Bone-targeted carbonic anhydrase inhibitors: effect of a proinhibitor on bone resorption in vitro

Many investigations have indicated a functional role for carbonic anhydrase in the mediation of hormone-stimulated bone resorption. These studies depend heavily on the use of heterocyclic sulfonamide inhibitors of carbonic anhydrase. These drugs have effects on many tissues other than bone, and some of these effects confound the interpretation of studies of the role of carbonic acid in bone metabolism. A novel, "bone-targeted" sulfonamide has been produced to obviate these extraosseous effects. This compound (designated WP-1) is the combination of tetracycline and acetazolamide, such that the acetazolamide is not an active inhibitor. Hydrolysis of WP-1 yields an active carbonic anhydrase inhibitor. WP-1 has a marked affinity for bone mineral, allowing deposition of the drug in bone. At a concentration of 10(-5) M, WP-1 attenuates parathyroid hormone stimulated net release of calcium from neonatal rat calvaria in culture. WP-1 is the first member of a class of drugs which may prove useful as pharmacological probes in the study of bone metabolism.     

Histomorphometric identification of carbonic anhydrase in fetal rat bone embedded in glycolmethacrylate

Carbonic anhydrase was identified in bone-resorbing cells present in sections of fetal rat femur embedded in glycolmethacrylate. Using a slight modification of the Hansson's histochemical method, we demonstrated that most chondroclasts (91.8-95.4%) and osteoclasts (95.1-96.3%) display a positive histochemical reaction for carbonic anhydrase. This staining was consistently inhibited in the presence of very low concentrations (10(-6), 10(-7) M) of the specific inhibitor acetazolamide. The number of chondroclasts reacting for carbonic anhydrase was identical to the number of acid phosphatase-stained chondroclasts determined on adjacent sections. A large majority of osteoclasts (96.3%) stained for carbonic anhydrase and for acid phosphatase (97.2%), with more osteoclasts reacting for the latter enzyme than the former (76.8 +/- 8.5 (SD) vs 85.3 +/- 9.2 cells/mm2 of endosteal bone; p less than 0.01). The observation that acetazolamide at a concentration as low as 10(-7) M inhibited Hansson's reaction, together with our histomorphometric results, validates the use of histochemical staining for carbonic anhydrase to evaluate activity of bone-resorbing cells identified in plastic-embedded fetal bone tissue.     

The p-nitrophenyl phosphatase activity of ubiquitin from bovine erythrocytes

Ubiquitin, a unique protein with esterase and carbonic anhydrase activity, has been found to have also a p-nitrophenyl phosphatase activity. This phosphomonoesterase activity of ubiquitin has an acidic pH optimum; its true substrate appears to be the phosphomonoanion, with a Km of 1.8 X 10(-3) M. It is competitively inhibited by the typical acid phosphatase inhibitors, arsenate (Ki = 1.3 X 10(-3) M), molybdate (Ki = 1.2 X 10(-6) M), and phosphate (Ki = 1.4 X 10(-3) M). These inhibitors have no effect on the CO2 hydration and p-nitrophenyl acetate esterase activities of the ubiquitin. Acetazolamide slightly inhibited the p-nitrophenyl phosphatase activity.     

Bone resorption and prostaglandin production by mouse calvaria in vitro: response to exogenous prostaglandins and their precursor fatty acids

Mouse calvaria were maintained in organ culture for 96 h and endogenous prostaglandin production and active bone resorption (45Ca release) measured. After a lag phase of 12 h, active resorption increased over the 96 h period. The amounts of prostaglandins released into the culture medium (measured by radioimmunoassay) were highest in the first 24 h of culture. Unless these were removed by preculturing for 24 h, or suppressed by indomethacin, no response to exogenous PGE2, or prostaglandin precursors could be demonstrated. Bone resorption was stimulated after preculture by both PGE2 and PGF2 alpha in a dose-dependent manner (10-8M-10-5M), with PGE2 being the more potent. Collagen synthesis was unaffected by PGF2 alpha, whereas PGE2 (10-5M) had an inhibitory effect. Eicosatrienoic acid did not stimulate bone resorption at lower concentrations (10-7M-1-5M), but was inhibitory at 10-4M. Arachidonic acid also inhibited resorption at 10-4m, but at lower concentrations (10-7M-10-5M) increased active resorption. This was concomitant with a rise in PGE2 and PGF2 alpha levels, PGE2 production being significantly higher than PGF2 alpha. The effects of PGE2 (10-8M) and PGF2 alpha (10-8M) appeared additive; there was no evidence of synergistic or antagonistic effects when varying ratios of PGE2: PGF2 alpha were employed.     

