Bioscience Reports, Vol. 12, No. 5, 1992
HYPOTHESIS
A Hypothesis for the Local Control of Osteoclast Function by Ca 2+, Nitric Oxide and Free Radicals A. S. M. Towhidul Alam, 1 Christopher L.-H. Huang, z David R. Blake, 3 and Mone Zaidi 1'4 Recieved July 20, 1992. Several important conclusions have recently emerged from in vitro studies on the resorptive cell of bone, the osteoclast. First, it has been established that osteoclast function is modulated locally, by changes in the local concentration of Ca 2+ caused by hydroxyapatite dissolution. It is thought that activation by Ca z+ of a surface membrane Ca 2+ receptor mediates these effects, hence providing a feedback control. Second, a number of molecules produced locally by the endothelial cell, with Which the osteoclast is in intimate contact, have been found to affect bone resorption profoundly. For instance, the autocoid nitric oxide strongly inhibits bone resorption. Finally, reactive oxygen species have been found to aid bone resorption and enhance osteoclastic activity directly. Here, we will attempt to integrate these control mechanisms into a unified hypothesis for the local control of bone resorption. KEY WORDS: osteoclast; Ca 2+ receptor; peroxide; nitric oxide.
INTRODUCTION The osteoclast is a cell unique in its ability to resorb bone. Excessive osteoclastic activity leads to high levels of bone destruction in osteoporosis, Paget's bone disease and rheumatoid arthritis. Despite its central role in the development of major bone and joint diseases, we are still relatively ignorant of the normal ~Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London SW17 ORE, U.K. 2 The Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, U.K. 3 Bone and Joint Research Unit, The London Hospital Medical College, London EC1 2AD, U.K. 4 To whom correspondence should be addressed. 369 0144-8463/92/100(/-0369506.50/09 1992 Plenum Publishing Corporation
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functioning of the osteoclast and of its regulation. However, the considerable progress made in the last decade has resulted mainly from the introduction of new methods for monitoring the activity of isolated osteoclasts. We now have a much better understanding of osteoclast control. The cell biology of calcitonin, a potent inhibitor of bone resorption is extensively reviewed elsewhere (1-4). In addition, osteoclast activity is controlled by several bone- and endothelium-derived factors. Prostaglandins are the most well known, and their actions on bone formation and resorption are now fairly well characterized (review: 5). Furthermore, the osteoclast is influenced by a complex matrix of cytokines including various interleukins and colony-stimulating factors. Cytokines mainly regulate osteoclast formation and recrutiment, but may influence osteoclast function indirectly, via the osteoblast. There are numberous excellent reviews on the important role of cytokines in bone biology (6-8). We will concentrate here on less-well-known and recently established factors involved in local osteoclast control, in particular, Ca 2§ nitric oxide (NO) and free radicals.
C a 2+
A N D ITS SURFACE M E M B R A N E RECEPTOR
In 1989, we (10) and others (11) reported that the exposure of osteoclasts to millimolar concentrations of Ca 2+ (9) led to an immediate rise of cytosolic [Ca 2+] (10-12) resulting in rapid cell retraction (13, 14). This was followed by a marked inhibition of bone resorption (10-16) accompanied by a reduction in the secretion of resorptive enzymes (10, 14, 15). The morphological and functional responses seen in osteoclasts when exposed to elevated extracellular [Ca 2+] may represent the basis for a short-range control mechanism (10, 13). It has been demonstrated recently that a range of divalent and trivalent cations of the alkaline earth, transition and lanthanide series mimics the effect of elevated extracellular [Ca 2+] in producing osteoclastic inhibition (17-20). These include Mg 2+, Ba 2+ (17), Ni 2+ (18), Cd 2+ (19) and La 3+ (20). A sensitivity to di- and trivalent cations is consistent with the existence of a surface membrane cation receptor, popularly termed the Ca 2+ receptor. It is therefore unlikely that the reported effects of extracellular [Ca 2+] are exerted via a mechanism regulated by passive alterations in cytosolic [Ca2+]. Instead, there is now a growing list of cells capable of responding to changes in the concentration of extracellular Ca 2+ using a surface membrane receptor-like entity (13, 21). A recent study with the cytotrophoblast has suggested that a Ca 2+ receptor-like molecule may be a 500 kD protein (22). In the osteoclast, Ca 2+ receptor expression can also be induced under certain physiological conditions. For example, freshly isolated osteoclasts from egg-laying birds do not exhibit a sensitivity to elevated extracellular [Ca 2+] (23). It is likely that the Ca 2+ receptor is heavily downregulated during the egg-laying cycle, but can be induced to re-appear by culturing osteoclasts away from the high Ca 2+ microenvironment of bone. Thus, in contrast to freshly isolated cells, chicken or quail osteoclasts cultured for up to 7 days in 1 mM-[Ca 2+] on plastic substrate do respond to elevated extracellular [Ca 2+] (16, 23). The somewhat temporary
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absence of a mechanism that could potentially switch-off bone resorption would be consistent with the physiological requirement for high levels of bone resorption during egg shell calcification (24). Indeed, the avian osteoclast model may eventually prove useful for studying the regulation of expression of the putative Ca 2+ receptor. Cytosolic [Ca 2+] elevation has been implicated as being the intracellular signal mediating the functional inhibition seen in response to extracellular [Ca 2+] elevation (10, 11). The evidence relates to the observation that the functional effects of extracellular [Ca 2+] elevation and those of the Ca 2+ ionophore, ionomycin, are additive (17). Furthermore, agents such as ionic perchlorate (25), verapamil (26), Ni 2+ (18), Cd 2+ (19) and La 3+ (20) that elevate cytosolic [Ca 2+] inhibit osteoclastic bone resorption. It is notable that whilst only a transient elevation of cytosolic [Ca 2+] is required for causing an ~nhibition of bone resorption, a sustained elevation is essential for reducing secretory activity. The reason for this difference is unclear. Whilst the effects of elevated extracellular [Ca 2+] on osteoclast function now appear to be fairly well established, there is relatively less information on the associated mechanisms of cytosolic [Ca 2+] elevation, in vitro investigations aimed at precisely defining intracellular signalling pathways in the osteoclast have been difficult to perform as Ca 2+ functions both as extracellutar regulator and intracelluar second messenger. Nevertheless, we have provided evidence that activation of the Ca 2+ receptor triggers a rapid mobilization of Ca 2+ from intracellular stores (18, 19). This is followed by the transmembrane flux of Ca 2+ through a unique Ca 2+ receptor-operated Ca 2+ channel (27). In order to achieve a distinction between intracellular Ca 2+ mobilization and extracellular Ca 2+ entry, we required a means of cellular activation that was independent of the presence of extracellular Ca 2+. Thus, in line with earlier studies on parathyroid cells (28, 29) we explored the use of an alternative divalent cation, Ni 2+, to activate the osteoclast Ca 2+ receptor (I8). We studied osteoclasts in vitro in a flow chamber, and followed their cytosolic [Ca 2+] through fura 2 fluorescence signals, using procedures described on earlier occasions (18). Ni 2+ application to osteoclasts in Ca2+-free, EGTAcontaining solution with less than 5 nM-[Ca 2+] permitted normal cytosolic [Ca 2+] responses. As would be expected, therefore, the prior depletion of intracellular Ca 2+ stores using ionomycin as ionophore, abolished cytosolic [Ca 2+] transients in response to Ni 2+. These results have clearly indicated that Ni2+-induced cytosolic [Ca ~+] transients are primarily due to intracellular Ca 2+ redistribution. Furthermore, the cytosolic [Ca 2+] responses to Ni 2+ displayed all characteristics of a receptor-mediated action (18). Firstly, the response amplitude to the application of N~2+ was concentration-dependent. Secondly, repeated administration of Ni 2+ yielded progressively reduced amplitudes of cytosolic [Ca 2+] responses. Finally, such desensitization was dependent upon the concentration of Ni 2+ used for conditioning the cells, For example, the reduction in the cytosolic [Ca 2+] response elicited by a test concentration of Ni 2+ at 5000 #M, that would ordinarily produce a maximal response, varied directly with the concentration of Ni 2+ employed in a conditioning exposure.
