Lysyl oxidase (LOX) is a copper-containing amine oxidase known to catalyze the covalent cross-linking of fibrillar collagens and elastin at peptidyl lysine residues. In addition, its involvement in cancer, wound healing, cell motility, chemotaxis, and differentiation reflect a remarkable functional diversity of LOX. To investigate novel mechanisms of LOX regulation and function, we performed a yeast two-hybrid screen to identify LOX-interacting proteins. Three overlapping positive clones were identified as C-terminal fragments of fibronectin (FN). Glutathione S-transferase pull-downs and solid phase binding assays confirmed this interaction. LOX binds to the cellular form of FN (cFN) with a dissociation constant (K d ) of 2.5 nM. This was comparable with our measured K d of LOX binding to tropoelastin (1.9 nM) and type I collagen (5.2 nM), but LOX demonstrated a much lower binding affinity for the plasma form of FN (pFN). Immunofluorescent microscopy revealed co-localization of FN and LOX in normal human tissues, where these proteins may interact in vivo. LOX enzymatic activity assays showed that cFN does not seem to be a substrate of LOX. However, cFN can act as a scaffold for enzymatically active 30-kDa LOX. Furthermore, in FN-null mouse embryonic fibroblasts, we observed dramatically decreased proteolytic processing of the 45-kDa LOX proenzyme to the 30-kDa active form, with a corresponding decrease in LOX enzyme activity. Our results suggest that the FN matrix may provide specific microenvironments to regulate LOX catalytic activity.Lysyl oxidase (LOX), 1 a copper-dependent amine oxidase, catalyzes the oxidative deamination of peptidyl lysine residues in collagen and elastin molecules to ␦-aminoadipic-␤-semialdehyde or allysine, which can then spontaneously condense with neighboring amino groups or other peptidyl aldehydes to form covalent cross-links in fibrillar collagens and elastin (1, 2). The covalent cross-linking of these extracellular matrix (ECM) proteins by LOX is essential to the formation of insoluble collagen and elastic fibers and for normal mammalian development (3, 4). In many human pathologies, including cardiovascular disease, fibrosis, and cancer, LOX expression and activity have been demonstrated to be altered as compared with normal conditions (1, 2).The human LOX protein is synthesized as a prepro-enzyme of 417 amino acids with an N-terminal signal peptide sequence for secretion. During protein trafficking in the Golgi, a copper atom is incorporated at the copper-binding site (residues 286 -296) (5-7), which allows the formation of a lysyl-tyrosylquinone cofactor derived from Tyr-355 and Lys-320 residues (8). After secretion into the extracellular space, proteolytic cleavage between residues Gly-168 and Asp-169 removes the N-terminal propeptide, yielding an active enzyme of 30 kDa (9 -11). This processing is mainly accomplished by the procollagen C-proteinase bone morphogenic protein-1 (BMP-1), and to a lesser degree, by mammalian Tolloid-like 1 protein (12, 13). However, it is not know...

Malignant mesothelioma is strongly associated with asbestos exposure. Among asbestos fibers, crocidolite is considered the most and chrysotile the least oncogenic. Chrysotile accounts for more than 90% of the asbestos used worldwide, but its capacity to induce malignant mesothelioma is still debated. We found that chrysotile and crocidolite exposures have similar effects on human mesothelial cells. Morphological and molecular alterations suggestive of epithelial-mesenchymal transition, such as E-cadherin down-regulation and β-catenin phosphorylation followed by nuclear translocation, were induced by both chrysotile and crocidolite. Gene expression profiling revealed high-mobility group box-1 protein (HMGB1) as a key regulator of the transcriptional alterations induced by both types of asbestos. Crocidolite and chrysotile induced differential expression of 438 out of 28,869 genes interrogated by oligonucleotide microarrays. Out of these 438 genes, 57 were associated with inflammatory and immune response and cancer, and 14 were HMGB1 targeted genes. Crocidolite-induced gene alterations were sustained, whereas chrysotile-induced gene alterations returned to background levels within 5 weeks. Similarly, HMGB1 release in vivo progressively increased for 10 or more weeks after crocidolite exposure, but returned to background levels within 8 weeks after chrysotile exposure. Continuous administration of chrysotile was required for sustained high serum levels of HMGB1. These data support the hypothesis that differences in biopersistence influence the biological activities of these two asbestos fibers.

The extracellular matrix (ECM) plays a critical role during the development and invasion of primary brain tumours. However, the function of ECM components and signalling between a permissive ECM and invasive astrocytes is not fully understood. We have recently reported the ECM enzyme, lysyl oxidase (LOX), in the central nervous system and observed up-regulation of LOX in anaplastic astrocytoma cells. While the catalytic function of LOX is essential for cross-linking of ECM proteins, we also reported that LOX induced invasive and metastatic properties in breast tumour epithelial cells through hydrogen peroxide-mediated FAK/Src activation. In this study, we tested the hypothesis that active LOX is expressed in anaplastic astrocytes and promotes FAK activation and invasive/migratory behaviour. Results demonstrate that increased expression and activity of LOX positively correlated with invasive phenotype of malignant astrocytoma cell lines. Immunohistochemistry detected increased LOX within tumour cells and ECM in grade I-IV astrocytic neoplasm compared with normal brain and coincidence of increased LOX with the loss of glial fibrillary acidic protein in higher-grade tumours. Increased active LOX in invasive astrocytes was accompanied by phosphorylation of FAK[Tyr576] and paxillin[Tyr118]; furthermore, both FAK and paxillin tyrosine phosphorylation were diminished by beta-aminopropionitrile inhibition of LOX activity and depletion of H(2)O(2) via catalase treatment. Additionally, we provide evidence that in astrocytes, LOX is likely processed by bone morphogenic protein-1 and LOX activity might be further stimulated by the expression of fibronectin in these cells. These results demonstrate an important LOX-mediated mechanism that promotes migratory/invasive behaviour of malignant astrocytes.

Cu-dependent lysyl oxidase (LOX) plays a catalytic activity-related, primary role in the assembly of the extracellular matrix (ECM), a dynamic structural and regulatory framework which is essential for cell fate, differentiation and communication during development, tissue maintenance and repair. LOX, additionally, plays both activity-dependent and independent extracellular, intracellular and nuclear roles that fulfill significant functions in normal tissues, and contribute to vascular, cardiac, pulmonary, dermal, placenta, diaphragm, kidney and pelvic floor disorders. LOX activities have also been recognized in glioblastoma, diabetic neovascularization, osteogenic differentiation, bone matrix formation, ligament remodeling, polycystic ovary syndrome, fetal membrane rupture and tumor progression and metastasis. In an inflammatory context, LOX plays a role in diminishing pluripotent mesenchymal cell pools which are relevant to the pathology of diabetes, osteoporosis and rheumatoid arthritis. Most of these conditions involve mechanisms with complex cell and tissue type-specific interactions of LOX with signaling pathways, not only as a regulatory target, but also as an active player, including LOX-mediated alterations of cell surface receptor functions and mutual regulatory activities within signaling loops. In this review, we aim to provide insight into the diverse ways in which LOX participates in signaling events, and explore the mechanistic details and functional significance of the regulatory and cross-regulatory interactions of LOX with the EGFR, PDGF, VEGF, TGF-β, mechano-transduction, inflammatory and steroid signaling pathways.

Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S-S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S-S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S-S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S-S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein.