Integrin binding site within the gC1q domain orchestrates EMILIN-1-induced lymphangiogenesis.

Lymphatic vessels (LVs) play a pivotal role in the control of tissue homeostasis and also have emerged as important regulators of immunity, inflammation and tumor metastasis. EMILIN-1 is the first ECM protein identified as a structural modulator of the growth and maintenance of LV; accordingly, Emilin1-/- mice display lymphatic morphological alterations leading to functional defects as mild lymphedema, leakage and compromised lymph drainage. Many EMILIN-1 functions are exerted by the binding of its gC1q domain with the E933 residue of α4 and α9ß1 integrins. To investigate the specific regulatory role of this domain on lymphangiogenesis, we generated a transgenic mouse model expressing an E933A-mutated EMILIN-1 (E1-E933A), unable to interact with α4 or α9 integrin. The mutant resulted in abnormal LV architecture with dense, tortuous and irregular networks; moreover, the number of anchoring filaments was reduced and collector valves had aberrant narrowed structures. E933A mutation also affected lymphatic function in lymphangiography assays and made the transgenic mice more prone to lymph node metastases. The finding that the gC1q/integrin interaction is crucial for a correct lymphangiogenesis response was confirmed and reinforced by functional in vitro tubulogenesis assays. In addition, ex vivo thoracic-duct ring assays revealed that E1-E933A-derived lymphatic endothelial cells had a severe reduction in sprouting capacity and were unable to organize into capillary-like structures. All these data provide evidence that the novel "regulatory structural" role of EMILIN-1 in the lymphangiogenic process is played by the integrin binding site within its gC1q domain.

Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4ß1 integrin interaction, cell adhesion and anti-proliferative activity.

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 α4ß1 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 α4ß1 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 α4ß1 integrin after NE treatment. Functional studies showed that NE impaired the EMILIN1-α4ß1 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.

Local inhibition of elastase reduces EMILIN1 cleavage reactivating lymphatic vessel function in a mouse lymphoedema model.

Lymphatic vasculature critically depends on the connections of lymphatic endothelial cells with the extracellular matrix (ECM), which are mediated by anchoring filaments (AFs). The ECM protein EMILIN1 is a component of AFs and is involved in the regulation of lymphatic vessel functions: accordingly, Emilin1(-/-) mice display lymphatic vascular morphological alterations, leading to functional defects such as mild lymphoedema, lymph leakage and compromised lymph drainage. In the present study, using a mouse post-surgical tail lymphoedema model, we show that the acute phase of acquired lymphoedema correlates with EMILIN1 degradation due to neutrophil elastase (NE) released by infiltrating neutrophils. As a consequence, the intercellular junctions of lymphatic endothelial cells are weakened and drainage to regional lymph nodes is severely affected. The local administration of sivelestat, a specific NE inhibitor, prevents EMILIN1 degradation and reduces lymphoedema, restoring a normal lymphatic functionality. The finding that, in human secondary lymphoedema samples, we also detected cleaved EMILIN1 with the typical bands of an NE-dependent pattern of fragmentation establishes a rationale for a powerful strategy that targets NE inhibition. In conclusion, the attempts to block EMILIN1 degradation locally represent the basis for a novel 'ECM' pharmacological approach to assessing new lymphoedema treatments.

Functional osteoclastogenesis: the baseline variability in blood donor precursors is not associated with age and gender.

Mononuclear osteoclast precursors circulate in the monocyte fraction of peripheral blood and form multinuclear cells with all osteoclastic phenotypic characteristics when cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kB ligand (RANKL). The method to obtain osteoclast precursors from peripheral blood is simple but the number of recovered osteoclasts is often largely insufficient for functional analyses. The original aim of this study was to develop a rapid and efficient method that could overcome the donor variability and enrich the osteoclast precursors from a small volume of peripheral blood as a basis for future clinical studies to correlate the differentiation potential of circulating osteoclast precursors with bone lesions in cancer patients. We improved the efficiency of osteoclastogenesis by reducing isolation and purification times and overcame the use of flow cytometry and immunomagnetic purification procedures. In our culture system the osteoclast number was increased several-fold and the precursors were able to reach a full differentiation within seven days of culture. Both age as well as gender differences in osteoclastogenesis efficiency were no longer evident by processing limited volume blood samples with this simple and rapid method.

Neutrophil elastase-dependent cleavage compromises the tumor suppressor role of EMILIN1.