A fast screening method for histochemical localization of carbonic anhydrase. Application to kidney, skeletal muscle, and thrombocytes

A simple method for histochemical localization of carbonic anhydrase using 5-dimethyl-amino-naphthalene-1-sulfonamide (DNSA) is described. Cryosections of tissues, or cell smears, are incubated in 3 to 10 X 10(-5) M DNSA and viewed in a fluorescence microscope. Upon excitation with ultraviolet light, sites of carbonic anhydrase localization can be identified by an intense blue fluorescence, which is due to the emission of blue light (lambda max = 470 nm) by carbonic anhydrase-DNSA complexes. This fluorescence can be largely suppressed by simultaneous incubation with 1 X 10(-4) to 2 X 10(-3) M concentrations of nonfluorescent carbonic anhydrase inhibitors, displacing DNSA from its binding site on the enzyme. Application of the method to kidney, skeletal muscle, and thrombocytes yields patterns of carbonic anhydrase localization that are in good agreement with results that have been obtained with a variety of other techniques.     

Sarcolemmal carbonic anhydrase in red and white rabbit skeletal muscle

Sarcolemmal vesicles of white and red skeletal muscles of the rabbit were prepared by consecutive density gradient centrifugations in sucrose and dextran according to Seiler and Fleischer (1982, J. Biol. Chem. 257, 13,862-13,871). White and red muscle membrane fractions enriched in sarcolemma were characterized by high ouabain-sensitive Na+, K(+)-ATPase, by high Mg2(+)-ATPase activity, and by a high cholesterol content. Ca2(+)-ATPase activity, a marker enzyme for sarcoplasmic reticulum, was not detectable in the highly purified white and red muscle sarcolemmal fractions. White and red muscle sarcolemmal fractions exhibited no significant differences with regard to Na+, K(+)-ATPase, Mg2(+)-ATPase, and cholesterol. Specific activity of carbonic anhydrase in white muscle sarcolemmal fractions was 38 U.ml/mg and was 17.6 U.ml/mg in red muscle sarcolemma. Inhibition properties of sarcolemmal carbonic anhydrase were analyzed for acetazolamide, chlorzolamide, and cyanate. White muscle sarcolemmal carbonic anhydrase is characterized by inhibition constants, KI, toward acetazolamide of 4.6 X 10(-8) M, toward chlorzolamide of 0.75 X 10(-8) M, and toward cyanate of 1.3 X 10(-4) M. Red muscle sarcolemmal carbonic anhydrase is characterized by KI values toward acetazolamide of 8.1 X 10(-8) M, toward chlorzolamide of 6.3 X 10(-8) M, and toward cyanate of 0.81 X 10(-4) M. In contrast to the high specific carbonic anhydrase activities in sarcolemma, carbonic anhydrase activity in sarcoplasmic reticulum from white muscle varied between values of only 0.7 and 3.3 U.ml/mg. Carbonic anhydrase of red muscle sarcoplasmic reticulum ranged from 2.4 to 3.7 U.ml/mg.     

Prostaglandin-related bone resorption in cultured neonatal mouse calvaria: evaluation of biopotency of nonsteroidal anti-inflammatory drugs

