Elastin Microfibril Interface-located Protein Research Articles

Emilin2 marks the target region for mesenchymal cell accumulation in bone regeneration

BackgroundRegeneration of injured tissue is dependent on stem/progenitor cells, which can undergo proliferation and maturation processes to replace the lost cells and extracellular matrix (ECM). Bone has a higher regenerative capacity than other tissues, with abundant mesenchymal progenitor cells in the bone marrow, periosteum, and surrounding muscle. However, the treatment of bone fractures is not always successful; a marked number of clinical case reports have described nonunion or delayed healing for various reasons. Supplementation of exogenous stem cells by stem cell therapy is anticipated to improve treatment outcomes; however, there are several drawbacks including the need for special devices for the expansion of stem cells outside the body, low rate of cell viability in the body after transplantation, and oncological complications. The use of endogenous stem/progenitor cells, instead of exogenous cells, would be a possible solution, but it is unclear how these cells migrate towards the injury site.MethodsThe chemoattractant capacity of the elastin microfibril interface located protein 2 (Emilin2), generated by macrophages, was identified by the migration assay and LC–MS/MS. The functions of Emilin2 in bone regeneration were further studied using Emilin2–/– mice.ResultsThe results show that in response to bone injury, there was an increase in Emilin2, an ECM protein. Produced by macrophages, Emilin2 exhibited chemoattractant properties towards mesenchymal cells. Emilin2–/– mice underwent delayed bone regeneration, with a decrease in mesenchymal cells after injury. Local administration of recombinant Emilin2 protein enhanced bone regeneration.ConclusionEmilin2 plays a crucial role in bone regeneration by increasing mesenchymal cells. Therefore, Emilin2 can be used for the treatment of bone fracture by recruiting endogenous progenitor cells.

Elastin microfibril interface-located protein 1 in fibroblasts is regulated by amphiregulin and interleukin-1α produced by keratinocytes.

The structure of elastic fibres changes with ageing. Elastin microfibril interface-located protein 1 (EMILIN-1) is known to contribute to structural changes in elastic fibres. EMILIN-1 is one of the components of elastic fibres and also colocalizes with oxytalan fibres near the epidermis. Therefore, EMILIN-1 may be affected by epidermal-dermal interactions. The purpose of this study is to identify the key factors involved in epidermal-dermal interactions during the structural degeneration of elastic fibres. Keratinocytes and fibroblasts were co-cultured, and changes in elastic fibre-related proteins were evaluated. Additionally, cytokine arrays were used to identify the factors involved in epidermal-dermal interactions. EMILIN-1 expression in fibroblasts was increased in the presence of keratinocytes, and its expression decreased when keratinocytes were stressed. Amphiregulin (AREG) and interleukin-1α (IL-1α) were identified as the keratinocyte-derived cytokines that influence the production of EMILIN-1, which is secreted by the fibroblasts. EMILIN-1 expression was promoted by AREG and decreased by IL-1α via an increase in cathepsin K (a catabolic enzyme). AREG and IL-1α were associated with changes in EMILIN-1 levels in fibroblasts. The findings suggest that the suppression of IL-1α expression and promotion of AREG expression in the epidermis could be a new approach that prevents the wrinkles and sagging caused by the structural changes in elastic fibres.

The role of EMILIN-1 in the osteo/odontogenic differentiation of dental pulp stem cells

BackgroundHuman dental pulp stem cells (hDPSCs) may be the best choice for self-repair and regeneration of teeth and maxillofacial bone tissue due to their homogeneous tissue origin, high proliferation and differentiation rates, and no obvious ethical restrictions. Recently, several studies have shown that extracellular matrix (ECM) proteins can effectively regulate the proliferation and differentiation fate of mesenchymal stem cells (MSCs). However, the role of elastin microfibril interface-located protein-1 (EMILIN-1), a new ECM glycoprotein, in osteo/odontogenic differentiation of hDPSCs has not been reported. The aim of this study was to explore the effect of EMILIN-1 during osteo/odontogenic differentiation of hDPSCs.MethodshDPSCs were cultured in osteo/odontogenic induction medium. qPCR and Western blot analysis were performed to detect osteo/odonto-specific genes/proteins expression as well as the expression of EMILIN-1. After knockdown of Emilin-1 in hDPSCs with small interfering RNA and exogenous addition of recombinant human EMILIN-1 protein (rhEMILIN-1), Cell Counting Kit-8 assay, alkaline phosphatase staining, alizarin red S staining, qPCR and Western blot were performed to examine the effect of EMILIN-1 on proliferation and osteo/odontogenic differentiation of hDPSCs.ResultsDuring the osteo/odontogenic induction of hDPSCs, the expression of osteo/odonto-specific genes/proteins increased, as did EMILIN-1 protein levels. More notably, knockdown of Emilin-1 decreased hDPSCs proliferation and osteo/odontogenic differentiation, whereas exogenous addition of rhEMILIN-1 increased them.ConclusionsThese findings suggested that EMILIN-1 is essential for the osteo/odontogenic differentiation of hDPSCs, which may provide new insights for teeth and bone tissue regeneration.