Proteolysis of the extracellular matrix (ECM) is a key event in tumor growth and progression. The breakdown of ECM can lead to the generation of bioactive fragments that promote cell growth and spread. EMILIN1, a multidomain glycoprotein expressed in several tissues, exerts a crucial regulatory function through the engagement of α4/α9 integrins. Unlike the majority of ECM molecules that elicit a proliferative program, the signals emitting from EMILIN1 engaged by α4/α9ß1 integrins are antiproliferative. In this study, aimed to demonstrate if the suppressor role of EMILIN1 was related to its structural integrity, we tested the possibility that EMILIN1 could be specifically cleaved. Among the proteolytic enzymes released in the tumor microenvironment we showed that neutrophil elastase cleaved EMILIN1 in three/four major fragments. The consequence of this proteolytic process was the impairment of its anti-proliferative role. Accordingly, EMILIN1 was digested in sarcomas and ovarian cancers. Sarcoma specimens were infiltrated by neutrophils (PMNs) and stained positively for elastase. The present findings highlight the peculiar activity of PMN elastase in disabling EMILIN1 suppressor function.

EMILIN1/α9ß1 integrin interaction is crucial in lymphatic valve formation and maintenance.

Lymphatic vasculature plays a crucial role in the maintenance of tissue interstitial fluid balance. The role of functional collecting lymphatic vessels in lymph transport has been recently highlighted in pathologies leading to lymphedema, for which treatments are currently unavailable. Intraluminal valves are of paramount importance in this process. However, valve formation and maturation have not been entirely elucidated yet, in particular, the role played by the extracellular matrix (ECM). We hypothesized that EMILIN1, an ECM multidomain glycoprotein, regulates lymphatic valve formation and maintenance. Using a mouse knockout model, we show that in the absence of EMILIN1, mice exhibit defects in lymphatic valve structure and in lymph flow. By applying morphometric in vitro and in vivo functional assays, we conclude that this impaired phenotype depends on the lack of α9ß1 integrin engagement, the specific lymphatic endothelial cell receptor for EMILIN1, and the ensuing derangement of cell proliferation and migration. Our data demonstrate a fundamental role for EMILIN1-integrin α9 interaction in lymphatic vasculature, especially in lymphatic valve formation and maintenance, and underline the importance of this ECM component in displaying a regulatory function in proliferation and acting as a "guiding" molecule in migration of lymphatic endothelial cells.

An EMILIN1-negative microenvironment promotes tumor cell proliferation and lymph node invasion.

The evidence that EMILIN1 (Elastic Microfibril Interface Located proteIN) deficiency in Emilin1(-/-) mice caused dermal and epidermal hyperproliferation and an abnormal lymphatic phenotype prompted us to hypothesize the involvement of this extracellular matrix component in tumor development and in lymphatic metastasis. Using the 12-dimethylbenz(α)anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) two-stage model of skin carcinogenesis, we found that Emilin1(-/-) mice presented an accelerated formation, a higher incidence, and the development of a larger number of tumors compared with their wild-type littermates. EMILIN1-negative tumors showed more Ki67-positive proliferating cells and higher levels of pErk1/2. In these tumors, PTEN expression was lower. Emilin1(-/-) mice displayed enhanced lymphangiogenesis both in the tumor and in the sentinel lymph nodes. Accordingly, tumor growth and lymph node metastasis of transplanted syngenic tumors were also increased in Emilin1(-/-) mice. In vitro transmigration assays through lymphatic endothelial cells showed that EMILIN1 deficiency greatly facilitated tumor cell trafficking. Overall, these data established that EMILIN1 exerts a protective role in tumor growth, in tumor lymphatic vessel formation, as well as in metastatic spread to lymph nodes and reinforced the importance of its presence in the microenvironment to determine the tumor phenotype.

MMP-13 stimulates osteoclast differentiation and activation in tumour breast bone metastases.