The objective of this study was the development of an assay based on suppression of endogenous prostaglandin synthesis in cultured neonatal mouse calvaria for evaluation of the biopotency of nonsteroidal anti-inflammatory drugs in bone. In preliminary trials, osteolytic activity due to spontaneous prostaglandin production over a 72 h culture period was found highly variable, and could not be stabilized by addition of the common precursor arachidonic acid to the culture medium. Eventually, continuous exposure of mouse calvaria to moderate concentrations of thrombin (greater than or equal to 14 U/ml medium) proved to be satisfactory to achieve stable rates of bone resorption through continuous stimulation of prostaglandin synthesis from endogenous sources. Notably, the extent of net calcium release into the medium was highly reproducible in different experiments. As an example for possible applications of the bioassay, the ability of acemetacin to interfere with prostaglandin synthesis in bone, which had not been assessed before, was evaluated in a comparative assay with indomethacin and acetylsalicylic acid. While 1 X 10(-8) M acemetacin appeared to augment thrombin-induced bone resorption, as did 5 X 10(-6) M acetylsalicylic acid, a dose-dependent inhibition of calcium release was observed between 10(-7)-10(-5) M acemetacin. In this respect, the biopotency of indomethacin was 50 times higher than that of acemetacin and exceeded that of acetylsalicylic acid by a factor of more than 2000. These data could be useful for the appraisal of multiple effects of the investigated drugs on prostaglandin-related bone turnover.     

Hormonal regulation of acid phosphatase release by osteoclasts disaggregated from neonatal rat bone

Osteoclasts disaggregated from neonatal rat long bones and incubated on plastic or glass substrates were found to release a considerable proportion of tartrate-resistant acid phosphatase into culture supernatants. Enzyme release was detectable in the supernatant medium of cultures containing as few as ten cells after 1 hr of incubation and proceeded in a linear manner for the ensuing 6 hr. Calcitonin (1 pg/ml) and cytochalasin B (5 micrograms/ml) inhibited release into the supernatant, suggesting that release represents enzyme secretion. Prostaglandin E1 induced transient inhibition followed by recovery; parathyroid hormone and 1,25(OH)2 vitamin D3 were without influence. Acid phosphatase release in these cultures shows a pattern of hormone responsiveness that coincides with the effects of these hormones on bone resorption by isolated osteoclasts. The extent of acid phosphatase release and its regulation by calciotropic hormones imply a central role for acid hydrolase secretion in osteoclastic bone resorption. The experimental system described in this study may facilitate analysis of the pharmacological hormonal and cellular regulation of osteoclastic function.     

Inhibition of carbonic anhydrases in type I muscle fibers influences contractility

We tested the effects of inhibiting the carbonic anhydrase activity of rat soleus and extensor digitorum longus muscles on the isometric contractile properties and the resistance to fatigue. SOL and EDL muscles from female rats were incubated in vitro in the presence of methazolamide, a specific inhibitor of carbonic anhydrase, before determining their contractile properties. Methazolamide had no effects on the contractile properties of the soleus muscle (10(-5) or 10(-3) M) and extensor digitorum longus (10(-3) M), except for the half-relaxation time of the soleus muscle which increased significantly. Values for half-relaxation time were significantly increased with both concentrations of the inhibitor. Muscles were then submitted to a fatigue protocol lasting 30 min. During the fatigue test, no significant difference was observed between control and 10(-5) M methazolamide soleus muscles. In presence of 10(-3) M methazolamide however, the soleus muscle showed a significantly increased resistance to fatigue compared with control preparations. No significant effect was observed with the extensor digitorum longus muscle exposed to 10(-3) M methazolamide. Results are discussed in terms of the presence of two different isoforms of carbonic anhydrase that may be associated with calcium uptake and energy metabolic processes, respectively.     

Dissociation of organic acid secretion from macrophage mediated bone resorption

A prevailing concept in the literature on bone resorption suggests that the removal of calcium crystals from the bone matrix is the result of the secretion of lactic and/or citric acid. In the present study, we have reassessed this concept using an in vitro bone resorption system consisting of thioglycolate elicited rat peritoneal macrophages co-cultured, for up to 96 hours, with devitalized ⁴⁵Ca-labeled bone particles. In these combined cultures, medium lactate concentration increased linearly for the first 48 hours of culture and remained at a plateau thereafter. Medium citrate concentration, on the other hand, remained constant and at very low levels throughout incubation. In contrast to both citrate and lactate, bone resorption, measured as ⁴⁵Ca release, began a few hours after the onset of culture and increased at a constant rate for the balance of the 96-hour culture period. Alteration of resorptive activity by the addition of 10^?6M cortisol (which stimulates ⁴⁵Ca release) or the interposition of a filter between cells and bone (which inhibits resorption) was not paralleled by similar shifts in lactate or citrate concentration. These experiments indicate that mobilization of the bone mineral can occur in the absence of a concurrent, generalized release of lactic and citric acid by sesorbing cells. On the other hand, the data do not exclude a possible role for these compounds under circumstances where they are secreteo into a “closed” compartment at the cell-bone interface or, in the case of lactate, during the initial period of resorptive activity.     