EMILIN1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.

Elastin microfibril interface-located protein 1 and its catabolic enzyme, cathepsin K, regulate the age-related structure of elastic fibers in the skin.

The elastic fiber structure becomes shorter, thicker, and curved with age. Nonetheless, the proteins and catabolic enzymes influencing the maintenance of and change in the three-dimensional (3D) structure of elastic fibers remain unknown. This study aimed to identify the proteins involved in the maintenance and degeneration of elastic fiber structures. We performed a combined 3D structural analysis using tissue decolorization technology and mRNA abundance and comprehensive protein expression of tissue-derived cells. The relationship between the proteins was evaluated. Elastin microfibril interface-located protein 1 (EMILIN-1) and cathepsin K (CTSK) were implicated in structural changes in elastic fibers with aging. EMILIN-1 and CTSK levels were highly correlated and changed with age. CTSK was identified as the degrading enzyme of EMILIN-1. CTSK fragmented the otherwise linearly existing dermal elastic fiber structure, with more evident changes in oxytalan fibers. EMILIN-1 expression in fibroblasts was increased by co-culturing with keratinocytes. Furthermore, CTSK expression was increased by UV stress in keratinocytes, resulting in decreased EMILIN-1 expression. Using our new assessment strategy, we observed that EMILIN-1 and CTSK are highly linked to changes in the elastic fiber structure with aging. These results indicate that suppressing CTSK expression and increasing EMILIN-1 expression might be an effective approach to prevent elastic fiber morphological changes that lead to wrinkles and sagging. Furthermore, EMILIN-1 in the dermis increases due to interaction with the epidermis, which could provide a new target for the therapeutic care of elastic fibers (including preservation of oxytalan fibers) in epidermis-dermis interaction.

Human amniotic epithelial cell transplantation improves scar remodeling in a rabbit model of acute vocal fold injury: a pilot study

ObjectiveTo gain insight into the molecular mechanisms underlying the early stages of vocal fold extracellular matrix (ECM) remodeling after a mid-membranous injury resulting from the use of human amniotic epithelial cells (hAEC), as a novel regenerative medicine cell-based therapy.MethodsVocal folds of six female, New Zealand White rabbits were bilaterally injured. Three rabbits had immediate bilateral direct injection of 1 × 106 hAEC in 100 µl of saline solution (hAEC) and three with 100 µl of saline solution (controls, CTR). Rabbits were euthanized 6 weeks after injury. Proteomic analyses (in-gel trypsin protein digestion, LC–MS/MS, protein identification using Proteome Discoverer and the Uniprot Oryctolagus cuniculus (Rabbit) proteome) and histological analyses were performed.ResultshAEC treatment significantly increased the expression of ECM proteins, elastin microfibril interface-located protein 1 (EMILIN-1) and myocilin that are primarily involved in elastogenesis of blood vessels and granulation tissue. A reactome pathway analysis showed increased activity of the anchoring fibril formation by collagen I and laminin, providing mechanical stability and activation of cell signaling pathways regulating cell function. hAEC increased the abundance of keratin 1 indicating accelerated induction of the differentiation programming of the basal epithelial cells and, thereby, improved barrier function. Lastly, upregulation of Rab GDP dissociation inhibitor indicates that hAEC activate the vesicle endocytic and exocytic pathways, supporting the exosome-mediated activation of cell–matrix and cell-to-cell interactions.ConclusionsThis pilot study suggests that injection of hAEC into an injured rabbit vocal fold favorably alters ECM composition creating a microenvironment that accelerates differentiation of regenerated epithelium and promotes stabilization of new blood vessels indicative of accelerated and improved repair.

Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.

Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.

