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Assessment of the host immune response pattern is of increasing importance as highly prognostic and diagnostic, in immune-related diseases and in some types of cancer. Chronic inflammation is a major hallmark in colon cancer formation, but, despite the extent of local inflammatory infiltrate has been demonstrated to be extremely informative, its evaluation is not routinely assessed due to the complexity and limitations of classical immunohistochemistry (IHC). In the last years, technological advance helped in bypassing technical limits, setting up multiplex IHC (mIHC) based on tyramide signal amplification (TSA) method and designing software suited to aid pathologists in cell scoring analysis. Several studies verified the efficacy of this method, but they were restricted to the analysis of human samples. In the era of translational medicine the use of animal models to depict human pathologies, in a more complete and complex approach, is really crucial. Nevertheless, the optimization and validation of this method to species other than human is still poor. We took advantage of Multispectral Imaging System to identify the immunoprofile of Dextran Sulphate Sodium (DSS)-treated mouse colon. We optimized a protocol to sequentially stain formalin fixed paraffin embedded murine colon samples for CD3, CD8a, CD4, and CD4R5B0 antigens. With this approach we obtained a detailed lymphocyte profile, while preserving the morphological tissue context, generally lost with techniques like gene expression profiling or flow cytometry. This study, comparing the results obtained by mIHC with immunophenotyping performed with cytofluorimetric and standard IHC methods validates the potentiality and the applicability of this innovative approach.

Colon cancer is one of the first tumor types where a functional link between inflammation and tumor onset has been described; however, the microenvironmental cues affecting colon cancer progression are poorly understood. Here we demonstrate that the expression of the ECM molecule EMILIN-1 halts the development of AOM-DSS induced tumors. In fact, upon AOM-DSS treatment the Emilin1-/- (E1-/-) mice were characterized by a higher tumor incidence, bigger adenomas and less survival. Similar results were obtained with the E933A EMILIN-1 (E1-E933A) transgenic mouse model, expressing a mutant EMILIN-1 unable to interact with α4/α9β1 integrins. Interestingly, upon chronic treatment with DSS, E1-/- and E1-E933A mice were characterized by the presence of increased inflammatory infiltrates, higher colitis scores and more severe mucosal injury respect to the wild type (E1+/+) mice. Since alterations of the intestinal lymphatic network are a well-established feature of human inflammatory bowel disease and EMILIN-1 is a key structural element in the maintenance of the integrity of lymphatic vessels, we assessed the lymphatic vasculature in this context. The analyses revealed that both E1-/- and E1-E933A mice displayed a higher density of LYVE-1 positive vessels; however, their functionality was severely compromised after colitis induction. Taken together, these results suggest that the loss of EMILIN-1 expression may cause the reduction of the inflammatory resolution during colon cancer progression due to a decreased lymph flow and impaired inflammatory cell drainage.

// Teresa Maria Elisa Modica 1 , Orlando Maiorani 1 , Giulio Sartori 1 , Eliana Pivetta 1 , Roberto Doliana 1 , Alessandra Capuano 1 , Alfonso Colombatti 1 , Paola Spessotto 1 1 Department of Translational Research, Experimental Oncology 2 Division, CRO Aviano, National Cancer Institute, Aviano, PN 33081, Italy. Correspondence to: Alfonso Colombatti, email: acolombatti@cro.it Keywords: extracellular matrix, gC1q domain, α4β1 integrin, RAS-ERK pathway, proliferation Abbreviations: ECM, extracellular matrix Received: October 26, 2016 Accepted: January 09, 2017 Published: February 03, 2017 ABSTRACT The extracellular matrix plays a fundamental role in physiological and pathological proliferation. It exerts its function through a signal cascade starting from the integrins that take direct contact with matrix constituents most of which behave as pro-proliferative clues. On the contrary, EMILIN1, a glycoprotein interacting with the α4β1 integrin through its gC1q domain, plays a paradigmatic anti-proliferative role. Here, we demonstrate that the EMILIN1-α4 interaction de-activates the MAPK pathway through HRas. Epithelial cells expressing endogenous α4 integrin and persistently plated on gC1q inhibited pERK1/2 increasing HRasGTP and especially the HRasGTP ubiquitinated form (HRasGTP-Ub). The drug salirasib reversed this effect. In addition, only the gC1q-ligated α4 integrin chain co-immunoprecipitated the ubiquitinated HRas. Only epithelial cells transfected with the wild type form of the α4 integrin chain showed the EMILIN1/α4β1/HRas/pERK1/2 link, whereas cells transfected with a α4 integrin chain carrying a truncated cytoplasmic tail had no effect. In this study we unveiled the pathway activated by the gC1q domain of EMILIN1 through α4β1 integrin engagement and leading to the decrease of proliferation in an epithelial system.