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Thus, Ni 2+ proved to be an effective tool for perturbing cellular activation systems in osteoclasts, in vitro. It was possible to apply a range of experimental constraints, including external Ca 2+ deprivation in particular, to elucidate some basic properties of the transduction system triggered by the divalent cation, Ni 2+. We have also recently suggested that Ca z+ influx, triggered in response to Ca 2+ receptor activation, occurs via a unique Ca 2+ receptor-operated Ca 2+ channel (27). In reaching this conclusion, we followed lines which have in the past allowed for the prediction of receptor-operated Ca 2+ channels in a variety of eukaryotic cells (30). First, Ca 2+ influx following receptor activation has been found to be sensitive to changes in the electrochemical gradient across the cell membrane (30). Hence, a change in the electrical term of this gradient, caused by a change in membrane voltage, modulates Ca 2+ influx. We found that cell membrane depolarisation using 100mMo[K +] attenuated the peak cytosolic [Ca 2+] response to elevated extracellular [Ca2+], whilst hyperpolarisation significantly potentiated this response (27). Thus, in response to activation of the CaZ+receptor, the magnitude of Ca 2+ influx was modulated by the electrical term of the electrochemical gradient. Secondly, receptor-operated Ca 2+ channels have been shown to possess a high degree of selectivity for divalent cations whose permeation properties follow a rank order of potency. Using this concept, Hallam and co-workers (1988) established a method for the quantification of Mn 2+ influx by utilising the ability of the cation to quench the fluorescence emitted by a CaZ+-sensitive fluorochrome, fura 2 (31). We have developed the use of a MgZ+-sensitive fluorochrome, magfura, to demonstrate Mg 2+ influx directly which we found was modulated by the extracellular [Ca 2+] (27). This provided clear evidence that receptor activation by Ca 2+ could cause the permeation of a different divalent cation, Mg 2+. The results described thus clearly argue for a fundamental biophysical separation of the processes of cation-induced receptor activation and cation permeation, although receptor and channel may be two parts of a single structural entity.
SMALL MOLECULES FROM ENDOTHELIAL CELLS
We have recently been interested in the role of the endothelial cell in osteoclast control. A number of molecules produced by the endothelium have been shown to have profound effects on bone resorption. In particular, we have recently demonstrated that NO (32) and endothelin (33) are potent resorption inhibitors, whilst the reactive oxygen species, hydrogen peroxide (H202), is a powerful stimulator of bone resorption (34). These recent revelations raise a clear possibility that the endothelial cell, found in abundance in bone marrow, may have a primary role in osteoclast control. Furthermore, osteoclasts are in close proximity to vascular endothelial cells, particularly in the "cutting cones" of cortical bone (35). NO, a short-lived autocoid (tl/2 = 3 to 50 s), is a product of L-arginine metabolism in the vascular endothelium. The autocoid is a potent endogenous vasodilator and also mediates the relaxation induced by several vasodilators,
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including acetylcholine and bradykinin (36). Despite its primary action in blood flow regulation, the effects of NO are much more diverse. For example, NO appears to play an important role in intercellular communication in neutrophils, brain tissue, renal epithelial cells, mast cells and autonomic nerves (review: 37). Our observation that NO and the NO-generating drug, 3 morpholinosyndnonimine (or SIN-l) strongly inhibit osteoclastic bone resorption by isolated osteoclasts is consistent with earlier studies that have shown similar effects of nitro-containing vasodilators on resorption of bone in organic culture (38). The latter compounds have also been found to inhibit parathyroid hormone- and vitamin D3-stimulated bone resorption. However, unlike previous studies, ours has used single isolated cells to demonstrate potent direct effects of NO on osteoclast spreading. On exposure to NO or SIN-l, osteoclasts were shown to undergo marked retraction~ the R effect, without an action on margin ruffling (32). In contrast, other endothelial cell-derived molecules, namely endothelin (33) and prostacyclin (39), selectively inhibit cell margin ruffling, the Q effect. The basis for NO action in most cell types has been attributed to an increase in intracellular cyclic GMP. This is due to the activation by NO, of a soluble guanylate cyclase (37, 40). In contrast, in the osteoclast, the di-esterase-resistant cell-permeant analogues, dibutyryl and 8-bromo cyclic GMP were found not to mimic the effects of NO on osteoclast spreading and bone resorption (32). This makes a role for cyclic GMP in the action of NO on the osteoclast rather unlikely. Similarly, an important role for cyclic AMP has also been excluded on the basis of a lack of effects of forskolin, a cyclic AMP agonist, on cell spreading. Furthermore, NO has been found not to elevate cytosolic [Ca2+], ruling out mediation by intracellular [Ca 2+] (32). NO is indeed known to act through mechanisms not involving cyclic GMP, for example, by activating of cytosolic ADP-ribosyltransferase (41). The autocoid also blocks fibroblast proliferation and mitogenesis (42) and enhances macrophage cytostatic activity by inducing the destruction of iron-containing enzymes. The latter results from intracellular nitrosyl-iron complex formation (43). Superoxide anions ( O 2 ) can react with, and remove, local NO and this may explain the reported action of O f in enhancing fibroblastic proliferation and mitogenesis at low concentrations (44). In case of the osteoclast, certain other alternatives would need to be examined. These include possible actions of NO on substrate adhesion molecules or on the osteoclast cytoskeleton exerted either directly, or via an action on kinases or cellular mechanoproteins, such as kinesin (45). The rapid retraction of osteoclasts in response to NO application, indicating cell detachment or de-adhesion, as observed on time-lapse video recordings (32); is consistent with these possiblities. Several reactive oxygen species are also generated from endothelial cells (46-49). One such species, H202, was found to produce potent stimulatory effects on osteoclast motility and bone resorption at nanomolar concentrations (34). H202 produced bursts of cytoplasmic motile activity resulting in increased numbers of osteoclastic excavations in the bone resorption assay. The size of individual excavations remained unaffected.