Cardiac Malformations in EMILIN2 Deficient Mice

Background: EMILIN2, elastin microfibril interface located protein 2, is expressed during cardiovascular development, by cardiac stem cells, and in adult animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial or complete deletions of this chromosome region have cardiac malformations. The objective of this study was to evaluate whether the Emilin2 deficient mice have cardiac defects. Methods and results: Cardiac malformations were assessed in adult Emilin2, Emilin1 (EMILIN2 homologue) deficient mice, and Emilin2:Emilin1 double deficient mice. The primary malformation in Emilin2 deficient mice was ventricular septal defects. Although EMILIN1, is also expressed during cardiovascular development,the primary cardiac anomalies in Emilin1 deficient mice were valve insufficiency (pulmonary, aortic and mitral). Only half of the expected Emilin2:Emilin1 double deficient offspring were weaned, indicating embryonic and perinatal lethality. Surviving Emilin2:Emilin1 double deficient mice had both ventricular septal defects and regurgitation of valves. There was a trend for anterior wall thinning and left ventricular dilation in Emilin2:Emilin1 double deficient mice. In embryonic Emilin2:Emilin1 double deficient mice complete arterioventricular canal defects and ventricular septal defects were identified. A potential mechanism for development of cardiac defects in Emilin2 deficiency may be reduced mobilization of stem cells during heart development. Conclusions: Emilin2 deficient mice express cardiac anomalies, primarily ventricular septal defects, and Emilin1 mice exhibit primarily valve abnormalities including pulmonary regurgitation, whereas in Emilin2: Emilin1 double deficient mice, both anomalies occurred, suggesting that Emilin2 and Emilin1 have differential roles in cardiac development. Emilin2 deficient mice have cardiac malformations similar to individuals with deletions of the short arm of Chromosome 18. Emilin2 deficient mice may be an informative model to investigate the consequences of cardiac anomalies in subjects with deletions of the short arm of Chromosome 18, 18p

Matrix metalloproteinase-9-dependent mechanisms of reduced contractility and increased stiffness in the aging heart.

Matrix metalloproteinases (MMPs) collectively degrade all extracellular matrix (ECM) proteins. Of the MMPs, MMP-9 has the strongest link to the development of cardiac dysfunction. Aging associates with increased MMP-9 expression in the left ventricle (LV) and reduced cardiac function. We investigated the effect of MMP-9 deletion on the cardiac ECM in aged animals. We used male and female middle-aged (10- to16-month old) and old (20- to 24-month old) wild-type (WT) and MMP-9 null mice (n = 6/genotype/age). LVs were decellularized to remove highly abundant mitochondrial proteins that could mask identification of relative lower abundant components, analyzed by shotgun proteomics, and proteins of interest validated by immunoblot. Elastin microfibril interface-located protein 1 (EMILIN-1) decreased with age in WT (p < 0.05), but not in MMP-9 null. EMILIN-1 promotes integrin-dependent cell adhesion and EMILIN-1 deficiency has been associated with vascular stiffening. Talin-2, a cytoskeletal protein, was elevated with age in WT (p < 0.05), and MMP-9 deficiency blunted this increase. Talin-2 is highly expressed in adult cardiac myocytes, transduces mechanical force to the ECM, and is activated by increases in substrate stiffness. Our results suggest that MMP-9 deletion may reduce age-related myocardial stiffness, which may explain improved cardiac function in MMP-9 null animals. We identified age-related changes in the cardiac proteome that are MMP-9 dependent, suggesting MMP-9 as a possible therapeutic target for the aging patient.

In vitro elastogenesis: instructing human vascular smooth muscle cells to generate an elastic fiber-containing extracellular matrix scaffold

Elastic fibers are essential for the proper function of organs including cardiovascular tissues such as heart valves and blood vessels. Although (tropo)elastin production in a tissue-engineered construct has previously been described, the assembly to functional elastic fibers in vitro using human cells has been highly challenging. In the present study, we seeded primary isolated human vascular smooth muscle cells (VSMCs) onto 3D electrospun scaffolds and exposed them to defined laminar shear stress using a customized bioreactor system. Increased elastin expression followed by elastin deposition onto the electrospun scaffolds, as well as on newly formed fibers, was observed after six days. Most interestingly, we identified the successful deposition of elastogenesis-associated proteins, including fibrillin-1 and -2, fibulin-4 and -5, fibronectin, elastin microfibril interface located protein 1 (EMILIN-1) and lysyl oxidase (LOX) within our engineered constructs. Ultrastructural analyses revealed a developing extracellular matrix (ECM) similar to native human fetal tissue, which is composed of collagens, microfibrils and elastin. To conclude, the combination of a novel dynamic flow bioreactor and an electrospun hybrid polymer scaffold allowed the production and assembly of an elastic fiber-containing ECM.

