Functional sites of chemically modified tetracyclines: inhibition of the oxidative activation of human neutrophil and chicken osteoclast pro-matrix metalloproteinases.

OBJECTIVE: We studied the relative ability of 6 different chemically modified non-antimicrobial analogs of tetracycline (CMT) to inhibit human and chicken matrix metalloproteinases (MMP) in vitro. The ability of tetracycline and its analogs to inhibit MMP appears to depend on the Ca++/Zn++ binding site at C11 (carbonyl oxygen) and C12 (OH group) of the molecule, which is lacking in CMT-5, the pyrazole derivative of tetracycline. This significant property of CMT-5 was used to differentiate between the effects of CMT on already active MMP versus the oxidative activation of latent MMP (pro-MMP). METHODS: Cultured chicken osteoclast conditioned medium and purified human neutrophil progelatinase (MMP-9) and pro-collagenase (MMP-8) were assayed for proteinase activities using gelatin and collagen, respectively. The pro-MMP were activated either by preincubation with 1 mM aminophenylmercuric acetate (APMA) or 100 microM sodium hypochlorite (NaOCI). CMT were added either to the preincubation mixtures together with NaOCl or after activation of pro-MMP with NaOCl. RESULTS: All CMT tested, except CMT-5, inhibited APMA or NaOCl activated pro-MMP. However, CMT-5 (like the other CMT), inhibited the oxidative activation of pro-MMP by NaOCl when added together by scavenging the reactive oxygen species. The degradation of type-I collagen by chicken osteoclast conditioned medium was probably due to MMP-2 and/or MMP-13. CONCLUSION: Oxidative activation of pro-MMP may be crucial during soft tissue/bone destruction in the inflammatory diseases, including the arthritides. Our results indicate that the Ca++/Zn++ binding site of CMT is not essential for inhibition of the oxidative activation of pro-MMP.

Effects of tetracyclines on bone metabolism.

The anti-resorptive properties of tetracyclines (TCs) and their non-antimicrobial, chemically modified analogues (CMTs) have enormous therapeutic potential in medicine and dentistry. Osseous destructive diseases associated with excessive mammalian collagenase (matrix metalloproteinase) activity and collagen breakdown include malignancy, arthritis, and periodontitis. However, apart from the significant antimatrix metalloproteinase effects of TCs, TCs/CMTs are also potent inhibitors of osteoclast function (i.e., anti-resorptive). Thus, TCs can affect several parameters of osteoclast function and consequently inhibit bone resorption by (1) altering intracellular calcium concentration and interacting with the putative calcium receptor; (2) decreasing ruffled border area; (3) diminishing acid production; (4) diminishing the secretion of lysosomal cysteine proteinases (cathepsins); (5) inducing cell retraction by affecting podosomes; (6) inhibiting osteoclast gelatinase activity; (7) selectively inhibiting osteoclast ontogeny or development; and (8) inducing apoptosis or programmed cell death of osteoclasts. TCs/CMTs, as anti-resorptive drugs, may act similarly to bisphosphonates and primarily affect osteoclast function.

The nonantimicrobial properties of tetracycline for the treatment of periodontal disease.

Tetracyclines have nonantimicrobial properties that appear to modulate host response. In that regard, tetracyclines and their nonantimicrobial chemically modified analogues (chemically modified tetracycline molecules [CMTs]) inhibit the extracellular activity of mammalian neutrophil and osteoblast collagenases. The activity of this matrix metalloproteinase appears crucial in the destruction of collagen. Apart from its anticollagenase effect, tetracyclines are also potent inhibitors of osteoclast function. Several recent studies have also addressed the therapeutic potential of tetracyclines and CMTs in periodontal disease. These drugs reduced excessive gingival collagenase activity and severity of periodontal breakdown in rats infected with Porphyromonas gingivalis and in diabetic rats. CMT was not associated with the emergence of resistant microorganisms. In human double-blind clinical trials, low-dose doxycycline therapy substantially reduced collagenase activity in the gingival and crevicular fluid, and prevented the loss of attachment in adult periodontitis without the emergence of doxycycline-resistant microorganisms. Tetracyclines and CMTs have enormous therapeutic potential because these drugs can inhibit the activity of matrix metalloproteinases as well as osteoclast function, and thus prevent the degradation of osseous connective tissues in periodontal as well as arthritic diseases.