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The mechanism of osteoclastic stimulation by H202 is not yet known. One possibility is that basal cyclic AMP formation is reduced. In theory, this should enhance osteoclast motility, as elevated cyclic AMP levels potently inhibit osteoclast motility (50, 51). To our knowledge, there are no reports demonstrating an action of H202 on cyclic AMP levels. Our studies also indicate that H202 does not reduce cytosolic [Ca 2+] in isolated osteoclasts to account for its observed stimulatory effects on bone resorption (34). It would nevertheless seem attractive to examine the role of the multisubunit transcription factor, NF-KB, in H202-induced osteoclastic activation. H202 has been found to activate NF-KB by releasing IKB from its complex with p65 and p50 (52). The latter heterodimeric protein is a DNA-binding factor best characterized in cells of the immune system, including macrophages and monocytes (53). If NF-KB is found in osteoclasts, its modulation by H202, and any relationships to osteoclastic activation, including any interactions with inflamatory cytokines, should be urgently investigated.
A UNIFIED HYPOTHESIS F O R LOCAL OSTEOCLAST C O N T R O L Bone resorption is the result of individually controlled, but successive steps (review: 6). The first step involves the generation and vascular dissemination of osteoclasts. This is followed by osteoclast activation that occurs on contact with mineral. The digestion of bone matrix and dissolUtion of mineral closely follows. Whilst we have a fairly precise knowledge of temporal relationships that culminate in the resorption of bone, we are relatively less aware of events that result in the termination of the resorptive episode. In the following paragraphs, we describe the resorptive event with specific reference to recently discovered potential control mechanisms of the termination and recovery stages (Fig. 1). The relationships described are admittedly hypothetical.
The Pre-Resorptive Stage This immediate pre-resorptive stage results in the formation of a highly specialized microenvironment. A sealing zone of close adhesion forms between the bone-apposed surface of osteoclast and the bone substrate. This stage is dependent critically upon the adhesion of an osteoclast to bone and subsequently, upon its cytoplasmic spreading. Once such a resorptive microenvironment has formed, the osteoclast plasma membrane within it is thrown into complex folds, the 'ruffled border'. The later specialization is the site where most activities essential for bone resorption appear to reside.
The Resorption Stage In this phase, enzymes, protons and free radicals are poured into the resorptive microenvironment. Within this highly acidic microenvironment (pH-3 to 4), hydrolases and cysteine-proteinases digest protein matrix, whilst bone
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Pre-resorptive
- -
.~o~ Ho~
, ~ f ~
Fig. 1. The bone resorptive episode and its local control: a hypothetical scheme based upon new data. Abbreviations: H202hydrogen peroxide; O2---superoxide radicals; HO~--reactive intermediate species.
mineral or hydroxyapatite is dissolved by the action of the secreted acid. In addition, free radicals, mainly 0 2 , are thought to aid the digestion of bone collagen. Factors leading to osteoclast detachment or retraction will inhibit resorption by interfering with the maintenance of the highly acidic resorptive' microenvironment. It is notable that during this stage, the osteoclast generates, and is consequently exposed to high concentrations of ambient Ca 2+. In the resorptive hemivacuole, these levels are estimated to be in the region of between 8 and 40 mM (9).
Tile Termination Stage Completion of the resorptive episode appears to be closely followed by osteoclast detachment (or de-adhesion) and retraction. We suggest that the latter responses are triggered by a change in the ambient [Ca 2+1 that developes locally during resorption. There is compelling evidence that this ambient [Ca 2+] change is monitored by a surface membrane Ca receptor on the osteoclast (see above) (10-20). In vitro adhesion assays (54) and time-lapse studies (14) have revealed that activation of the osteoclast Ca 2+ receptor by elevated ambient [Ca 2+] leads
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to a failure of adhesion and to dramatic pseudopodial and podosomal retraction in attached cells. The cell spread area can reduce by up to 60% at 15raM extracellular [Ca a+] (13). Cell retraction and de-adhesion may thus provide the means whereby ambient [Ca 2+] acutely regulates bone resorption by interfering with the cell-bone seal and by diminshing the overall area of cell contact with the bone. By preventing further bone resorption, this will minimize the build-up of Ca 2+ in the resorptive hemivacuole. Whilst such a mechanism may explain what is likely to be a subtle, minute-to-minute dynamic control, the observed inhibition of secretory activity in response to elevated ambient [Ca 2+] (15) may influence resorption in the longer term and in instances where the local concentration of Ca 2+ becomes very high. Osteoclastic retraction may also result from an increased exposure to NO produced locally (Fig. 2). It is not known however, as to whether such an effect can be exerted from within the resorptive hemivacuole. If NO were to be synthesized in the osteoclast and secreted together with O~-, then an interesting set of reactions could take place. O~- may react with NO to yield the peroxynitrite anion, O N O O - , which upon further protonation would form peroxynitrous acid, O N O O H . Upon homolysis, peroxynitrous acid would produce further radicals (55, 56). Whether such reactive oxygen species have a role in osteoclast regulation is presently unknown.
~12" ~
..~-N0 F'-(
OONO'-I~OONOH-I~NO2+HO']
~*
L02- H202..,,,~
Fig. 2. A detailed hypothetical scheme of local osteoclast control. The reactive oxygen species, HaO 2 is likely to provide an excitatory signal following the resorptive episode, and by inducing increased osteoclast motility, would allow the cell to move away and resorb at a different location. Abbreviations: N O - n i t r i c oxide; H202--hydrogen peroxide; O2---superoxide radicals; O N O O --peroxynitrite radical; ONOOH-peroxynitrous acid; HO--hydroxyl radical; HO~--reactive intermediate species; CT---calcitonin; ET--endothelin; P T H - parathyroid hormone; Vit D--vitamin D3; PG--prostag|adin; GF--growth factors; Cyto--cytokines; PGI2--prostacyclin; oc-osteoclast; ob--osteoblast; ec--endothelial cell.
Local OsteoclastControl
377 The Recovery Stage
It is notable that whilst osteoclasts undergo actue retraction in response to elevated extracellular [Ca2+], their pseudopodial motile activities appear not to cease (57). The cells therefore retain the ability to move away from the resorption site and to resorb at a different location. This process could be aided by the local production of the stimulatory reactive oxygen species, H202 (Fig. 2). This may ensue from the ambient pH change caused by the disruption of the osteoclastbone seal in response to an elevated extracellular [Ca2+]. A pH change will allow the O2- secreted locally by the osteoclast (58-60) (and by other cells) to undergo initial protonation to form the reactive intermediate radical species, HO2, and upon further protonation, to generate H202. Production of the longer-lived membrane-permeant species, H202 may at this point, provide an appropriate excitatory signal that could reverse the acute osteoclastic inhibition resulting from an exposure to elevated ambient [Ca 2§ (10, 11, 15). Enhanced motility induced by H202 would thus allow an osteoclast to move away from the lacuna and resorb elsewhere. This hypothesis would be consistent with our in vitro studies showing marked effects of H202 on the motility of isolated osteoclasts (34). Relevance to Bone and Joint Disease The potent stimulatory effects of H202 on bone resorption appear to have direct implications in bone and joint pathology. The generation of rective oxygen species may be particularly important in the bone resorption that occurs in association with inflammatory diseases, such as in rheumatoid arthritis. For example, H202 is released into chronically inflamed joint cavities by activated synovial neutrophils or as a result of hypoxic-reperfusion injury to the endothelium, generated by intermittent pressure changes in the mobile and inflamed joint. Calculated H202 production from activated neutrophils may often be at rates in excess of 500 nmol/min (61, 62); these concentrations clearly would be in line with those required for bone destruction. An efficient source of reactive oxygen species during episodes of hypoxicreperfusion injury appears to be the enzyme, xanthine oxidase. The latter enzyme has been localized to both normal and rheumatoid synovia (63) and to the endothelium (64). The generation of reactive oxygen species from synovial tissue has also been confirmed by electron spin resonance spectroscopy. In addition, free radical generation from synovial tissue is reduced by inhibitors of xanthine oxidase, implicating the enzyme as being their major source (65). Clearly, the inhibition of osteoclastic activity is a major therapeutic aim in osteolytic diseases of the bone and joints. Two major areas of drug development could follow, one using a strategy to inhibit the production and action of reactive species, and another using agents that could accomplish the release of NO at sites of excessive bone resorption. Drugs that inhibit reactive oxygen species and their production have been shown to be active inhibitors of bone resorption in model systems (58). It may be difficult to succeed with the second strategy, as marked hypotension is known to result from the systemic administration of osteoclast-
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inhibitory doses of drugs, such as SIN-1 (32). However, a promising approach that clearly deserves study, is the development of novel NO-generating agents that could be targetted to sites of potential bone destruction. Finally, the generation of reactive oxygen species has also been implicated in the ageing process, particularly in relation to neurological, vascular and dermal changes (66). It would be interesting to determine whether radical-induced changes could also relate to senescent bone cells and hence, for example, to the development of osteoporosis.
ACKNOWLEDGEMENTS The work described above was supported, in part, by grants from The Arthritis and Rheumatism Council, U.K. (Project Grant Z1 and Z2 to M.Z. and Programme and Project Grants to D.R.B.), The Medical Res. Council, U.K. (to M.Z.), The Leverhulme Trust, U.K. (to M.Z.), Sandoz Foundation for Gerontological Res., Basel, Switzerland (to M.Z.) and The Royal Society (to C.L.-H.). A.S.M.T.A. is a recipient of the Overseas Res. Scholars Award from the Council of Vice Chancellors and Principals of the Universities of the United Kingdom. The authors are grateful to Mr. W. Smith (Cambridge) for skilled assistance. REFERENCES 1. Zaidi, M., Breimer, L. H. and MacIntyre, I. (1987) Quart. J. Exp. Physiol. 72:371-408. 2. Breimer, L. H., MacIntyre, I. and Zaidi, M. (1988) Biochem. J. 255:377-390. 3. Zaidi, M., Moonga, B. S., Bevis, P. J. R., Bascal, Z. A. and Breimer, L. H. (1990) Crit. Rev. Clin. Lab. Sci. 28:109-174. 4. Zaidi, M., Moonga, B. S., Bevis, P. J. R., Alam, A. S. M. T., Legon, S., Wimalawansa, S. J., MacIntyre, I., Breimer, L. H. (1991) In: Vitamins and Hormones (Volume 46) (Ed. G. D. Aurbach) New York, Academic Press. pp. 87-164. 5. Chambers, T. J. (1988) Ciba Foundation Symposium 136:92-107. 6. Vaes, G. (1988) Clin. Orthop. 231:239-271. 7. Roodman, G. D. (1991) Crit. Rev. Oral Biol. and Med. 2:389-409. 8. Zaidi, M., Alam, A. S. M. T., Shankar, V. S., Bax, B. E., Bax, C. M. R., Moonga, B. S., Bevis, P. J. R., Stevens, C., Blake, D. R. and Huang, C. L.-H. (1992) Biol. Rev. (ln press). 9. Silver, I. A., Murrills, R. J. and Etherington, D. J. (1988) Exp. Cell Res. 175:266-276. 10. Zaidi, M., Datta, H. K., Patchell, A., Moonga, B. S. and MacIntyre, I. (1989) Biochem. Biophys. Res. Commun. 163:1461-1465. 11. Malgaroli, A., Meldolesi, J., Zambonin-Zallone, A. and Teti, A. (1989) J. Biol. Chem. 264:14342-14347. 12. Donahue, H. J., Ijima, K., Goligorsky, M. S., Rubin, C. T. and Rifkin, B. R. (1992) J. Bone Min. Res. (In press). 13. Zaidi, M. (1990)Biosci. Rep. 10:493-507. 14. Datta, H. K., MacIntyre, I. and Zaidi, M. (1989) Biosci. Rep. 9:247-251. 15. Moonga, B. S., Moss, D. W., Patchell, A. and Zaidi, M. (1990) J. Physiol. 429:29-45. 16. Miyauchi, A., Hruska, K. A., Greenfield, E. M., Duncan, R., Alvarez, J., Barattolo, R., Colucci, S., Zambonin-Zallone, A., Teitelbaum, A. L. and Teti, A. (1990) J. Cell Biol. 111: 2543-2552. 17. Zaidi, M., Kerby, J., Huang, C. L.-H., Alam, A. S. M. T., Rathod, H., Chambers, T. J. and Moonga, B. S. (1991) J. Cell. Physiol. 149:422-427.