Distinct profiles of oxidative stress–related and matrix proteins in adult bone and soft tissue osteosarcoma and desmoid tumors: A proteomics study

Osteosarcomas rarely occur in older adults. Proteomics has not been reported to date in osteosarcoma occurring in the older adult population. This proteomic investigation was conducted to identify differentially expressed proteins in osteosarcoma occurring in various backgrounds from older adults. Desmoid tumors, known to recur locally but not metastasize, were also analyzed. Protein digests isolated from formalin-fixed, paraffin-embedded tumor tissue specimen representing 14 primary osteosarcomas of soft tissue and bone and 18 desmoid tumors were analyzed by high-resolution liquid chromatography-tandem mass spectrometry for protein identification and relative quantification by spectral counting. Elevated abundance levels of several proteins including heat shock protein 90 (HSP90), elastin microfibril interface-located protein 1, and clusterin were identified in osteosarcoma with slight differences in proteomic profiles. Desmoids had an abundance of collagen II and periostin only. The findings were confirmed by immunohistochemical staining for HSP90 and clusterin in the experimental samples and additionally in 16 posttherapy conventional osteosarcomas in tissue microarrays constructed from heterogeneous sarcomas and benign lesions. All osteosarcomas were positive for HSP90 and clusterin to a variable extent. One case of well-differentiated parosteal osteosarcoma was negative. Thirty of 75 other high-grade sarcomas including cases of chondrosarcoma were positive for HSP90. Low-grade and benign lesions and scars and 18 desmoid tumors had little or no expression of these proteins. HSP90 and clusterin represent candidate markers of aggressiveness in osteosarcoma occurring in older adults and may be indicative of drug resistance.

CNGA3 is expressed in inner ear hair cells and binds to an intracellular C-terminus domain of EMILIN1

The molecular characteristics of CNG (cyclic nucleotide-gated) channels in auditory/vestibular hair cells are largely unknown, unlike those of CNG mediating sensory transduction in vision and olfaction. In the present study we report the full-length sequence for three CNGA3 variants in a hair cell preparation from the trout saccule with high identity to CNGA3 in olfactory receptor neurons/cone photoreceptors. A custom antibody targeting the N-terminal sequence immunolocalized CNGA3 to the stereocilia and subcuticular plate region of saccular hair cells. The cytoplasmic C-terminus of CNGA3 was found by yeast two-hybrid analysis to bind the C-terminus of EMILIN1 (elastin microfibril interface-located protein 1) in both the vestibular hair cell model and rat organ of Corti. Specific binding between CNGA3 and EMILIN1 was confirmed with surface plasmon resonance analysis, predicting dependence on Ca2+ with Kd=1.6×10-6 M for trout hair cell proteins and Kd=2.7×10-7 M for organ of Corti proteins at 68 μM Ca2+. Pull-down assays indicated that the binding to organ of Corti CNGA3 was attributable to the EMILIN1 intracellular sequence that follows a predicted transmembrane domain in the C-terminus. Saccular hair cells also express the transcript for PDE6C (phosphodiesterase 6C), which in cone photoreceptors regulates the degradation of cGMP used to gate CNGA3 in phototransduction. Taken together, the evidence supports the existence in saccular hair cells of a molecular pathway linking CNGA3, its binding partner EMILIN1 (and β1 integrin) and cGMP-specific PDE6C, which is potentially replicated in cochlear outer hair cells, given stereociliary immunolocalizations of CNGA3, EMILIN1 and PDE6C.

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

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.

EMILIN2 (Elastin microfibril interface located protein), potential modifier of thrombosis

BackgroundElastin microfibril interface located protein 2 (EMILIN2) is an extracellular glycoprotein associated with cardiovascular development. While other EMILIN proteins are reported to play a role in elastogenesis and coagulation, little is known about EMILIN2 function in the cardiovascular system. The objective of this study was to determine whether EMILIN2 could play a role in thrombosis.ResultsEMILIN2 mRNA was expressed in 8 wk old C57BL/6J mice in lung, heart, aorta and bone marrow, with the highest expression in bone marrow. In mouse cells, EMILIN2 mRNA expression in macrophages was higher than expression in endothelial cells and fibroblasts. EMILIN2 was identified with cells and extracellular matrix by immunohistochemistry in the carotid and aorta. After carotid ferric chloride injury, EMILIN2 was abundantly expressed in the thrombus and inhibition of EMILIN2 increased platelet de-aggregation after ADP-stimulated platelet aggregation.ConclusionsThese results suggest EMILIN2 could play a role in thrombosis as a constituent of the vessel wall and/or a component of the thrombus.

