Faculty Bibliography

Previous studies demonstrated that tetracyclines (TCs) inhibited Type I (interstitial) and Type IV collagenases from different mammalian sources, but there are no studies of TCs effect on osteoblast collagenase (C'ase). The present study assessed the effect of TCs on C'ase activity from osteosarcoma cells. Semiconfluent UMR 106-01 cells were treated with minocycline or chemically modified tetracycline (CMT) at 10 micrograms/ml in the presence or absence of bovine parathyroid hormone, b-PTH-(1-34), at 10(-7)M for 24, 48, 72 and 96 hours. Media were collected at each time point and assayed following concentration, destruction of TIMP by reduction/alkylation, activation with p-aminophenylmercuric acetate (APMA), and incubation with 3H-methylated collagen substrate (approximately 100,000 dpm) at 27 degrees C for 18 hours. Collagenase activity from media was also analyzed by SDS-PAGE and fluorography. b-PTH appeared to stimulate C'ase 60-fold compared to controls; minocycline and CMT reduced PTH stimulation approximately 65% and 90%, respectively. Moreover, TCs incubated with partially purified osteoblastic collagenase directly, inhibited its activity in vitro as indicated by a lack of degradation to collagen alpha A chains. Therefore, TCs ability to inhibit bone resorption in organ culture, reported previously, may be due, in part, to reduced osteoblast collagenase activity

Phenytoin (PHT), a widely used anticonvulsant, has been shown to inhibit bone resorption in rodent organ cultures. The drug also has complex effects on bone metabolism including chronic clinical symptoms of osteomalacia. However, the precise mechanism of PHT action in bone is still unclear. Neutral collagenases that specifically cleave native collagen have been implicated in the turnover of connective tissue. The effect of PHT was assessed on collagenase and gelatinase activities from UMR 106-01 rat osteoblastic osteosarcoma cells. Semiconfluent cells were treated with PHT (50 and 10 micrograms/ml) in the presence of bovine parathyroid hormone, b-PTH-(1-34), at 10(-7) M for 24, 48, 72 and 96 h. The media were assayed following concentration, APMA activation, and incubation with native or denatured [3H]-methyl collagen substrate (approximately 100,000 dpm) at 27 degrees C for 18 h and 35 degrees C for 2 h, respectively. Enzyme activities were presented as primary counts per minute for each time point and calculated as % activity of PTH at 10(-7) M. Parathyroid hormone (10(-7) M) stimulated collagenase activity (approximately 65-fold) and gelatinase activity (approximately 400-fold). PHT (50 micrograms/ml) reduced the PTH-stimulated collagenase activity by 18-53% and the gelatinase activity by 58-72%. SDS PAGE and fluorography following PHT treatment indicated a PHT-induced partial inhibition of PTH-stimulated degradation to alpha A chains of Type I collagen. Phenytoin may inhibit bone resorption through its action on the transcription, synthesis, and/or secretion of the collagenolytic enzymes, collagenase and gelatinase

5,5-diphenylhydantoin (Phenytoin, PHT), a widely used anticonvulsant, is also a vitamin K antagonist and disrupts bone metabolism, leading to osteomalacia. The vitamin K-dependently synthesized protein, osteocalcin, has been implicated as a key regulatory protein in bone resorption. The purpose of the present study was to determine whether PHT had an effect on osteocalcin secretion. Cells were grown to confluence in Ham's F-12 nutrient mixture, and treated with 1,25 (OH)2 vitamin D3 (2.6 microM to 2.6 pM) or PHT (5-100 micrograms/mL) for either 24 or 48 h of pretreatment. The media were then discarded, replaced with fresh media and test reagents, and quantitated for osteocalcin by radioimmunoassay at 0, 4, and 8 h secretion time points. Results were statistically analyzed by the Student's two-tailed t test. Controls showed a nearly linear secretion rate of osteocalcin, reaching 8-9 ng/10(6) cells by 8 h. Vitamin D3 (2.6 nM) maximally stimulated secretion nearly two-fold after 24 or 48 h of pretreatment in comparison to controls. PHT alone (25-100 micrograms/mL) exerted an inhibitory effect, which appeared dose-dependent and was most evident at 4 and 8 h. PHT (50 micrograms/mL) had a significant effect, in the presence of a range of vitamin D3 concentrations (2.6 microM to 2.6 pM), after 48 h of pretreatment. A maximal PHT dose of 100 micrograms/mL had no effect on either the viability or the numbers of cultured cells. These data indicate that PHT affects osteocalcin secretion from osteoblastic rat osteosarcoma (ROS 17/2.8) cells