periostin in promotion of fibronectin secretion from the ER.
-
Expression, Purification and Characterization of Soluble Recombinant Periostin Protein Produced by Escherichia coli
Journal of Biochemistry, 2009Co-Authors: Issei Takayama, Akira KudoAbstract:: Periostin is a matricellular protein participating in the tissue remodelling of damaged cardiac tissue after acute myocardial infarction and of the periodontal ligament in mice. However, further studies on the periostin protein have been limited by the intrinsic difficulty of purifying this protein produced in Escherichia coli due to its insolubility. Here, we demonstrate the expression of recombinant periostin protein with high solubility and monodispersity in E. coli. Periostin is composed of an amino-terminal EMI Domain, a tandem repeat of 4 fas1 Domains (RD1-4), and a carboxyl-terminal region (CTR). We expressed the RD4-CTR region tagged with GST at amino-terminal and 6x Histidine at carboxyl-terminal end in E. coli. The recombinant protein was purified by using GSH-Sepharose and nickel chelation affinity chromatography, followed by gel filtration chromatography. The RD4-CTR protein exhibited high solubility and monodispersity. On average, 9.1 mg of purified RD4-CTR was routinely obtained from 1 L of culture media. Furthermore, the RD4-CTR was biochEMIcally active, because it bound to the RD1-4, the same as intact periostin protein that had been purified from mammalian cells. Our results should enable us to produce the periostin recombinant protein in large quantities and facilitate future studies on functional and structural analyses of periostin.
Giorgio M. Bressan - One of the best experts on this subject based on the ideXlab platform.
-
Overlapping, complementary and site-specific expression pattern of genes of the EMILIN/Multimerin family
Matrix Biology, 2020Co-Authors: Paola Braghetta, Paolo Bonaldo, Miriam Zanetti, Alessandra Ferrari, Paola De Gemmis, Dino Volpin, Giorgio M. BressanAbstract:The EDEN gene superfamily comprises genes that contain the EMI Domain, a structural motif recently identified in proteins of the extracellular matrix. We report here the detailed expression pattern of genes of the EMILIN/Multimerin family, the most numerous group of EDEN superfamily, during mouse development. In situ hybridization has revealed that the EMILIN/Multimerin genes are particularly expressed in the cardio-vascular system and in mesenchymal cells. In general, the territories of expression of each gene are partially overlapping or complementary with that of other members of the family and, usually, more than one gene of the family is active in different tissues, consistent with the possibility of functional compensation. The analysis is particularly relevant in the interpretation of gene targeting experiments.
-
EMILIN-3, peculiar member of elastin microfibril interface-located protein (EMILIN) family, has distinct expression pattern, forms oligomeric assemblies, and serves as transforming growth factor β (TGF-β) antagonist.
Journal of Biological Chemistry, 2012Co-Authors: Alvise Schiavinato, Ann-kathrin A. Becker, Miriam Zanetti, Diana Corallo, Martina Milanetto, Dario Bizzotto, Giorgio M. Bressan, Marija Guljelmovic, Mats Paulsson, Raimund WagenerAbstract:Abstract EMILIN-3 is a glycoprotein of the extracellular matrix belonging to a family that contains a characteristic N-terminal cysteine-rich EMI Domain. Currently, EMILIN-3 is the least characterized member of the elastin microfibril interface-located protein (EMILIN)/Multimerin family. Using RNA, immunohistochEMIcal, and protein chEMIstry approaches, we carried out a detailed characterization of the expression and biochEMIcal properties of EMILIN-3 in mouse. During embryonic and postnatal development, EMILIN-3 showed a peculiar and dynamic pattern of gene expression and protein distribution. EMILIN-3 mRNA was first detected at E8.5–E9.5 in the tail bud and in the primitive gut, and at later stages it became abundant in the developing gonads and osteogenic mesenchyme. Interestingly and in contrast to other EMILIN/Multimerin genes, EMILIN-3 was not found in the cardiovascular system. Despite the absence of the globular C1q Domain, immunoprecipitation and Western blot analyses demonstrated that EMILIN-3 forms disulfide-bonded homotrimers and higher order oligomers. Circular dichroism spectroscopy indicated that the most C-terminal part of EMILIN-3 has a substantial α-helical content and forms coiled coil structures involved in EMILIN-3 homo-oligomerization. Transfection experiments with recombinant constructs showed that the EMI Domain contributes to the higher order self-assembly but was dispensable for homotrimer formation. EMILIN-3 was found to bind heparin with high affinity, a property mediated by the EMI Domain, thus revealing a new function for this Domain that may contribute to the interaction of EMILIN-3 with other extracellular matrix and/or cell surface molecules. Finally, in vitro experiments showed that EMILIN-3 is able to function as an extracellular regulator of the activity of TGF-β ligands.