β-Criptoxanthin stimulates bone formation and inhibits bone resorption in tissue culture in vitro

The effect of beta-cryptoxanthin, which is greatly present in fruits, has not been clarified so far on bone metabolism. The effect of beta-cryptoxanthin on bone formation and bone resorption was investigated in tissue culture in vitro. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-8)-10(-5) M beta-cryptoxanthin. The presence of beta-cryptoxanthin (10(-6) or 10(-5) M) caused a significant increase in calcium content, alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal and metaphyseal tissues. These increases were completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. beta-Carotene (10(-6) or 10(-5) M) or xantine (10(-6) or 10(-5) M) had no effect on the diaphyseal and metaphyseal calcium contents. The bone-resorbing factors parathyroid hormone (1-34) (PTH; 10(-7) M) or prostaglandin E2 (PGE2; 10(-5) M) caused a significant decrease in calcium content in the diaphyseal and metaphyseal tissues. The decrease in bone calcium content induced by PTH or PGE2 was completely inhibited by beta-cryptoxanthin (10(-8)-10(-6) M). In addition, beta-cryptoxanthin (10(-8)-10(-6) M) completely inhibited the PTH (10(-7) M)- or PGE, (10(-5) M)-induced increase in medium glucose consumption and lactic acid production by diaphyseal and metaphyseal tissues. The inhibitory effect of beta-cryptoxanthin (10(-7) M) on PTH (10(-7) M)- or PGE2 (10(-5) M)-stimulated decrease in the diaphyseal calcium content was significantly prevented in the presence of 10(-3) M vanadate, an inhibitor of protein tyrosine phosphatase. Vanadate (10(-3) M) did not have a significant effect on calcium content and lactic acid production in control bone tissues. The present study demonstrates that beta-cryptoxanthin has a direct stimulatory effect on bone formation and an inhibitory effect on bone resorption in tissue culture in vitro.     

Inhibitory effect of okadaic acid on bone resorption in neonatal mouse calvaria in vitro. Protein dephosphorylation as an important regulatory mechanism in the bone resorption process.

Okadaic acid (OA), a potent inhibitor of protein phosphatase type 1 and protein phosphatase type 2A was studied for its effect on bone resorption in neonatal mouse calvaria. OA (0.01 to 1000 ng/ml) had no effect on the basal bone resorption rate, except at 1000 ng/ml, were a small inhibitory effect was observed. Resorption stimulated by parathyroid hormone (10(-8) M) was abolished in the presence of OA, half maximal inhibition being observed at 1 ng/ml. However, at 50 ng/ml or higher, OA significantly increased lactate dehydrogenase activity in the medium, indicating a cytotoxic effect at these concentrations. Similar inhibitory effects were observed when bone resorption was stimulated by 1,25-dihydroxycholecalciferol (10(-8) M) or prostaglandin E2 (10(-6) M). From this it is concluded that protein dephosphorylation may represent an important regulatory mechanism in the bone resorption process.     

Role of hemoglobin in proton transfer to the active site of carbonic anhydrase.

The binding of bovine oxyhemoglobin to bovine carbonic anhydrase with a dissociation constant between 10(-5) and 10(-7) M has been determined by countercurrent distribution using aqueous, biphasic polymer systems. This result provides an explanation for the very efficient proton transfer between hemoglobin and carbonic anhydrase, a transfer which enhances the catalytic activity of carbonic anhydrase as measured by 18O exchange between bicarbonate and water at chemical equilibrium (Silverman, D. N., Tu, C. K., and Wynns, G. C. (1978) J. Biol. Chem, 253, 2563-2567). Two rate constants describing 18O exchange activity of carbonic anhydrase at pH 7.5 show saturation behavior when plotted against hemoglobin concentration consistent with a dissociation constant of 2.5 X 10(-6) M between bovine hemoglobin and carbonic anhydrase. Interpretation of these rate constants in terms of a two-step model for 18O exchange indicates that hemoglobin enhances the rate of exchange from carbonic anhydrase of water containing the oxygen abstracted from bicarbonate, but does not affect the catalytic interconversion of CO2 and HCO3- at chemical equilibrium.     

