2D-PAGE reveals the absence of collagenase production in normal human osteoblasts in culture

Normal human skin fibroblast primary cell lines secrete over 50 proteins into culture medium. These have been mapped previously using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and this technique has now been used to investigate extracellular protein secretion by human osteoblasts in vitro. We report the mapping of a number of consistent markers specific to the osteoblast. In particular, one protein chain with posttranslational modifications was found to be unique to the osteoblast extracellular protein map. The absence of the N- and O-glycoforms of collagenase from the osteoblast profile in this study concurs with findings reported using the immunoprecipitation functional assay and Northern blot analysis. The use of 2-D PAGE in phenotypic assessment provides a more complete analysis than the standard range of single-parameter tests for osteoblasts. Mapping of extracellular and cellular proteins in addition to bone matrix protein analysis will allow a comprehensive analysis of normal osteoblast function. This technique may also be applied to the study of osteoblasts in relation to bone disease and in assessing the phenotypic shift within a normal osteoblast culture.

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Two‐dimensional polyacrylamide gel electrophoresis reveals differences between osteoblast and fibroblast extracellular proteins

Hankey

Nicholas

Hughes

1992

Electrophoresis

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Reactive oxygen species activate and tetracyclines inhibit rat osteoblast collagenase

Ramamurthy

Vernillo

Greenwald

et al. 1993

Journal of Bone and Mineral Research

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Recent studies have demonstrated that tetracyclines (TCs) scavenge reactive oxygen species (ROS). Hypochlorous acid (HOCl), an ROS produced by neutrophils, has been shown to activate neutrophil procollagenase. The objective of the present study was to determine whether (1) HOCl also activated osteoblast procollagenase and (2) TCs inhibited this enzyme in the presence of HOCl. HOCl (5 microM) activated the proenzyme approximately sixfold (P < 0.01) from the medium of PTH-treated UMR-106-01 osteoblastic osteosarcoma cells as determined by functional collagenase assay (3H-methyl-labeled collagen substrate). Doxycycline (50-400 microM) and chemically modified tetracycline, CMT-1 (100-400 microM), significantly inhibited collagenase activity 50-90% and 40-80%, respectively, in the presence of 5 microM HOCl. Concentrations of 6-25 microM doxycycline and 10-50 microM CMT-1 had no significant effect. Furthermore, an excess concentration of cation (50 mM CaCl2 or 50 microM ZnCl2) added to the incubation mixtures containing either doxycycline or CMT-1 did not restore collagenase activity, as demonstrated by SDS-PAGE-fluorography. These data suggested that TCs reduced available HOCl and thus prevented the hypochlorous acid conversion of the osteoblast proenzyme to active collagenase. TCs may have therapeutic potential in the treatment of periodontitis and other diseases by several mechanisms that inhibit pathologic collagen breakdown.

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Modulation by Soluble Factors of Gelatinase Activities Released by Osteoblastic Cells

Damiens¹,

Fortun²,

Charrier³

et al. 2000

Cytokine

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2D-PAGE reveals the absence of collagenase production in normal human osteoblasts in culture

Cited by 5 publications

References 2 publications

Two‐dimensional polyacrylamide gel electrophoresis reveals differences between osteoblast and fibroblast extracellular proteins

Two‐dimensional polyacrylamide gel electrophoresis reveals differences between osteoblast and fibroblast extracellular proteins

Reactive oxygen species activate and tetracyclines inhibit rat osteoblast collagenase

Modulation by Soluble Factors of Gelatinase Activities Released by Osteoblastic Cells

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