Networked-based Characterization of Extracellular Matrix Proteins from Adult Mouse Pulmonary and Aortic Valves

A precise mixture of extracellular matrix (ECM) secreted by valvular cells forms a scaffold that lends the heart valve the exact mechanical and tensile strength needed for accurate hemodynamic performance. ECM proteins are a key component of valvular endothelial cell (VEC)-valvular interstitial cell (VIC) communication essential for maintenance of the valve structure. This study reports the healthy adult pulmonary and aortic valve proteomes characterized by LC-MS/MS, resulting in 2710 proteins expressed by 1513 genes, including over 300 abundant ECM proteins. Surprisingly, this study defines a distinct proteome for each semilunar valve. Protein-protein networking (PPN) was used as a tool to direct selection of proteomic candidates for biological investigation. Local PPN for nidogen 1 (Nid1), biglycan (Bgn), elastin microfibril interface-located protein 1 (Emilin-1), and milk fat globule-EGF factor 8 protein (Mfge8) were enriched with proteins essential to valve function and produced biological functions highly relevant to valve biology. Immunofluorescent investigations demonstrated that these proteins are functionally distributed within the pulmonary and aortic valve structure, indicative of important contribution to valve function. This study yields new insight into protein expression contributing to valvular maintenance and health and provides a platform for unbiased assessment of protein alterations during disease processes.

EMILIN-1 regulates the amount of oxytalan fiber formation in periodontal ligaments in vitro

The elastic system fibers comprise oxytalan, elaunin, and elastic fibers, differing in their relative microfibril and elastin contents. Among them, human periodontal ligament (PDL) contains only oxytalan fibers (pure microfibrils). Elastin microfibril interface-located protein-1 (EMILIN-1) is localized at the interface between microfibrils and elastin. We hypothesized that EMILIN-1 may contribute to the formation of oxytalan fibers. We used a small interfering RNA (siRNA) for EMILIN-1 in PDL cell culture to examine the extracellular deposition of fibrillin-1 (the major component of microfibrils). EMILIN-1 was labeled on microfibrils positive for fibrillin-1 and was colocalized with fibrillin-1 upon immunoprecipitation assay. EMILIN-1 suppression reduced the level of fibrillin-1 deposition to 23% of the control, and this was responsible for the diminution of fibrillin-1 deposition revealed by immunofluorescence. These results suggest that EMILIN-1 may regulate the formation of oxytalan fibers and play a role in their homeostasis.

Mouse chromosome 17 candidate modifier genes for thrombosis

Two overlapping quantitative trait loci (QTLs) for clot stability, Hmtb8 and Hmtb9, were identified on mouse chromosome 17 in an F2 intercross derived from C57BL/6J (B6) and B6-Chr17A/J (B6-Chr17) mouse strains. The intervals were in synteny with a QTL for thrombotic susceptibility on chromosome 18 in a human study, and there were 23 homologs between mouse and human. The objective of this study was to determine whether any of these genes in the syntenic region are likely candidates as modifiers for clot stability. Seven genes, Twsg1, Zfp161, Dlgap1, Ralbp1, Myom1, Rab31, and Emilin2, of the 23 genes with single nucleotide polymorphisms (SNPs) in the mRNA-UTR had differential expression in B6 and A/J mice. Dlgap1, Ralbp1, Myom1, and Emilin2 also had nonsynonymous SNPs. In addition, two other genes had nonsynonymous SNPs, Lama1 and Ndc80. Of these nine candidate genes, Emilin2 was selected for further analysis since other EMILIN (Elastin Microfibril Interface Located Protein) proteins have known functions in vascular structure and coagulation. Differences were found between B6 and A/J mice in vessel wall architecture and EMILIN2 protein in plasma, carotid vessel wall, and thrombi formed after ferric chloride injury. In B6-Chr17A/J mice both clot stability and Emilin2 mRNA expression were higher compared to those in B6 and A/J mice, suggesting the exposure of epistatic interactions. Although other homologous genes in the QTL region cannot be ruled out as causative genes, further investigation of Emilin2 as a candidate gene for thrombosis susceptibility is warranted.Electronic supplementary materialThe online version of this article (doi:10.1007/s00335-010-9274-6) contains supplementary material, which is available to authorized users.