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18. Zaidi, M., Shankar, V. S., Bax, C. M. R., Bax, B. E., Alam, A. S. M. T., Banerji, B., Bevis, P. J. R., Gill, J. S., Moonga, B. S., and Huang, C. L.-H. (1992b) In: Calcium Regulation and Bone Metabolism (Eds. D. V. Cohn and A. R. Tahjian, Jr.) Elsevier, Amsterdam. (In press.) 19. Shankar, V. S., Bax, C. M. R., Alam, A. S. M. T., Bax, B. E., Huang, C. L.-H. and Zaidi, M. (1992) Biochem. Biophys. Res. Commun. (In press.) 20. Shankar, V. S., Alam. A. S. M. T., Bax, C. M. R., Bax, B. E., Pazianas, M., Huang, C. L.-.H. and Zaidi, M. (1992) Biochem. Biophys. Res. Commun. 187:907-912 21. Brown, E. M. (1991) Physiol. Rev. 71:371-411. 22. Juhlin, C. R., Lundgren, S., Johansson, H., Lorentzen, Z., Rask, L., Larsson, E., Rastad, J., Akerstron G. and Klareskog, G. (1990) J. Biol. Chem. 265:8275-8280. 23. Bascal, Z. A., Moonga, B. S., Dacke, C. J. and Zaidi, M. (1992) Exp. Physiol. 77:501-504. 24. Dacke, C. J. (1979) Calcium Regulation in Submammalian Vertebrates. Academic Press, London. 25. Moonga, B. S., Datta, H. K., Bevis, P. J. R., Huang, C. L.-H., MacIntyre, I. and Zaidi, M. (1991) Exp. Physiol. 76: 923-933. 26. Zaidi, M., MacIntyre, I. and Datta, H. K. (1990) Biochem. Biophys. Res. Commun. 167: 807-812. 27. Bax, C. M. R. Shankar, V. S., Moonga, B. S., Huang, C. L.-H and Zaidi, M. (1992) Biochem. Biophys. Res. Commun. 183:619-625. 28. Nemeth, E. F. and Scarpa, A. (1987) Ann. N.Y. Acad. Sci. 493:542-551. 29. Nemeth, E. F., Wallace, J. and Scarpa, A. (1986) J. Biol. Chem~ 261:2668-2674. 30. Rink, T. J. (1988) Nature 334: 649-650. 31. HaUam, T. J., Jacob, R. and Merritt, J. E. (1988) Biochem. J. 225:179-184. 132. Maclntyre, I., Zaidi, M., Alam, A. S. M. T., Datta, H. K., Moonga, B. S., Lidbury, P. S., Hecker, M. and Vane, J. R. (1991) Proc. Natl. Acad. Sci..USA 88:2936-2940. 33. Alam, A. S. M. T., Gallagher, A., Shankar, V. S., Ghatei, M. A., Datta, H. K., Huang, C. L.-H., Moonga, B. S., Chambers, T. J., Bloom, S. R. and Zaidi, M. (1992) Endocrinol. 130: 3617-3624. 34. Bax, B. E., Alam, A. S. M. T., Banerji, B., Bax, C. M. R., Bevis, P. J. R., Stevens, C. R., Moonga, B. S., Blake, D. R. and Zaidi, M. (1992) Biochem. Biophys. Res. Commun. 183:1153-1158. 35. Owen, M. (1988) J. Cell Sci. 10:6,3-76. 36. Moncada, S., Palmer, R. M. J. and Higgs, E. A. (1988) Hypertension 12:365-372. 37. Ignarro, L. J. (1988) Circ. Res. 65:1-21. 38. Ibbotson, K. J. and D'Souza, S. M. (1989) J. Bone Min. Res. 4:S-204. ,39. Chambers, T. J., McSheehy, P. M. J., Thomson, B. M. and Fuller, K. (1985) Endocrinot. 116: 234-239. 40. Katsuki, S., Arnold, W., Mittal, C. and Murad, F. (1977) J. Cyclic Nucleotide Res. 3:23-35. 41. Brune, B. and Lapetina, E. G. (1989) J. Biol. Chem. 264:8455-8461. 42. Garg, U. C. and Hassid, A. (1990) Biochem. Biophys. Res. Commun. 171:474-479. 43. Lancaster, J. R. Jr. and Hibbs, J. B. Jr. (i990) Proc. Natl. Acad. Sci. USA 87:1223-1227. 44. Burdon, R. H., Gil, V. and Rice-Evans, C. (1989) Free Radical Res. Commun. 7:149-159. 45. Yang, J. T., Saxton, W. M., Stewart, R. J., Raft, E. C. and Goldstein, L. S. B. (1990) Science 249: 42-47. 46. Rosen, G. M. and Freeman, B. A. (1984) Proc. Natl. Acad. Sci. USA 81:726-732. 47. Matsubara, T. and Ziff, M. (1986) J. Immunol. 137:3295-3301. 48. Matsubara, T. and Ziff, M. (1986) J. Cell. Physiol. 127:207-211. 49. Gorog, P., Pearson, J. D. and Kakkfir, V. V. (1988) Atherosclerosis 72:19-27. 50. Zaidi, M., Datta, H. K., Moonga, B. S. and MacIntyre, I. (1990) J. Endocrinol. 126:473-481. 51. Alam, A. S. M. T., Moonga, B. S., Huang, C. L.-H. and Zaidi, M. (1991) Biochem. Biophys. Res. Commun. 179:134-139. 52. Schreck, R., Reiber, P. and Baeurele, P. A. (1991) E M B O J. 10:2247-2258. 53. Baeuerle, P. A. and Baltimore, D. (1991) In: Molecular Aspects of Cellular Regulation, Hormone Control and Regulation of Gene Transcroption (Eds. P. Cohen and J. G. Foukes) Amsterdam~ Elsevier/North Holland Biomedical Press, pp 409-432. 54. Zaidi, M., Datta, H. K., Patchell, A., Moonga, B. S., Moss, D. W., Makgoba, M. W. and MacIntyre, I. (1989) J. Bone Min. Res. 4:$200. 55. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A. and Freeman, B. A. (1990) Proc. Natl. Acad. Sci. USA 17:1604-1620. 56. Yang, G., Candy, T. E. G., Boaro, M., Wilkin, H. E., Jones, P., Nazhat, M. B., Saadatla-Nazhat, R. A. and Blake, D. R. (1992) Free Radicals Biol. Med. 12:327-330.
