3765

Lysyl oxidase (LOX), an extracellular copper-dependent amine-oxidase, has been demonstrated to be involved in diverse functions such as extracellular matrix (ECM) stabilization, developmental and cell growth regulation, and cell motility. LOX has long been known to catalyze the lysine-derived covalent cross-links of collagen and elastic fibers, but it seems unlikely that this traditional role alone would explain its wide range of biological functions. Indeed, novel LOX substrates, such as growth hormones, have been recently identified and suggest that LOX still has unidentified molecular mechanisms of action. In addition, little is known about how LOX is targeted to specific extracellular microenvironments and how its proteolytic activation is regulated. To elucidate possible novel mechanisms of action and regulation for the LOX enzyme, we performed a yeast two-hybrid screen with the 32-kD active form of LOX as bait. From the results of the screen, we identified the extracellular matrix protein fibronectin (FN) as a potential LOX-binding protein. FN has been previously implicated in many aspects of tumor formation, angiogenesis, and metastasis. Given LOX’s emerging role in breast cancer, especially that of promoting epithelial plasticity and invasion, we were interested in verifying this interaction and determining its biological significance. We performed GST pull-down experiments, co-immunoprecipitations, and solid phase binding assays to verify and characterize the interactions of LOX with FN. The results show that LOX preferentially bound to the cellular form of FN (cFN) as compared to the plasma form (pFN). We found that cFN was a poor substrate for LOX’s amine oxidase activity, but that active LOX, bound to a cFN matrix, remained enzymatically active towards third party molecules. Most surprisingly, we found that FN seems critical for the proteolytic activation of the LOX enzyme. In FN-null fibroblasts, LOX processing and catalytic activity was dramatically reduced to 5-10% of normal conditions. These data suggest that the extracellular FN matrix may act as scaffolding to regulate and guide LOX’s crosslinking activity in specific microenvironments.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]