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.

Heritable connective tissue diseases are a highly heterogeneous family of over 200 disorders that affect the extracellular matrix. While the genetic basis of several disorders is established, the etiology has not been discovered for a large portion of patients, likely due to rare yet undiscovered disease genes. By performing trio‐exome sequencing of a 55‐year‐old male proband presenting with multiple symptoms indicative of a connective disorder, we identified a heterozygous missense alteration in exon 1 of the Elastin Microfibril Interfacer 1 (EMILIN1) gene, c.64G>A (p.A22T). The proband presented with ascending and descending aortic aneurysms, bilateral lower leg and foot sensorimotor peripheral neuropathy, arthropathy, and increased skin elasticity. Sanger sequencing confirmed that the EMILIN1 alteration, which maps around the signal peptide cleavage site, segregated with disease in the affected proband, mother, and son. The impaired secretion of EMILIN‐1 in cells transfected with the mutant p.A22T coincided with abnormal protein accumulation within the endoplasmic reticulum. In skin biopsy of the proband, we detected less EMILIN‐1 with disorganized and abnormal coarse fibrils, aggregated deposits underneath the epidermis basal lamina, and dermal cells apoptosis. These findings collectively suggest that EMILIN1 may represent a new disease gene associated with an autosomal‐dominant connective tissue disorder.

EMILIN1, a homo-trimeric adhesive ECM glycoprotein, interacts with the α4β1 integrin through its gC1q domain. Uniquely among the C1q family members, the EMILIN1 gC1q presents only nine-stranded β-sandwich fold and the missing strand is substituted by a disordered 19-residue long segment spanning from Y927 to G945 at the apex of the gC1q domain. This unstructured loop exposes to the solvent the acidic residue E933, which plays a key role in the α4β1 integrin mediated interaction. Here, we experimentally determined that the three E933 residues (one from each monomer) are all required for ligand binding. By docking the NMR structure of the gC1q to a virtual α4β1 crystal structure based on the known structures of α4β7 and α5β1 integrins we built a model of α4β1-gC1q complex where three E933 residues are smoothly forced to coordinate the Mg2+ ion at the βI MIDAS site of the integrin. By bringing the three E933 close in space, the trimeric supramolecular organization of gC1q allows the formation of a proper 3D geometry and suggests a quaternary-structure-dependent mode of interaction. Furthermore, we experimentally identified R904 as a synergistic residue for cell adhesion. Accordingly, the model showed that this residue is able to form potential stabilizing intra-chain salt bridges with residues E928 and E930. This mode of interaction likely accounts for a more stable and durable α4β1-gC1q interaction in comparison with the prototypic CS1 ligand. To our knowledge, this is the first report describing the simultaneous involvement of all the three acidic residues of a trimeric ligand in the formation of a dimeric complex with the integrin βI domain.

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 extracellular matrix glycoprotein EMILIN1 exerts a wide range of functions mainly associated with its gC1q domain. Besides providing functional significance for adhesion and migration, the direct interaction between α4β1 integrin and EMILIN1-gC1q regulates cell proliferation, transducing net anti-proliferative effects. We have previously demonstrated that EMILIN1 degradation by neutrophil elastase (NE) is a specific mechanism leading to the loss of functions disabling its regulatory properties. In this study we further analysed the proteolytic activity of NE, MMP-3, MMP-9, and MT1-MMP on EMILIN1 and found that MMP-3 and MT1-MMP partially cleaved EMILIN1 but without affecting the functional properties associated with the gC1q domain, whereas NE was able to fully impair the interaction of gC1q with the α4β1 integrin by cleaving this domain outside of the E933 integrin binding site. By a site direct mutagenesis approach we mapped the bond between S913 and R914 residues and selected the NE-resistant R914W mutant still able to interact with the α4β1 integrin after NE treatment. Functional studies showed that NE impaired the EMILIN1-α4β1 integrin interaction by cleaving the gC1q domain in a region crucial for its proper structural conformation, paving the way to better understand NE effects on EMILIN1-cell interaction in pathological context.

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.