Isolation and Characterization of 2-Nitroimidazole Produced by Streptomyces Species as an Inhibitor of Both Carbonic Anhydrase and Shell Formation in the Barnacle Balanus amphitrite

Carbonic anhydrase is thought to be involved in the process of calcium carbonate deposition in calcified tissues of many organisms. Barnacles form hard calcified shells for protection against predation, and represent a class of marine-fouling animals. In order to inhibit barnacle growth by inhibiting shell formation, we searched for carbonic anhydrase inhibitors from microbial secondary metabolites. A simple assay for assessing carbonic-anhydrase-inhibiting activity was developed. Screening of many microorganisms isolated from soil with this assay resulted in a microbial strain that produced a carbonic anhydrase inhibitor. This strain was identified as Streptomyces eurocidicus mf294. The inhibitor was isolated through 4 purification steps and identified as 2-nitroimidazole on the basis of spectroscopic data. 2-Nitroimidazole inhibited barnacle carbonic anhydrase dose-dependently and complete inhibition was reached at the concentration of 1 x 10(-5) M. 2-Nitroimidazole did not affect settlement or metamorphosis of barnacle larvae, but inhibited shell formation at concentrations higher than 1 x 10(-4) M. These findings strongly support the idea that carbonic anhydrase is involved in calcification.     

Actions of recombinant interleukin 1, interleukin 2, and interferon-gamma on bone resorption in vitro

Human peripheral blood cells, when cultured in vitro, release bone-resorbing factors, which have been called osteoclast-activating factors (OAF) but remain unidentified. We showed previously that a monocyte product, similar to interleukin 1 (IL 1), is a powerful stimulator of bone resorption in vitro. However, the possibility remained that other immune cell products may contribute to OAF activity. We have therefore tested three recombinant cytokines; IL 1, interleukin 2 (IL 2), and interferon-gamma (IFN-gamma) for their activity in a neonatal mouse bone resorption assay. We report here that purified recombinant murine IL 1 is a potent and powerful stimulator of bone resorption in vitro, active over a concentration range of 0.14 to 33 U/ml (1.3 X 10(-12) to 3.1 X 10(-10) M). IL 1-stimulated bone resorption was unaffected by cyclooxygenase inhibition but was inhibited by calcitonin and IFN-gamma. IL 2 had no effect on bone resorption.     

Gamma interferon inhibits basal and interleukin 1-induced prostaglandin production and bone resorption in neonatal mouse calvaria

Production of the osteolytic arachidonic acid metabolites, prostaglandin (PG) E2, PGI2 and PGF2 alpha, by neonatal mouse calvariae was quantitated by gas chromatography/mass spectrometry. Mouse recombinant interleukin 1 (rIL-1) raised medium levels of PGE2 and PGI2 (measured as 6-keto-PGF1 alpha) in the dose range tested (1.0-10.0 U/ml culture medium), while an effect on PGF2 was only observed at 10 U/ml. Bone resorption in response to rIL-1 reached a plateau at 3.0 U/ml. Mouse recombinant gamma-interferon (rIFN-gamma) between 100-500 U/ml suppressed basal PG synthesis and spontaneous resorption of cultured bone. In addition, IFN-gamma at 100 U/ml prevented stimulation of PG synthesis by 3.0 U/ml rIL-1 and thereby reduced the bone resorbing activity of the cytokine by at least 60%. 5 X 10(-7) M indomethacin was equally effective in suppression of PG synthesis and bone resorption. The present study provides evidence that IFN-gamma inhibits PG synthesis and consequently resorption of cultured bone.     