Reactive oxygen species activate and tetracyclines inhibit rat osteoblast collagenase.

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.

Blocking periodontal disease progression by inhibiting tissue-destructive enzymes: a potential therapeutic role for tetracyclines and their chemically-modified analogs.

Tetracyclines (TCs) have wide therapeutic usage as antimicrobial agents; these drugs (e.g., minocycline, doxycycline) remain useful as adjuncts in periodontal therapy. However, TCs also have non-antimicrobial properties which appear to modulate host response. In that regard, TCs and their chemically-modified analogs (CMTs) have been shown to inhibit the activity of the matrix metalloproteinase (MMP), collagenase. The activity of this enzyme appears crucial in the destruction of the major structural protein of connective tissues, collagen. Such pathologic collagenolysis may be a common denominator in tissue destructive diseases such as rheumatoid and osteoarthritis, diabetes mellitus, bullous dermatologic diseases, corneal ulcers, and periodontitis. The mechanisms by which TCs affect and, possibly, diminish bone resorption (a key event in the pathogenesis of periodontal and other diseases) are not yet understood. However, a number of possibilities remain open for investigation including the following: TCs may 1) directly inhibit the activity of extracellular collagenase and other MMPs such as gelatinase; 2) prevent the activation of its proenzyme by scavenging reactive oxygen species generated by other cell types (e.g. PMNs, osteoclasts); 3) inhibit the secretion of other collagenolytic enzymes (i.e. lysosomal cathepsins); and 4) directly affect other aspects of osteoclast structure and function. Several recent studies have also addressed the therapeutic potential of TCs and CMTs in periodontal disease. These drugs reduced excessive gingival collagenase activity and severity of periodontal breakdown in rats infected with Porphyromonas gingivalis and in diabetic rats. Furthermore, the latter drug (CMT) was not associated with the emergence of TC-resistant microorganisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone regulation of matrix degrading enzymes in rat osteoblastic osteosarcoma 17/2.8 cells.

The present study was designed to further understand the role of PTH on the secretion of the neutral metalloproteinases, collagenase and gelatinase, from the rat osteosarcoma clonal cell line, ROS 17/2.8. Semiconfluent cells were treated with bovine parathyroid hormone, b-PTH-(1-34) at 100 nM-0.01 nM for 24-96 hours and pooled, concentrated media were analyzed by functional assay for collagenase (3H-methyl collagen) and gelatinase (3H-methyl gelatin). Collagenase activity significantly decreased (P less than 0.01) in the PTH conditioned media in a dose-dependent manner before (98-64%) and after (91-39%) reduction and alkylation. SDS-PAGE and fluorography apparently showed the most degradation to alpha A chains in collagen with controls, whereas this substrate remained intact with PTH (100 nM). PTH (100 nM) media also showed neutral gelatinase activity approximately 2% compared to control before and after reduction and alkylation (P less than 0.01). Significant amounts of an inhibitor to collagenase and gelatinase might have been secreted at 1 nM and 0.01 nM PTH, since collagenase and gelatinase activities were greater after reduction and alkylation. Reduction and alkylation likely destroyed these significant amounts of inhibitor. Polymorphonuclear leukocyte collagenase activity was also inhibited 80% by PTH conditioned media, but not by control. However, upon reduction and alkylation which destroyed inhibitor, the PTH treated media showed only a 14% inhibition against polymorphonuclear leukocyte collagenase (P less than 0.01). PTH appeared to downregulate neutral metalloproteinase activities through its effects on an inhibitor. This downregulation may represent a specific phenotypic response to PTH in ROS 17/2.8 cells.

Cathepsin B and L activities in isolated osteoclasts.

Cathepsin B and L activities were examined with chicken osteoclasts isolated by sequential filtration and inhibitors were added to disaggregated rat osteoclasts on cortical bovine bone. Z-Phe-Phe-CHN2, a selective inhibitor of cathepsin L, at 1, 5, and 10 microM, inhibited bone resorption by rat osteoclasts 50, 85, and 100 per cent and, in chicken osteoclasts, cathepsin L activity was comparably inhibited. Cathepsin L in avian osteoclasts was also 25-fold higher than cathepsin B. Chicken osteoclasts treated with Z-Phe-Ala-CHN2, a generalized cysteine proteinase inhibitor, had both cathepsins inhibited to the same extent. Cathepsin L may play a key role in resorption.

