Eosinophils have been associated with fibrosis. To investigate their direct role in fibrosis, human peripheral blood eosinophil sonicate was added to human lung or dermal fibroblasts, and proliferation ([ 3 H]thymidine) and collagen synthesis ([ 3 H]proline) were evaluated. Proliferation was enhanced significantly in the monolayers in a dosedependent manner. The activity of the eosinophil fibrogenic factor(s) remained unaltered when heated (56°C, 30 min). Supernatants of cultured eosinophils (20 min or 18 hr) also enhanced lung fibroblast proliferation, indicating that the preformed mitogenic factor(s) can be released both promptly and with a long kinetic. Eosinophils significantly decreased collagen production in lung fibroblasts while increasing it in dermal fibroblasts. However, eosinophils containing matrix metalloproteinase 9 (zymography) in latent form and tissue inhibitors of metalloproteinases 1 and 2 (reverse zymography) did not inf luence either fibroblast matrix metalloproteinases or tissue inhibitors of metalloproteinases. Eosinophil sonicate added to skin and lung fibroblasts in tridimensional collagen lattices significantly enhanced lattice contraction. Transforming growth factor ␤ (TGF-␤) is a major fibrogenic cytokine produced by eosinophils. Therefore, to assess its role, eosinophil sonicate was preincubated with anti-TGF-␤ neutralizing antibodies. This treatment partially inhibited proliferation of lung and collagen synthesis of dermal fibroblasts and suppressed the stimulation of lattice contraction, indicating the fibrogenic role of eosinophil-associated TGF-␤. In conclusion, we have shown that eosinophils act as direct modulatory cells in fibroblast proliferation, collagen synthesis, and lattice contraction, in part, through TGF-␤. These data corroborate the importance of eosinophils in skin and lung fibrosis.

We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mM lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII ␤-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.The extracellular matrix protein elastin is responsible for the elastic properties of tissues such as lung, skin, and large arteries (1-3). Due to its numerous cross-links and the extreme hydrophobicity of its tropoelastin chains, elastin is highly resistant to proteolysis. However, during inflammatory disorders, proteinases secreted from polymorphonuclear neutrophils, such as elastase, cathepsin G, and gelatinase B may cause significant elastolysis (4).It has been established that peptides derived from elastin or from the hydrophobic domains of tropoelastin interact with cells via a cell surface-resided 67-kDa elastin-binding protein identical to an enzymatically inactive, alternatively spliced form of ␤-galactosidase (5). The binding of elastin peptides to the elastin-binding protein (EBP) 1 has been shown to be responsible for chemotaxis to the peptides (6 -12), stimulation of cell proliferation (13-16), ions flux modifications (17, 18), vasorelaxation (19 -22), and enzymes secretion (23,24).Matrix metall...

Degradation of the cartilage proteoglycan aggrecan is a key early event in the development of osteoarthritis. Adamalysin with thrombospondin motifs (ADAMTS) -4 and ADAMTS-5 are considered to be the main enzymes responsible for aggrecan breakdown, making them attractive drugs targets. Here we show that calcium pentosan polysulfate (CaPPS), a chemically sulfated xylanopyranose from beechwood, is a multifaceted exosite inhibitor of the aggrecanases and protects cartilage against aggrecan degradation. CaPPS interacts with the noncatalytic spacer domain of ADAMTS-4 and the cysteine-rich domain of ADAMTS-5, blocking activity against their natural substrate aggrecan with inhibitory concentration 50 values of 10-40 nM but only weakly inhibiting hydrolysis of a nonglycosylated recombinant protein substrate. In addition, CaPPS increased cartilage levels of tissue inhibitor of metalloproteinases-3 (TIMP-3), an endogenous inhibitor of ADAMTS-4 and -5. This was due to the ability of CaPPS to block endocytosis of TIMP-3 mediated by low-density lipoprotein receptor-related protein. CaPPS also increased the affinity of TIMP-3 for ADAMTS-4 and -5 by more than 100-fold, improving the efficacy of TIMP-3 as an aggrecanase inhibitor. Studies with TIMP-3-null mouse cartilage indicated that CaPPS inhibition of aggrecan degradation is TIMP-3 dependent. These unique properties make CaPPS a prototypic disease-modifying agent for osteoarthritis.

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The scavenger receptor low-density lipoprotein receptor-related protein 1 (LRP-1) mediates the clearance of a variety of biological molecules from the pericellular environment, including proteinases which degrade the extracellular matrix in cancer progression. However, its accurate functions remain poorly explored and highly controversial. Here we show that LRP-1 silencing by RNA interference results in a drastic inhibition of cell invasion despite a strong stimulation of pericellular matrix metalloproteinase 2 and urokinase-type plasminogen activator proteolytic activities. Cell migration in both two and three dimensions is decreased by LRP-1 silencing. LRP-1-silenced carcinoma cells, which are characterized by major cytoskeleton rearrangements, display atypical overspread morphology with a lack of membrane extensions. LRP-1 silencing accelerates cell attachment, inhibits cell-substrate deadhesion, and induces the accumulation, at the cell periphery, of abundant talin-containing focal adhesion complexes deprived of FAK and paxillin. We conclude that in addition to its role in ligand binding and endocytosis, LRP-1 regulates cytoskeletal organization and adhesive complex turnover in malignant cells by modulating the focal complex composition, thereby promoting invasion.

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Supplementary key words toxin • His-mCherry-NT-lysenin • lateral membrane heterogeneity • vital confocal imaging • membrane tension • cholesterol • temperatureLipids at the outer leafl et of the mammalian plasma membrane are mainly composed of: i ) SM, the most abundant sphingolipid (SL), based on a ceramide backbone and bearing a phosphocholine polar head; ii ) glycosphingolipids (GSLs), another group of SLs bearing various sugars instead of phosphocholine, from simple glucosylceramide (GlcCer) to complex GSLs such as GM1 [for a review, see ( 1 )]; iii ) phosphatidylcholine (PC), the major glycerophospholipid, sharing the same phosphocholine polar head as SM; and iv ) nonpolar cholesterol. Lipid bilayers are no longer considered as a homogenous solvent for membrane proteins ( 2 ), but are now represented with lateral heterogeneity at two different scales of time and space: i ) transient nanometric "lipid rafts", defi ned as small clusters enriched in SLs, sterol, and GPI-anchored proteins ( 3, 4 ); versus ii ) submicrometric/mesoscale domains ( 5-15 ). These larger and more stable domains are well-characterized on artifi cial vesicles ( 16, 17 ), but their relevance for living cells has been questioned ( 18,19 ).The occurrence in living cells of submicrometric/mesoscale domains was fi rst inferred from unexpected behavior in fl uorescence recovery after photobleaching (FRAP)

Background: Tissue inhibitor of metalloproteinases-3 (TIMP-3) is endocytosed, but its regulatory mechanism is not well understood. Results: TIMP-3 endocytosis occurs mainly through low density lipoprotein receptor-related protein-1 (LRP-1), but shed sLRP-1 binds TIMP-3. Conclusion: TIMP-3-sLRP-1 complexes are retained extracellularly with metalloproteinase inhibitory activity. Significance: LRP-1 is the master regulator of extracellular levels of TIMP-3.