INTRODUCTION: The increased bone degradation in osteolytic metastases depends on stimulation of mature osteoclasts and on continuous differentiation of new pre-osteoclasts. Metalloproteinases (MMP)-13 is expressed in a broad range of primary malignant tumours and it is emerging as a novel biomarker. Recent data suggest a direct role of MMP-13 in dissolving bone matrix complementing the activity of MMP-9 and other enzymes. Tumour-microenvironment interactions alter gene expression in malignant breast tumour cells promoting osteolytic bone metastasis. Gene expression profiles revealed that MMP-13 was among the up-regulated genes in tumour-bone interface and its abrogation reduced bone erosion. The precise mechanism remained not fully understood. Our purpose was to further investigate the mechanistic role of MMP-13 in bone osteolytic lesions. METHODS: MDA-MB-231 breast cancer cells that express MMP-13 were used as a model for in vitro and in vivo experiments. Conditioned media from MDA-MB-231 cells were added to peripheral blood mononuclear cultures to monitor pre-osteoclast differentiation and activation. Bone erosion was evaluated after injection of MMP-13-silenced MDA-MB-231 cells into nude mice femurs. RESULTS: MMP-13 was co-expressed by human breast tumour bone metastases with its activator MT1-MMP. MMP-13 was up-regulated in breast cancer cells after in vitro stimulation with IL-8 and was responsible for increased bone resorption and osteoclastogenesis, both of which were reduced by MMP inhibitors. We hypothesized that MMP-13 might be directly involved in the loop promoting pre-osteoclast differentiation and activity. We obtained further evidence for a direct role of MMP-13 in bone metastasis by a silencing approach: conditioned media from MDA-MB-231 after MMP-13 abrogation or co-cultivation of silenced cells with pre-osteoclast were unable to increase pre-osteoclast differentiation and resorption activity. MMP-13 activated pre-MMP-9 and promoted the cleavage of galectin-3, a suppressor of osteoclastogenesis, thus contributing to pre-osteoclast differentiation. Accordingly, MMP-13 abrogation in tumour cells injected into the femurs of nude mice reduced the differentiation of TRAP positive cells in bone marrow and within the tumour mass as well as bone erosion. CONCLUSIONS: These results indicate that within the inflammatory bone microenvironment MMP-13 production was up-regulated in breast tumour cells leading to increased pre-osteoclast differentiation and their subsequent activation.

EMILIN1-α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation.

EMILIN1 promotes α4ß1 integrin-dependent cell adhesion and migration and reduces pro-transforming growth factor-ß processing. A knockout mouse model was used to unravel EMILIN1 functions in skin where the protein was abundantly expressed in the dermal stroma and where EMILIN1-positive fibrils reached the basal keratinocyte layer. Loss of EMILIN1 caused dermal and epidermal hyperproliferation and accelerated wound closure. We identified the direct engagement of EMILIN1 to α4ß1 and α9ß1 integrins as the mechanism underlying the homeostatic role exerted by EMILIN1. The lack of EMILIN1-α4/α9 integrin interaction was accompanied by activation of PI3K/Akt and Erk1/2 pathways as a result of the reduction of PTEN. The down-regulation of PTEN empowered Erk1/2 phosphorylation that in turn inhibited Smad2 signaling by phosphorylation of residues Ser245/250/255. These results highlight the important regulatory role of an extracellular matrix component in skin proliferation. In addition, EMILIN1 is identified as a novel ligand for keratinocyte α9ß1 integrin, suggesting prospective roles for this receptor-ligand pair in skin homeostasis.

Blood-derived human osteoclast resorption activity is impaired by Hyaluronan-CD44 engagement via a p38-dependent mechanism.

The control of bone resorption is crucial in osteolytic diseases. Once attached to bone, osteoclasts (OCs) initiate the resorption process through the activation of a complex cascade of morphological and biochemical changes. Hyaluronan (HA), an extracellular glycosaminoglycan long non-branching polysaccharide, is expressed in bone matrices. Here we demonstrate that HA counter-balances the erosion activity of human mature OCs by significantly reducing their degradative potential. HA treatment of fully differentiated OCs derived from human peripheral blood monocytes inhibited migration on collagen as well as bone resorption. HA-mediated effects were primarily due to TRAcP, MMP-9, and cathepsin K down-regulation and to the increased levels of TIMP-1, a natural MMP-9 inhibitor. Binding of HA to mature OCs was entirely mediated by CD44: function-blocking anti-CD44 antibodies fully abrogated HA effects, and the engagement of HA receptor caused a rapid de-phosphorylation of Ser325 in the CD44 cytoplasmic tail. The inhibitory action by HA was associated with a transient up-phosphorylation of Pyk2, a novel persistent phosphorylation of p38 and the down-regulation of NFATc1 transcription factor. Our results provide a direct evidence for the involvement of CD44 in the HA-dependent regulation of OC activity and suggest a signaling pathway that could be unique in OC function inhibition.

NG2 expression predicts the metastasis formation in soft-tissue sarcoma patients.

