The α4/α9 integrins directly engage the ECM glycoprotein EMILIN1 to inhibit skin cell proliferation upstream of TGF-β signaling.

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.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 intersti...

IntroductionThe 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.MethodsMDA-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.ResultsMMP-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.ConclusionsThese 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.

f 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 ␣9␤1 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 ␣9 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.