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57. Zaidi, M., Alam, A. S. M. T., Shanker, V., Bax, B. E., Moonga, B. S., Bevis, P. J. R., Pazianas, M. and Huang, C. L.-H. (1992) Eur. J. Biophys. (In press.) 58. Garrett, I. R., Boyce, B. F., Oreffo, R. O., Bonewald, L., Poser, J. and Mundy, G. R. (1990) J. Clin. Invest. 85: 632-639. 59. Key, L. L. Jr., Reis, W. L., Taylor, R. G., Hays, B. D. and Pitzer, B. R. (1990) Bone 11:115-119. 60. Oursler, M. J., Collin-Osdoby, P., Li, L., Schmitt, E. and Osdoby, P. (1991) J. Cell. Biochem. 46: 331-344. 61. Blake D. R., Allen, R. E. and Lunec, J. (1987) Brit. Med. Bull. 43:371-385. 62. Blake, D. R., Merry, P., Unsworth, J., Kidd, B. L., Outhwaite, F. M., Ballard, R., Morris, C. J., Gray, L. and Lunec, J. (1989) Lancet i:289-293. 63. Allen, R. E., Blake, D. R., Nazhat, J. B. and Jones, P. (1989) Lancet ii:282-283. 64. Allen, R. E., Owthwaite, J., Morris, C. J. and Blake, D. R. (1987) Ann. Rheum. Dis. 46: 843-845. 65. Stevens, C. R., Benboubetra, M., Harrison, R., Sahinoglu, T., Smith, E. C. and Blake, D. R. (1991) Ann. Rheum. Dis. 50:760-762. 66. Armstrong, D. (1983) In: Free Radicals in Molecular Biology, Aging and Disease. (Eds. D. Armstrong, R. S. Sohal, R. G. Cutler, and T. F. Slater) Raven Press, New York. pp 129-142.
HYPOTHESIS
A Hypothesis for the Local Control of Osteoclast Function by Ca 2+, Nitric Oxide and Free Radicals A. S. M. Towhidul Alam, 1 Christopher L.-H. Huang, z David R. Blake, 3 and Mone Zaidi 1'4 Recieved July 20, 1992. Several important conclusions have recently emerged from in vitro studies on the resorptive cell of bone, the osteoclast. First, it has been established that osteoclast function is modulated locally, by changes in the local concentration of Ca 2+ caused by hydroxyapatite dissolution. It is thought that activation by Ca z+ of a surface membrane Ca 2+ receptor mediates these effects, hence providing a feedback control. Second, a number of molecules produced locally by the endothelial cell, with Which the osteoclast is in intimate contact, have been found to affect bone resorption profoundly. For instance, the autocoid nitric oxide strongly inhibits bone resorption. Finally, reactive oxygen species have been found to aid bone resorption and enhance osteoclastic activity directly. Here, we will attempt to integrate these control mechanisms into a unified hypothesis for the local control of bone resorption. KEY WORDS: osteoclast; Ca 2+ receptor; peroxide; nitric oxide.
INTRODUCTION The osteoclast is a cell unique in its ability to resorb bone. Excessive osteoclastic activity leads to high levels of bone destruction in osteoporosis, Paget's bone disease and rheumatoid arthritis. Despite its central role in the development of major bone and joint diseases, we are still relatively ignorant of the normal ~Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London SW17 ORE, U.K. 2 The Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, U.K. 3 Bone and Joint Research Unit, The London Hospital Medical College, London EC1 2AD, U.K. 4 To whom correspondence should be addressed. 369 0144-8463/92/100(/-0369506.50/09 1992 Plenum Publishing Corporation
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functioning of the osteoclast and of its regulation. However, the considerable progress made in the last decade has resulted mainly from the introduction of new methods for monitoring the activity of isolated osteoclasts. We now have a much better understanding of osteoclast control. The cell biology of calcitonin, a potent inhibitor of bone resorption is extensively reviewed elsewhere (1-4). In addition, osteoclast activity is controlled by several bone- and endothelium-derived factors. Prostaglandins are the most well known, and their actions on bone formation and resorption are now fairly well characterized (review: 5). Furthermore, the osteoclast is influenced by a complex matrix of cytokines including various interleukins and colony-stimulating factors. Cytokines mainly regulate osteoclast formation and recrutiment, but may influence osteoclast function indirectly, via the osteoblast. There are numberous excellent reviews on the important role of cytokines in bone biology (6-8). We will concentrate here on less-well-known and recently established factors involved in local osteoclast control, in particular, Ca 2§ nitric oxide (NO) and free radicals.