The effect of tetracyclines on collagenase activity in UMR 106-01 rat osteoblastic osteosarcoma cells.

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.

The effect of phenytoin on collagenase and gelatinase activities in UMR 106-01 rat osteoblastic osteosarcoma cells.

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.

The effect of phenytoin on parathyroid hormone stimulated cAMP activity in cultured murine osteoblasts.

Cells were isolated by sequential collagenase digestion from the parietal segments of one day old mice (Swiss albino BNL strain) and characterized for osteoblast parameters by alkaline phosphatase histochemistry and bovine parathyroid hormone (bPTH-(1-34] induced cAMP activity (protein binding assay). Phenytoin (DPH) reduced PTH stimulated cAMP activity nearly 3-fold in the presence and nearly 1.5-fold in the absence of added calcium. In the absence of PTH, DPH exerted no significant effect. Bay-K-8644, a calcium channel activator, appeared to approximate the PTH stimulation of cAMP activity, even in the presence of DPH. This study demonstrates that DPH has a direct effect on PTH stimulated cAMP activity in cultured murine osteoblasts.

Phenytoin affects osteocalcin secretion from osteoblastic rat osteosarcoma 17/2.8 cells in culture.

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.

Calcitonin stimulates cAMP accumulation in chicken osteoclasts.

Calcitonin causes an increase in the accumulation of cAMP in mammalian osteoclasts leading to an inhibition of bone resorption. Increases in cAMP subsequent to calcitonin stimulation have not been detected in previous studies of chicken osteoclasts as the only source of large numbers of highly purified cells. In this report, we studied the effects of salmon calcitonin (sCT), bovine parathyroid hormone -(1-34) [(bPTH -(1-34)] and forskolin (FSK) on cAMP accumulation in freshly isolated osteoclasts obtained non-enzymatically from the metaphysis of 5-7 week old rachitogenic chickens. Parathyroid hormone did not stimulate the accumulation of intracellular cAMP. Calcitonin and forskolin treatment caused a nearly 2.5 and 3.5-fold increase in cAMP respectively. This study demonstrates that chicken osteoclasts respond to calcitonin with an increase in cAMP accumulation and that the rachitogenic chicken may be a valuable source of hormonally sensitive cells for the study of osteoclast biology.

Stimulation of collagen and glycosaminoglycan production by phenytoin 5,5-diphenylhydantoin in monolayer cultures of mesenchymal cells derived from embryonic chick sternae.

Cultures grown with or without phenytoin (PHT) at a concentration of 5 micrograms/ml from the fifth to the eighth day after plating were labelled with [14C]-proline (0.2 microCi/ml) from the sixth to eighth day. Collagenase digestion indicated that collagen content increased approx. 2-fold after PHT exposure. Increases in sulphated glycosaminoglycan product in response to PHT were approx. 1.5-fold; PHT also stimulated protein production. Both actinomycin D and cycloheximide blocked incorporation of [3H]-leucine, [3H]-proline, and H2(35)SO4 approx. 90 per cent with or without PHT. Continuous sucrose density-gradient fractionation indicated that PHT produced quantitative but not qualitative changes in cellular RNA.

Reduced sialylation of podocalyxin--the major sialoprotein of the rat kidney glomerulus--in aminonucleoside nephrosis.

In this study the sugar composition of podocalyxin was determined in puromycin aminonucleoside-treated (PAN) rats and controls. Podocalyxin from both control and PAN rats bound 125I-WGA and 125I-peanut lectin (the latter only after neuraminidase treatment) on nitrocellulose transfers. Purified podocalyxin from both control and PAN rats was found to contain sialic acid, Gal, GlcNac, and Man but lacked Fuc and GalNac by gas-liquid chromatography. In PAN rats the sialic acid content of podocalyxin was reduced from 4.5% to 1.5%, whereas the concentration of the other sugars (with the possible exception of Gal) was similar to that of controls. The density of podocalyxin on the epithelial cell surface was estimated after immunogold labeling with anti-podocalyxin IgG, and no differences were found between PAN rats and controls. These data indicate that the reduced total glomerular sialic acid content found in PAN is due to the combined effects of the decreased podocyte plasmalemmal surface area and the reduced sialic acid content of podocalyxin.