The Extracellular Matrix Glycoprotein Elastin Microfibril Interface Located Protein 2: A Dual Role in the Tumor Microenvironment

We have recently reported that elastin microfibril interface located protein 2 (EMILIN2), an extracellular matrix (ECM) glycoprotein, triggers cell death through a direct binding to death receptors. EMILIN2 thus influences cell viability through a mechanism that is unique for an ECM molecule. In the present work, we report an additional function for this molecule. First, we identify the region responsible for the proapoptotic effects, a 90-amino acid residue-long coiled-coil fragment toward the N-terminus of the molecule. The fragment recapitulates EMILIN2 proapoptotic mechanisms. In addition, using either the full molecule or the active fragment, for the first time, we demonstrate a significant antitumoral effect in vivo, likely due to a decrease in tumor cell viability. Unexpectedly, tumors treated with EMILIN2 or the deletion mutant display a significant increase of tumor angiogenesis. In view of this novel finding, the cotreatment of the growing tumors with an antiangiogenic drug led, in most cases, to a complete regression of tumor growth. These results grant further support to recent findings that pinpoint the microenvironment as an important regulator of cell fate under both physiological and pathological conditions and disclose the possibility of using EMILIN2 fragments as potent antineoplastic tools for cancer treatment.

The Solution Structure of EMILIN1 Globular C1q Domain Reveals a Disordered Insertion Necessary for Interaction with the α4β1 Integrin

The extracellular matrix protein EMILIN1 (elastin microfibril interface located protein 1) is implicated in maintaining blood pressure homeostasis via the N-terminal elastin microfibril interface domain and in trophoblast invasion of the uterine wall via the globular C1q (gC1q) domain. Here, we describe the first NMR-based homology model structure of the human 52-kDa homotrimer of the EMILIN1 gC1q domain. In contrast to all of the gC1q (crystal) structures solved to date, the 10-stranded beta-sandwich fold of the gC1q domain is reduced to nine beta strands with a consequent increase in the size of the central cavity lumen. An unstructured loop, resulting from an insertion unique to EMILIN1 and EMILIN2 family members and located at the trimer apex upstream of the missing strand, specifically engages the alpha4beta1 integrin. Using both Jurkat T and EA.hy926 endothelial cells as well as site-directed mutagenesis, we demonstrate that the ability of alpha4beta1 integrins to recognize the trimeric EMILIN1 gC1q domain mainly depends on a single glutamic acid residue (Glu(933)). Static and flow adhesion of T cells and haptotactic migration of endothelial cells on gC1q is fully dependent on this residue. Thus, EMILIN1 gC1q-alpha4beta1 represents a unique ligand/receptor system, with a requirement for a 3-fold arrangement of the interaction site.

Larger Pipe, Lower Resistance

STKE The pathogenesis of hypertension—a risk factor for heart disease, kidney disease, and stroke—is complex and poorly understood. Zacchigna et al. find that mice lacking elastin microfibril interface-located protein 1 (Emilin1), a secreted extracellular matrix protein expressed in the cardiovascular system, had high blood pressure in conjunction with decreased blood vessel diameter and increased peripheral resistance. Emilin1 contains a cysteine-rich domain, as do other proteins involved in the regulation of growth factor signaling, leading the authors to investigate the relationship between Emilin1 and transforming growth factor-β (TGF-β), which plays a critical role in vascular development and pathophysiology. Emilin1 blocked TGF-β signaling upstream of receptor activation and did not interfere with ligand/receptor binding or signaling in response to mature TGF-β1. Rather, Emilin1 bound to proTGF-β1, preventing its proteolytic processing and the production of biologically active TGF-β1. TGF-β signaling was enhanced in the aortic wall of the mice lacking Emilin1, and inactivation of one TGF-β1 allele in Emilin1 knockout mice restored normal blood vessel diameter and blood pressure. Thus, the authors conclude that Emilin1 acts to regulate blood pressure by modulating TGF-β processing and thus the availability of the biologically active form. — EMA Cell 124 , 929 (2006).