C a 2+
A N D ITS SURFACE M E M B R A N E RECEPTOR
In 1989, we (10) and others (11) reported that the exposure of osteoclasts to millimolar concentrations of Ca 2+ (9) led to an immediate rise of cytosolic [Ca 2+] (10-12) resulting in rapid cell retraction (13, 14). This was followed by a marked inhibition of bone resorption (10-16) accompanied by a reduction in the secretion of resorptive enzymes (10, 14, 15). The morphological and functional responses seen in osteoclasts when exposed to elevated extracellular [Ca 2+] may represent the basis for a short-range control mechanism (10, 13). It has been demonstrated recently that a range of divalent and trivalent cations of the alkaline earth, transition and lanthanide series mimics the effect of elevated extracellular [Ca 2+] in producing osteoclastic inhibition (17-20). These include Mg 2+, Ba 2+ (17), Ni 2+ (18), Cd 2+ (19) and La 3+ (20). A sensitivity to di- and trivalent cations is consistent with the existence of a surface membrane cation receptor, popularly termed the Ca 2+ receptor. It is therefore unlikely that the reported effects of extracellular [Ca 2+] are exerted via a mechanism regulated by passive alterations in cytosolic [Ca2+]. Instead, there is now a growing list of cells capable of responding to changes in the concentration of extracellular Ca 2+ using a surface membrane receptor-like entity (13, 21). A recent study with the cytotrophoblast has suggested that a Ca 2+ receptor-like molecule may be a 500 kD protein (22). In the osteoclast, Ca 2+ receptor expression can also be induced under certain physiological conditions. For example, freshly isolated osteoclasts from egg-laying birds do not exhibit a sensitivity to elevated extracellular [Ca 2+] (23). It is likely that the Ca 2+ receptor is heavily downregulated during the egg-laying cycle, but can be induced to re-appear by culturing osteoclasts away from the high Ca 2+ microenvironment of bone. Thus, in contrast to freshly isolated cells, chicken or quail osteoclasts cultured for up to 7 days in 1 mM-[Ca 2+] on plastic substrate do respond to elevated extracellular [Ca 2+] (16, 23). The somewhat temporary
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absence of a mechanism that could potentially switch-off bone resorption would be consistent with the physiological requirement for high levels of bone resorption during egg shell calcification (24). Indeed, the avian osteoclast model may eventually prove useful for studying the regulation of expression of the putative Ca 2+ receptor. Cytosolic [Ca 2+] elevation has been implicated as being the intracellular signal mediating the functional inhibition seen in response to extracellular [Ca 2+] elevation (10, 11). The evidence relates to the observation that the functional effects of extracellular [Ca 2+] elevation and those of the Ca 2+ ionophore, ionomycin, are additive (17). Furthermore, agents such as ionic perchlorate (25), verapamil (26), Ni 2+ (18), Cd 2+ (19) and La 3+ (20) that elevate cytosolic [Ca 2+] inhibit osteoclastic bone resorption. It is notable that whilst only a transient elevation of cytosolic [Ca 2+] is required for causing an ~nhibition of bone resorption, a sustained elevation is essential for reducing secretory activity. The reason for this difference is unclear. Whilst the effects of elevated extracellular [Ca 2+] on osteoclast function now appear to be fairly well established, there is relatively less information on the associated mechanisms of cytosolic [Ca 2+] elevation, in vitro investigations aimed at precisely defining intracellular signalling pathways in the osteoclast have been difficult to perform as Ca 2+ functions both as extracellutar regulator and intracelluar second messenger. Nevertheless, we have provided evidence that activation of the Ca 2+ receptor triggers a rapid mobilization of Ca 2+ from intracellular stores (18, 19). This is followed by the transmembrane flux of Ca 2+ through a unique Ca 2+ receptor-operated Ca 2+ channel (27). In order to achieve a distinction between intracellular Ca 2+ mobilization and extracellular Ca 2+ entry, we required a means of cellular activation that was independent of the presence of extracellular Ca 2+. Thus, in line with earlier studies on parathyroid cells (28, 29) we explored the use of an alternative divalent cation, Ni 2+, to activate the osteoclast Ca 2+ receptor (I8). We studied osteoclasts in vitro in a flow chamber, and followed their cytosolic [Ca 2+] through fura 2 fluorescence signals, using procedures described on earlier occasions (18). Ni 2+ application to osteoclasts in Ca2+-free, EGTAcontaining solution with less than 5 nM-[Ca 2+] permitted normal cytosolic [Ca 2+] responses. As would be expected, therefore, the prior depletion of intracellular Ca 2+ stores using ionomycin as ionophore, abolished cytosolic [Ca 2+] transients in response to Ni 2+. These results have clearly indicated that Ni2+-induced cytosolic [Ca ~+] transients are primarily due to intracellular Ca 2+ redistribution. Furthermore, the cytosolic [Ca 2+] responses to Ni 2+ displayed all characteristics of a receptor-mediated action (18). Firstly, the response amplitude to the application of N~2+ was concentration-dependent. Secondly, repeated administration of Ni 2+ yielded progressively reduced amplitudes of cytosolic [Ca 2+] responses. Finally, such desensitization was dependent upon the concentration of Ni 2+ used for conditioning the cells, For example, the reduction in the cytosolic [Ca 2+] response elicited by a test concentration of Ni 2+ at 5000 #M, that would ordinarily produce a maximal response, varied directly with the concentration of Ni 2+ employed in a conditioning exposure.
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Thus, Ni 2+ proved to be an effective tool for perturbing cellular activation systems in osteoclasts, in vitro. It was possible to apply a range of experimental constraints, including external Ca 2+ deprivation in particular, to elucidate some basic properties of the transduction system triggered by the divalent cation, Ni 2+. We have also recently suggested that Ca z+ influx, triggered in response to Ca 2+ receptor activation, occurs via a unique Ca 2+ receptor-operated Ca 2+ channel (27). In reaching this conclusion, we followed lines which have in the past allowed for the prediction of receptor-operated Ca 2+ channels in a variety of eukaryotic cells (30). First, Ca 2+ influx following receptor activation has been found to be sensitive to changes in the electrochemical gradient across the cell membrane (30). Hence, a change in the electrical term of this gradient, caused by a change in membrane voltage, modulates Ca 2+ influx. We found that cell membrane depolarisation using 100mMo[K +] attenuated the peak cytosolic [Ca 2+] response to elevated extracellular [Ca2+], whilst hyperpolarisation significantly potentiated this response (27). Thus, in response to activation of the CaZ+receptor, the magnitude of Ca 2+ influx was modulated by the electrical term of the electrochemical gradient. Secondly, receptor-operated Ca 2+ channels have been shown to possess a high degree of selectivity for divalent cations whose permeation properties follow a rank order of potency. Using this concept, Hallam and co-workers (1988) established a method for the quantification of Mn 2+ influx by utilising the ability of the cation to quench the fluorescence emitted by a CaZ+-sensitive fluorochrome, fura 2 (31). We have developed the use of a MgZ+-sensitive fluorochrome, magfura, to demonstrate Mg 2+ influx directly which we found was modulated by the extracellular [Ca 2+] (27). This provided clear evidence that receptor activation by Ca 2+ could cause the permeation of a different divalent cation, Mg 2+. The results described thus clearly argue for a fundamental biophysical separation of the processes of cation-induced receptor activation and cation permeation, although receptor and channel may be two parts of a single structural entity.
