The regulation of lysyl oxidase by bFGF and TGF-B1 in osteoblastic MC3T3-E1 Cells

Thesis (D.Sc.D.)--Boston University, Henry M. Goldman School of Graduate Dentistry, 1995 (Oral Biology).Includes bibliographical references: (leaves 57-73).Lysyl oxidase catalyzes the enzymatic step that is limiting for collagen and elastin crosslinking. As a crosslinked collagenous extracellular matrix is required for bone formation, mineralization and structural integrity, regulation of lysyl oxidase in osteoblastic cells, although not previously studied, is likely to have relevance to bone synthesis and repair. The goals of this study were to determine whether lysyl oxidase, like its type I collagen substrate, is regulated by basic fibroblast growth factor (bFGF) and transforming growth factor beta-1(TGF-[beta] 1) in osteoblastic MC3T3-E1 cells. We also established the degree of posttranscriptional control by bFGF. Steady-state lysyl oxidase mRNA levels were decreased to 30% of control after 24 hours of treatment with 1 nM and 10 nM bFGF. This regulation was time-dependent. Similarly, the COL1A1 mRNA level decreased to less than 10% of control after 24 hours of treatment. Media lysyl oxidase activity decreased in accordance with the steady-state mRNA changes in bFGF-treated cultures that were refed after 24 hours of growth factor treatment. Treatment of MC3T3-E1 cells with 0.01 – 0.1 nM bFGF for 24 hours, and treatment with 1 nM bFGF for up to 12 hours resulted in a modest stimulation of lysyl oxidase gene expression and enzyme activity. Fifty per cent of the down-regulation of lysyl oxidase was shown to be posttranscriptional by measuring bFGF-dependent-differences in decay rates of lysyl oxidase mRNA in the presence of 5,6-dichloro- 1 [Beta] D-ribofuranoaylbenzimidazole. New protein synthesis was not required for the down-regulation by bFGF, but cycloheximide did increase constitutive lysyl oxidase mRNA levels 2.5-fold. GAPDH or COL1A1 mRNA levels were not affected by cycloheximide. TGF-[Beta] 1 increased the levels of steady-state lysyl oxidase mRNA, proenzyme synthesis and secretion, and enzyme activity in a dose- and time-dependent manner. A discrepancy between the synthesis of lysyl oxidase proenzyme and enzyme activity indicated that the posttranstational maturation of lysyl oxidase may limit full activation of lysyl oxidase enzyme activity. TGF-[Beta] 1-mediated effects on steady-state lysyl oxidase mRNA levels are greater than th e effects on steady-state COL1A1 mRNA levels, suggesting that lysyl oxidase is a major target for TGF-[Beta]1 in this cell culture model. Mechanisms of regulation of lysyl oxidase and the possible role of lysyl oxidase activity in controlling collagen accuImulation in mineralized tissues are discussed