PRETRANSLATIONAL AND POSTTRANSLATIONAL REGULATION OF LYSYL OXIDASE BYTRANSFORMING GROWTH-FACTOR-BETA-1 IN OSTEOBLASTIC MC3T3-E1 CELLS

The final enzymatic step required for collagen crosslinking is the ext racellular oxidative deamination of peptidyl-lysine and -hydroxylysine residues by lysyl oxidase. A cross-linked collagenous extracellular m atrix is required for bone formation. The goals of this study were to compare the transforming growth factor (TGF)-beta 1 regulation of lysy l oxidase enzyme activity and steady state mRNA levels to changes in C OL1A1 mRNA levels in MC3T3-E1 osteoblastic cells. TGF-beta 1 increased steady state lysyl oxidase and COL1A1 mRNA levels in a dose- and time -dependent manner. The increase in lysyl oxidase mRNA levels was trans ient, peaking at 12 h and 8.8 times controls in cells treated with 400 pM TGF-beta 1. COL1A1 steady state mRNA levels increased maximally to 3.5-fold of controls. Development of increased lysyl oxidase enzyme a ctivity was delayed and was of slightly lower magnitude than the incre ase in its mRNA levels. This suggested limiting post translational pro cessing of lysyl oxidase proenzyme. Pulse-labeling/immunoprecipitation studies demonstrated slow proenzyme secretion and proteolytic process ing. Development and application of an independent assay for lysyl oxi dase proenzyme proteolytic processing activity verified its proportion ately lower stimulation by 400 pM TGF-beta 1. Thus, lysyl oxidase regu lation by TGF-beta 1 in osteoblastic cell cultures occurs at both pre- and post-translational levels. This regulation is consistent with inc reased production of a collagenous extracellular matrix.