Lysyl oxidase (LOX) is an extracellular amine oxidase that mediates the formation of collagen fibers. Thus far, five LOX family genes [LOX, lysyl oxidase-like (LOXL)1, LOXL2, LOXL3 and LOXL4] have been identified in humans, each encoding the characteristic C-terminal domains that are required for amine oxidase activity. During osteoblastogenesis, collagen fibers function as a three-dimensional scaffold for organizing mineral deposition. In this study, to assess the functional roles of the LOX family members in osteoblastogenesis, we investigated the temporal expression of these genes as a function of phenotypic development during the osteoblast differentiation of primary cultured mouse calvaria cells. Of the LOX family members, only LOX was prominently expressed during osteoblast differentiation. LOX expression was highest on day 9 of differentiation, as shown by RT-PCR and western blot analysis. The expression pattern of collagen, type I, alpha 2 (COL1A2), which encodes the α2-chain of mouse collagen type I, was similar to that of LOX. The total amine oxidase activity of the differentiating calvaria cells exhibited a temporal pattern that paralleled LOX expression, reaching the highest level on day 9 of differentiation. We also noted that the inhibition of the amine oxidase activity of LOX significantly suppressed both mineral nodule formation and the expression of osteoblast marker genes during the differentiation of primary calvaria cells. Taken together, these findings suggest that the LOX-mediated organization of collagen fibers in the extracellular matrix is an important regulator of osteoblastogenesis.

Lysyl oxidase-variant 2 (LOX-v2) is a novel variant of LOX that functions as an amine oxidase for the formation of collagen and elastin fibrils in the extracellular matrix (ECM). LOX-v2 lacks the N-terminal prepropeptide region of LOX but contains the C-terminal domains required for amine oxidase activity. To study the cellular localization of LOX-v2, we generated a recombinant construct of LOX-v2 with an epitope tag at the C-terminus and then transfected the recombinant construct into HEK293 cells. Upon ectopic expression, LOX-v2 showed much higher expression in the nucleus than in the cytoplasm. In coimmunofluorescence staining with subnuclear structures, LOX-v2 colocalized with the promyelocytic leukemia-nuclear bodies (PML-NBs). Further, the ectopic expression of LOX-v2 increased global SUMOylation in the nucleus. PML-NBs have been implicated in various cellular activities, including transcriptional regulation, DNA repair, cell cycle control, anti-viral response, and apoptosis. Our findings strongly indicate that LOX-v2 may be subject to different cellular processing from what LOX undergoes, playing a distinct functional role in the PML-NBs, beyond the cross-linking of the structural proteins.

Lysyl oxidase-variant 2 (LOX-v2) is a novel variant of lysyl oxidase (LOX) that functions as an amine oxidase for the formation of lysine-mediated crosslinks found in collagen and elastin fibrils. In addition to the amine oxidase activity in the extracellular matrix, several novel functions, such as tumor suppression, tumor progression, chemotaxis, cellular senescence, and modification of histones, have been assigned to LOX. In recent years, it has been reported that LOX is also present in nuclear locations, suggesting a novel functional role of LOX in the nucleus. To test the amine oxidase activity of LOX and LOX-v2 to nuclear histone proteins, we expressed and purified LOX and LOX-v2 as recombinant forms and then assessed the amine oxidase activity toward histone H2A in in vitro peroxidase-coupled fluorometric assays. Both LOX and LOX-v2 proteins showed significant levels of amine oxidase activity toward histone H2A in a β-aminopropionitrile-inhibitable manner. In immunofluorescence staining after ectopic expression in cultured cells, LOX was observed in the perinuclear, cytoplasmic, and extracellular areas, whereas LOX-v2 was predominantly detected in the nucleoplasm with a punctuate pattern. These findings suggest that LOX-v2 may play a novel functional role in the nucleus through the amine oxidase activity to the nuclear histone proteins. Elucidation of the specific functional roles of LOX-v2, such as substrate specificity toward different types of nuclear proteins and detailed analysis on subnuclear localization, will provide a significant clue in understanding the diverse functional roles currently assigned to a single enzyme, LOX.