Enhanced expression levels of NG2 proteoglycan in presurgical original lesions of soft-tissue sarcoma (STS) patients defines with 55% probability the immediate (i.e., within 12 months postsurgery) risk in these individuals to develop postsurgical secondary lesions, independently of any other clinical trait. It, therefore, provides a molecular factor that alone prospects a particularly unfavorable clinical outcome in such patients. Evaluation of the timing of metastasis formation in patients with high and low levels of NG2 in their primitive lesions further stratified the patients in subsets with diverse lag phases in the occurrence of metastatic disease. In our cohort of high-grade STS cases, transcription of NG2 also showed a 81-fold amplification in metastatic lesions, when compared to primitive ones, and this gene overexpression was accompanied by an abundant but nonuniform in situ expression of its product. In a similar manner as seen in primitive lesions, patients with higher levels of metastatic NG2 encountered a significantly more dismal clinical course. Multivariate analysis asserted that in these individuals upregulation of NG2 represented an absolute independent prognostic parameter. Therefore, minimally invasive assessment of the transcription levels of the NG2 gene represents a parameter capable of predicting the arising of metastatic disease within a definite postsurgery time interval, and affords in adjunct in the definition of life expectance in STS patients.

Emilin1 deficiency causes structural and functional defects of lymphatic vasculature.

Lymphatic-vasculature function critically depends on extracellular matrix (ECM) and on its connections with lymphatic endothelial cells (LECs). However, the composition and the architecture of ECM have not been fully taken into consideration in studying the biology and the pathology of the lymphatic system. EMILIN1, an elastic microfibril-associated protein, is highly expressed by LECs in vitro and colocalizes with lymphatic vessels in several mouse tissues. A comparative study between WT and Emilin1-/- mice highlighted the fact that Emilin1 deficiency in both CD1 and C57BL/6 backgrounds results in hyperplasia, enlargement, and frequently an irregular pattern of superficial and visceral lymphatic vessels and in a significant reduction of anchoring filaments. Emilin1-deficient mice also develop larger lymphangiomas than WT mice. Lymphatic vascular morphological alterations are accompanied by functional defects, such as mild lymphedema, a highly significant drop in lymph drainage, and enhanced lymph leakage. Our findings demonstrate that EMILIN1 is involved in the regulation of the growth and in the maintenance of the integrity of lymphatic vessels, a fundamental requirement for efficient function. The phenotype displayed by Emilin1(-/-) mice is the first abnormal lymphatic phenotype associated with the deficiency of an ECM protein and identifies EMILIN1 as a novel local regulator of lymphangiogenesis.

Expression profiles in malignant fibrous histiocytomas: clues for differentiating 'spindle cell' and 'pleomorphic' subtypes.

We analysed 21 samples of malignant fibrous histiocytoma (MFH) distinguished into the two principal morphological categories ('spindle cell' and the 'pleomorphic' subtypes). The aim of our study was to verify if a distinction between the two subclasses of MFH in terms of expression/activation of protein profiles could support and extend the morphological criteria. For this purpose, we carried out an immunohistochemical and immunoblotting analysis of proteins that could be relevant in sarcoma biology and potential diagnostic and therapeutical targets such as matrix metalloproteinases (MMPs) and molecules related to adhesive and proliferative properties. Our analysis revealed that MMP-1, MMP-9 expression and p27(kip1) cytoplasmic localisation can be considered valid parameters in the classification and potential explanation of the aggressive behaviour of this non-homogeneous group of MFH.

Differential Expression of IRS-1 and IRS-2 in Uterine Leiomyosarcomas with Distinct Oncogenic Phenotypes: Lack of Correlation with Downstream Signaling Events.

PURPOSE: Insulin receptor substrates (IRSs) are essential for insulin-induced mitogenic effects on several cell types but they also are involved in cell transformation.We investigated whether the differential constitutive expression and potential distinct downstream signaling events of IRS-1 and IRS-2 might be related to discrete tumourigenic phenotypes of three human uterine leiomyosarcoma cell lines, one of which was specifically isolated for the present study. METHODS AND RESULTS: SK-UT-1B egressed effectively from a gellyfied Matrigel matrix and grew as did DMR cells in an anchorage-independent manner in agar and induced rapidly growing tumours in nude mice. On the contrary, SK-LMS-1 cells did not emigrate from Matrigel, neither grew in agar nor were they tumourigenic. IRS-2 was highly expressed in the more malignant cell lines, whereas IRS-1 was present only in SK-LMS-1 cells. However, upon insulin stimulation both IRS- 1 and IRS-2 were tyrosine phosphorylated with a similar kinetic in the respective cell lines; furthermore, after 1 min of insulin stimulation PI3-kinase associated with IRSs and after 2 min Shc was phosphorylated and associated with Grb2 with minor differences detectable among the various cell lines in the duration of phosphorylation and/or in their association irrespective of whether IRS-1 or IRS-2 were expressed. DISCUSSION: Our findings tend to exclude that the malignancy displayed by uterine leiomyosarcomas might be directly linked to the activation of distinct IRS-1- or IRS-2-dependent pathways.