-
The EMILIN/Multimerin Family
Frontiers in Immunology, 2012Co-Authors: Alfonso Colombatti, Roberto Doliana, Giorgio M. Bressan, Maurizio Mongiat, Paola Spessotto, Gennaro EspositoAbstract:EMILINs and Multimerins (EMILIN1, EMILIN2, Multimerin1, and Multimerin2) constitute a four member family that in addition to the shared C-terminus gC1q Domain typical of the gC1q/TNF superfamily members contain a N-terminus unique cysteine-rich EMI Domain. These glycoproteins are homotrimeric and assemble into high molecular weight multimers. They are predominantly expressed in the extracellular matrix and contribute to several cellular functions in part associated with the gC1q Domain and in part not yet assigned nor linked to other specific regions of the sequence. Among the latter is the control of arterial blood pressure, the inhibition of Bacillus anthracis cell cytotoxicity, the promotion of cell death, the proangiogenetic function and a role in platelet hemostasis. The focus of this review is to highlight the multiplicity of functions and Domains of the EMILIN/Multimerin family with a particular emphasis on the regulatory role played by the ligand-receptor interactions of the gC1q Domain.EMILIN1 is the most extensively studied member both from the structural and functional point of view. The structure of the gC1q of EMILIN1 solved by NMR highlights unique characteristics compared to other gC1q Domains: it shows a marked decrease of the contact surface of the trimeric assembly and while conserving the jelly-roll topology with two β-sheets of antiparallel strands it presents a nine-stranded β-sandwich fold instead of the usual ten-stranded fold. This is likely due to the insertion of nine residues that disrupt the ordered strand organization and forma a highly dynamic protruding loop.. In this loop the residue E933 is the site of interaction between gC1q and the α4β1 and α9β1 integrins, and contrary to integrin occupancy that usually upregulates cell growth, when gC1q is ligated by the integrin the cells reduce their proliferative activity.
-
EMIlin1 deficiency causes structural and functional defects of lymphatic vasculature
Molecular and Cellular Biology, 2008Co-Authors: Carla Danussi, Roberto Doliana, Giorgio M. Bressan, Paola Spessotto, Alessandra Petrucco, Bruna Wassermann, Patrizia Sabatelli, Monica Montesi, Alfonso ColombattiAbstract:Lymphatic and blood vascular systems have distinct structural characteristics that reflect their specific and complementary functions. The lymphatic vasculature represents a second circulatory system and maintains tissue fluid homeostasis; it plays a major role in the absorption of dietary fat and in immune response, transporting lymphocytes and antigen-presenting cells to regional lymph nodes; finally, it provides routes for tumor metastasis (8). The lymphatic system consists of a complex network of lymphatic capillaries, which are uniquely adapted for the uptake of protein-rich lymph from tissue interstitium, and collecting lymphatic vessels that transport lymph back to the blood vascular system. The latter are surrounded by a basement membrane and smooth muscle cells, which are less organized than in blood vessels and, in addition, have intraluminal valves, which prevent lymph backflow (26). By contrast, lymphatic capillaries are blind-end vessels, lined by a single thin layer of overlapping lymphatic endothelial cells (LECs) directly connected to the surrounding extracellular matrix (ECM) by means of anchoring elastic filaments (18). These structures play a fundamental role in lymphatic-vessel function and represent one of the main distinguishing features between lymphatic and blood capillaries. When interstitial fluid pressure increases, anchoring filaments exert tension on LECs, thereby widening the capillary lumen and opening the overlapping cell junctions, which enable fluid and macromolecule uptake and cell entry. It is thought that abnormalities of anchoring filaments may reduce adsorption from the interstitium and propulsion of lymph and cells and promote pathological conditions, such as lymphedema or diseases related to impaired immune responses (9, 18). Furthermore, the perivascular ECM plays an integral role in lymphatic-vessel function, as the fluid equilibrium is controlled by the cooperation of both lymphatic function and the ECM (40). The elasticity and hydration of a tissue is determined by the composition and organization of the ECM. Extensive and chronic degradation of the ECM eventually renders lymphatic vessels nonresponsive to the changes in the interstitium and therefore causes dysfunction (28, 30). Lymphatic and blood endothelial cells express different lineage-specific molecules involved in the regulation of their biological functions that are frequently used as distinguishing markers, such as vascular endothelial growth factor receptor 3 (VEGFR-3) (22), podoplanin (3), Prox-1 (44), LYVE-1 (1, 34), neuropilin 2 (46), CCL21 (24), and desmoplakin (12). Recently, a comparative microarray analysis of gene expression profiles of lymphatic and blood endothelial cells identified previously unknown lymphatic lineage genes, including macrophage mannose receptor 1, plakoglobin, the chemokine CCL20, the integrin α9β1 (19, 32), and EMILIN1 (33). EMILIN1 is an ECM glycoprotein associated with elastic fibers (4, 6) and composed of an N-terminal cysteine-rich Domain and the EMI Domain (11), followed by a coiled-coil structure, a short collagenous stalk, and a C-terminal gC1q Domain (7). EMILIN1 is particularly abundant in the walls of large blood vessels, such as the aorta (10), and has been implicated in multiple functions. EMILIN1 is involved in elastogenesis and in the maintenance of blood vascular cell morphology (48). It interacts with the α4β1 integrin through the gC1q1 Domain (36) and has strong adhesive and migratory properties for different cell types (10, 36, 37). EMILIN1, via the EMI Domain, regulates pro-transforming growth factor beta (TGF-β) maturation and is involved in blood pressure homeostasis (47). To directly investigate the physiological function of EMILIN1 in lymphatic vessels, we studied the effects of its absence in mice that had targeted deletions in the EMIlin1 gene (48). Here, we report that EMILIN1 is highly expressed by LECs in vitro and that it colocalizes with lymphatic vessels in several mouse tissues. Importantly, EMIlin1 deficiency results in hyperplasia and enlargement of lymphatic vessels and in a significant reduction of anchoring filaments compared to those of wild-type (WT) mice. The lymphatic vessels of EMIlin1−/− mice are functionally altered. We found that lack of EMILIN1 leads to a mild lymphedema associated with inefficient lymph drainage and increased leakage. In addition, EMIlin1−/− mice develop larger lymphangiomas than their WT littermates. Altogether, these findings demonstrate an important role of EMILIN1 in the structure-function relationship of lymphatic vessels and identify EMILIN1 as a lymphangiogenesis modulator.