Transmembrane-4-L-six-family-1 (TM4SF1) was originally described as a cancer cell protein. Here, we show that it is highly expressed in the vascular endothelium of human cancers and in a banded pattern in the filopodia of cultured endothelial cells (EC). TM4SF1 knockdown prevented filopodia formation, inhibited cell mobility, blocked cytokinesis, and rendered EC senescent. Integrin-A5 and integrin-B1 subunits gave a similar staining pattern and interacted constitutively with TM4SF1,
Lysyl oxidase (LO), an enzyme secreted by vascular smooth muscle cells (VSMC), initiates the covalent crosslinking of polypeptide chains within collagen and elastin. The present study reveals that purified LO strongly induces directional migration of VSMC in an in vitro assay system. LO-dependent chemotaxis, but not chemokinesis, was abolished by beta-aminopropionitrile, an active site inhibitor of LO, or by catalase, as well as by prior heat denaturation. This indicates that the H(2)O(2) product of amine oxidation by LO is critical to the expression of its chemotactic activity. The results indicate that the chemotactic response requires direct access between LO and a substrate molecule (or molecules) tightly associated with the VSMC. The addition of LO to VSMC elevated the levels of intracellular H(2)O(2), enhanced stress fiber formation, and focal adhesion assembly, is consistent with the induction of the chemotactic response.
Lysyl oxidase (EC 1.4.3.13) is unique among the mammalian copper amine oxidases by catalyzing a critical post-translational modification essential to the biogenesis of connective tissue matrices. This enzyme initiates covalent cross-linking between and within the molecular units of elastin and of collagen by oxidizing peptidyl lysine in these proteins to peptidyl ␣-aminoadipic-␦-semialdehyde (1, 2). The peptidyl aldehyde can then condense with neighboring ⑀-amino groups or peptidyl aldehydes to form the covalent cross-linkages found in fibrillar collagen and elastin. Lysyl oxidase contains a tightly bound copper cofactor as well as a covalently bound carbonyl prosthetic group recently identified as lysine tyrosylquinone (3).Lysyl oxidase catalyzes primary amine oxidation through a ping pong bi ter kinetic mechanism (4, 5). Following initial Schiff base formation with the LTQ 1 cofactor, the bound substrate undergoes rate-limiting, general base-facilitated ␣-proton abstraction (6). Electrons migrating from the resulting carbanion reduce the carbonyl cofactor, followed by hydrolysis of the product imine intermediate to release the aldehyde product. The reduced enzyme, retaining the amino function of the substrate, is reoxidized by molecular oxygen to produce hydrogen peroxide and ammonia, regenerating the oxidized enzyme and completing the catalytic cycle.The role of lysyl oxidase in the growth and repair of connective tissues has been well documented. Markedly increased levels of LO activity are observed in a variety of fibrotic diseases in which excess collagen is deposited in the affected tissues, as in models of atherosclerosis, hypertension, and liver and pulmonary fibrosis (2). The possibility that the development of fibrosis may be restricted by the specific suppression of lysyl oxidase activity has stimulated the search for selective and potent inhibitors of this enzyme. These efforts have identified mechanism-based and ground-state inhibitors, including -substituted haloethylamines (7), benzylamines substituted with electronegative para-substituents (8), and 1,2-diamines (9), each of which appear to inhibit as adducts of the carbonyl cofactor.In the present report, we describe our observations that homocysteine thiolactone and the oxygen and selenium lactone analogues of this compound are active site-directed, irreversible inhibitors of lysyl oxidase. The selenium and sulfur lactones are the most potent of these and are selective for lysyl oxidase, among mechanistically similar copper-dependent mammalian amine oxidases tested in the present study. Notably, HCTL occurs in mammalian systems as a metabolic by-product of methyl transfer from S-adenosylhomocysteine. Moreover, the accumulation of HCTL has been suggested to be related to mechanisms of carcinogenesis and atherogenesis (10 -12), whereas it has also been shown to thiolate proteins, including low density lipoproteins in vitro (13,14). Elucidation of interactions of these compounds with lysyl oxidase should increase options for the design of antifib...
Lysyl oxidase (LO) plays a central role in the crosslinking of collagen and elastin in the extracellular matrix. Here we demonstrate that basic fibroblast growth factor (bFGF), a polypeptide which regulates proliferation, differentiation, and migration of a variety of cell types, is a substrate of LO. The oxidation of lysine residues in bFGF by LO resulted in the covalent crosslinking of bFGF monomers to form dimers and higher order oligomers and dramatically altered its biological properties. Both the mitogenic potential and the nuclear localization of bFGF were markedly inhibited in the Swiss 3T3 cells upon its oxidation by LO. NIH 3T3 IgBNM 6-1 cells (6-1 cells) overexpress bFGF which participates in an autocrine mechanism accounting for the transformation of these cells into a tumorigenic state. Exposure of the 6-1 cells to nanomolar concentrations of LO in culture oxidized lysine and generated crosslinkages in bFGF within the cell and markedly reduced proliferative rates. The lack of LO expression has been correlated with hyperproliferative cell growth, while this enzyme has been identified as a suppressor of ras-induced tumorigenesis. The present results illustrate a mechanism by which LO can depress normal and transformed cell growth.
Lysyl oxidase (LO), a secreted protein, was recently identified within the nuclei of vascular smooth-muscle cells (SMC) and 3T3 fibroblasts. A possible pathway by which LO can enter cell nuclei was explored in the present study. SMC were incubated with purified 32-kDa bovine aorta LO that had been fluorescently labeled with rhodamine (TRITC-LO). TRITC-LO entered the cytosol and then rapidly concentrated within the nuclei of preconfluent cultures of these cells, whereas carbonic anhydrase, a protein of similar molecular weight and similarly labeled, did not enter the cells under these conditions. LO that had been reductively methylated at lysine residues with [(14)C]HCHO was also taken up into the cytosolic and nuclear compartments. Intracellular uptake and intracellular distribution were not altered by inhibiting LO activity with beta-aminopropionitrile. An excess of native LO but not of carbonic anhydrase competitively inhibited the uptake of the isotopically labeled enzyme. Thus, once secreted and proteolytically processed, mature LO can enter the cells and concentrate within nuclei in a manner that appears to be specific and independent of its catalytic activity.
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