Expression of typical osteoclast markers by PBMCs after PEG-induced fusion as a model for studying osteoclast differentiation

Bone is a metabolically active organ subjected to continuous remodeling process that involves resorption by osteoclast and subsequent formation by osteoblasts. Osteoclast involvement in this physiological event is regulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Fusion of mono-nuclear pre-osteoclasts is a critical event for osteoclast differentiation and for bone resorption. Here we show that PBMCs can be successfully fused with polyethylenglicol (PEG) in order to generated viable osteoclast-like cells that exhibit tartrate-resistant acid phosphatase (TRAP) and bone resorptive activities. PEG-fused PBMCs expressed additional markers compatible with osteoclastogenic differentiation such as carbonic anhydrase II (CAII), calcitonin receptor (CR), cathepsin K (Cat K), vacuolar ATPase (V-ATPase) subunit C1 (V-ATPase), integrin β3, RANK and cell surface aminopeptidase N/CD13. Actin redistribution in PEG-fused cells was found to be affected by cell cycle synchronization at G0/G1 or G2/M phases. PEG-induced fusion also led to expression of tyrosine kinases c-Src and Syk in their phosphorylated state. Scanning electron microscopy images showed morphological features typical of osteoclast-like cells. The results here shown allow concluding that PEG-induced fusion of PBMCs provides a suitable model system for understanding the mechanisms involved in osteoclastogenesis and for assaying new therapeutic strategies.     

Carbonic anhydrase III: the phosphatase activity is extrinsic

The carbonic anhydrases reversibly hydrate carbon dioxide to yield bicarbonate and hydrogen ion. They have a variety of physiological functions, although the specific roles of each of the 10 known isozymes are unclear. Carbonic anhydrase isozyme III is particularly rich in skeletal muscle and adipocytes, and it is unique among the isozymes in also exhibiting phosphatase activity. Previously published studies provided evidence that the phosphatase activity was intrinsic to carbonic anhydrase III, that it had specificity for tyrosine phosphate, and that activity was regulated by reversible glutathionylation of cysteine186. To study the mechanism of this phosphatase, we cloned and expressed the rat liver carbonic anhydrase III. The purified recombinant had the same specific activity as the carbonic anhydrase purified from rat liver, but it had virtually no phosphatase activity. We attempted to identify an activator of the phosphatase in rat liver and found a protein of approximately 14 kDa, the amount of which correlated with the phosphatase activity of the carbonic anhydrase III fractions. It was identified as liver fatty acid binding protein, which was then purified to test for activity as an activator of the phosphatase and for protein-protein interaction, but neither binding nor activation could be demonstrated. Immunoprecipitation experiments established that carbonic anhydrase III could be separated from the phosphatase activity. Finally, adding additional purification steps completely separated the phosphatase activity from the carbonic anhydrase activity. We conclude that the phosphatase activity previously considered to be intrinsic to carbonic anhydrase III is actually extrinsic. Thus, this isozyme exhibits only the carbon dioxide hydratase and esterase activities characteristic of the other mammalian isozymes, and the phosphatase previously shown to be activated by glutathionylation is not carbonic anhydrase III.     

Tumor necrosis factors alpha and beta induce osteoblastic cells to stimulate osteoclastic bone resorption

Antigen- or mitogen-stimulated leukocytes release bone-resorbing activity into culture supernatants in vitro. Among the agents likely to be present in such supernatants are monocyte-derived tumor necrosis factor (TNF-alpha) and lymphocyte-derived tumor necrosis factor (TNF-beta) (lymphotoxin), both of which have recently been shown to stimulate bone resorption in organ culture. To identify the mechanism of action of these agents, we compared bone resorption by isolated osteoclasts with bone resorption by osteoclasts cocultured with osteoblastic cells, and with bone resorption by osteoclasts incubated with supernatants from osteoblastic cells, in the presence and absence of recombinant TNF-alpha and TNF-beta. We found that neither TNF-alpha nor TNF-beta had any significant effect on bone resorption by isolated osteoclasts, but in the presence of osteoblasts the agents caused a twofold to threefold stimulation of bone resorption. A similar degree of stimulation was achieved by supernatants from osteoblasts incubated with TNF before addition to osteoclasts, compared with supernatants to which TNF were added after osteoblast incubation. These experiments suggest that TNF-alpha and TNF-beta stimulate bone resorption through a primary effect on osteoblastic cells, which are induced by TNF to produce a factor that stimulates osteoclastic resorption. Half-maximal stimulation of resorption occurred at 1.5 X 10(-10) M and 2.5 X 10(-10) M for TNF-alpha and TNF-beta, respectively. This degree of potency is comparable to that of parathyroid hormone, the major physiologic systemic regulator of bone resorption, and suggests that the TNF may exert a significant influence on osteoclastic bone resorption in vivo.