Molecular events that contribute to lysyl oxidase enzyme activity and insoluble collagen accumulation in osteosarcoma cell clones

Maximum collagen synthesis and maximum accumulation of insoluble collagen o ccur at different phenotypic stages in developing osteoblastic cell culture s. Insoluble collagen accumulation depends in part on the activity of extra cellular enzymes including procollagen N-proteinases, procollagen C-protein ase (derived from the BMP1 gene), and lysyl oxidase. In addition to its act ion on procollagen, procollagen C-proteinase processes prolysyl oxidase to mature 32-kDa lysyl oxidase. The regulation of extracellular activities tha t control insoluble collagen accumulation has not been studied extensively. The present study compares molecular events that control production of a c ollagenous mineralized extracellular matrix in vitro among five different m urine osteosarcoma cell clones derived from the same tumor, but which diffe r in their ability to produce an insoluble mineralized matrix. Levels of in soluble type I collagen, insoluble calcium, bone morphogenetic protein 1 (B MP-1), and lysyl oxidase expression, lysyl oxidase biosynthesis, lysyl oxid ase activity, and prolysyl oxidase processing activity were determined. Res ults surprisingly indicate that lysyl oxidase activity is not related close ly to lysyl oxidase messenger RNA (mRNA) levels among the different cell cl ones. However, it appears that BMP-l-dependent prolysyl oxidase processing could contribute to the observed lysyl oxidase activity. Highest collagen a nd BMP-1 mRNA levels, prolysyl oxidase processing activity, and lysyl oxida se activity occurred in a cell clone (K8) that showed the highest levels of insoluble collagen accumulation. Culture media from a cell clone (K37) tha t accumulates little insoluble collagen or calcium but expresses high level s of lysyl oxidase mRNA contained low molecular weight fragments of lysyl o xidase protein and showed low lysyl oxidase activity. By contrast the K14 c ell line exhibits relatively high lysyl oxidase activity and collagen accum ulation, but low levels of mature lysyl oxidase protein. Together, these st udies indicate that catabolic as well as anabolic activities are important in regulating insoluble collagen accumulation in osteoblastic cells. In add ition, results suggest that products of genes homologous to lysyl oxidase m ay contribute to observed lysyl oxidase activity.