Abstract
Tumor necrosis factor-alpha (TNF-alpha) inhibits osteoblast function in vitro by inhibiting collagen deposition. Studies generally support that TNF-alpha does not inhibit collagen biosynthesis by osteoblasts but that collagen deposition is in some way diminished. The study investigated TNF-alpha regulation of biosynthetic enzymes and proteins crucial for posttranslational extracellular collagen maturation in osteoblasts including procollagen C-proteinases, procollagen C-proteinase enhancer, and lysyl oxidase. The working hypothesis is that such regulation could inhibit collagen deposition by osteoblasts. We report that in phenotypically normal MC3T3-E1 osteoblasts, TNF-alpha decreases collagen deposition without decreasing collagen mRNA levels or procollagen protein synthesis. Analyses of the cell layers revealed that TNF-alpha diminished the levels of mature collagen cross-links, pyridinoline and deoxypyridinoline. Further analyses revealed that the mRNA expression for lysyl oxidase, the determining enzyme required for collagen cross-linking, is down-regulated by TNF-alpha in a concentration- and time-dependent manner by up to 50%. The decrease was accompanied by a significant reduction of lysyl oxidase protein levels and enzyme activity. By contrast, Northern and Western blotting studies revealed that procollagen C-proteinases bone morphogenic protein-1 and mammalians Tolloid and procollagen C-proteinase enhancer were expressed in MC3T3-E1 cells and not down-regulated. The data together demonstrate that TNF-alpha does not inhibit collagen synthesis but does inhibit the expression and activity of lysyl oxidase in osteoblasts, thereby contributing to perturbed collagen cross-linking and accumulation. These studies identify a novel mechanism in which proinflammatory cytokine modulation of an extracellular biosynthetic enzyme plays a determining role in the control of collagen accumulation by osteoblasts.
Highlights
Tumor necrosis factor-␣ (TNF-␣)1 is an inflammatory cytokine produced primarily by monocytes and macrophages and by a variety of mesen
Further analyses revealed that the mRNA expression for lysyl oxidase, the determining enzyme required for collagen cross-linking, is downregulated by TNF-␣ in a concentration- and time-dependent manner by up to 50%
The present studies demonstrate that, in the presence of TNF-␣, less collagen accumulates in the extracellular matrix of osteoblast cultures and that the deposited collagen contains diminished levels of mature collagen cross-links, Pyd and Dpd. These data support the proposition that these effects are caused in part by a significant down-regulation of lysyl oxidase expression and activity by TNF-␣
Summary
TNF-␣1 is an inflammatory cytokine produced primarily by monocytes and macrophages and by a variety of mesen-. The study investigated TNF-␣ regulation of biosynthetic enzymes and proteins crucial for posttranslational extracellular collagen maturation in osteoblasts including procollagen C-proteinases, procollagen C-proteinase enhancer, and lysyl oxidase. Further analyses revealed that the mRNA expression for lysyl oxidase, the determining enzyme required for collagen cross-linking, is downregulated by TNF-␣ in a concentration- and time-dependent manner by up to 50%.
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