SMALL MOLECULES FROM ENDOTHELIAL CELLS
We have recently been interested in the role of the endothelial cell in osteoclast control. A number of molecules produced by the endothelium have been shown to have profound effects on bone resorption. In particular, we have recently demonstrated that NO (32) and endothelin (33) are potent resorption inhibitors, whilst the reactive oxygen species, hydrogen peroxide (H202), is a powerful stimulator of bone resorption (34). These recent revelations raise a clear possibility that the endothelial cell, found in abundance in bone marrow, may have a primary role in osteoclast control. Furthermore, osteoclasts are in close proximity to vascular endothelial cells, particularly in the "cutting cones" of cortical bone (35). NO, a short-lived autocoid (tl/2 = 3 to 50 s), is a product of L-arginine metabolism in the vascular endothelium. The autocoid is a potent endogenous vasodilator and also mediates the relaxation induced by several vasodilators,
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including acetylcholine and bradykinin (36). Despite its primary action in blood flow regulation, the effects of NO are much more diverse. For example, NO appears to play an important role in intercellular communication in neutrophils, brain tissue, renal epithelial cells, mast cells and autonomic nerves (review: 37). Our observation that NO and the NO-generating drug, 3 morpholinosyndnonimine (or SIN-l) strongly inhibit osteoclastic bone resorption by isolated osteoclasts is consistent with earlier studies that have shown similar effects of nitro-containing vasodilators on resorption of bone in organic culture (38). The latter compounds have also been found to inhibit parathyroid hormone- and vitamin D3-stimulated bone resorption. However, unlike previous studies, ours has used single isolated cells to demonstrate potent direct effects of NO on osteoclast spreading. On exposure to NO or SIN-l, osteoclasts were shown to undergo marked retraction~ the R effect, without an action on margin ruffling (32). In contrast, other endothelial cell-derived molecules, namely endothelin (33) and prostacyclin (39), selectively inhibit cell margin ruffling, the Q effect. The basis for NO action in most cell types has been attributed to an increase in intracellular cyclic GMP. This is due to the activation by NO, of a soluble guanylate cyclase (37, 40). In contrast, in the osteoclast, the di-esterase-resistant cell-permeant analogues, dibutyryl and 8-bromo cyclic GMP were found not to mimic the effects of NO on osteoclast spreading and bone resorption (32). This makes a role for cyclic GMP in the action of NO on the osteoclast rather unlikely. Similarly, an important role for cyclic AMP has also been excluded on the basis of a lack of effects of forskolin, a cyclic AMP agonist, on cell spreading. Furthermore, NO has been found not to elevate cytosolic [Ca2+], ruling out mediation by intracellular [Ca 2+] (32). NO is indeed known to act through mechanisms not involving cyclic GMP, for example, by activating of cytosolic ADP-ribosyltransferase (41). The autocoid also blocks fibroblast proliferation and mitogenesis (42) and enhances macrophage cytostatic activity by inducing the destruction of iron-containing enzymes. The latter results from intracellular nitrosyl-iron complex formation (43). Superoxide anions ( O 2 ) can react with, and remove, local NO and this may explain the reported action of O f in enhancing fibroblastic proliferation and mitogenesis at low concentrations (44). In case of the osteoclast, certain other alternatives would need to be examined. These include possible actions of NO on substrate adhesion molecules or on the osteoclast cytoskeleton exerted either directly, or via an action on kinases or cellular mechanoproteins, such as kinesin (45). The rapid retraction of osteoclasts in response to NO application, indicating cell detachment or de-adhesion, as observed on time-lapse video recordings (32); is consistent with these possiblities. Several reactive oxygen species are also generated from endothelial cells (46-49). One such species, H202, was found to produce potent stimulatory effects on osteoclast motility and bone resorption at nanomolar concentrations (34). H202 produced bursts of cytoplasmic motile activity resulting in increased numbers of osteoclastic excavations in the bone resorption assay. The size of individual excavations remained unaffected.
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The mechanism of osteoclastic stimulation by H202 is not yet known. One possibility is that basal cyclic AMP formation is reduced. In theory, this should enhance osteoclast motility, as elevated cyclic AMP levels potently inhibit osteoclast motility (50, 51). To our knowledge, there are no reports demonstrating an action of H202 on cyclic AMP levels. Our studies also indicate that H202 does not reduce cytosolic [Ca 2+] in isolated osteoclasts to account for its observed stimulatory effects on bone resorption (34). It would nevertheless seem attractive to examine the role of the multisubunit transcription factor, NF-KB, in H202-induced osteoclastic activation. H202 has been found to activate NF-KB by releasing IKB from its complex with p65 and p50 (52). The latter heterodimeric protein is a DNA-binding factor best characterized in cells of the immune system, including macrophages and monocytes (53). If NF-KB is found in osteoclasts, its modulation by H202, and any relationships to osteoclastic activation, including any interactions with inflamatory cytokines, should be urgently investigated.
A UNIFIED HYPOTHESIS F O R LOCAL OSTEOCLAST C O N T R O L Bone resorption is the result of individually controlled, but successive steps (review: 6). The first step involves the generation and vascular dissemination of osteoclasts. This is followed by osteoclast activation that occurs on contact with mineral. The digestion of bone matrix and dissolUtion of mineral closely follows. Whilst we have a fairly precise knowledge of temporal relationships that culminate in the resorption of bone, we are relatively less aware of events that result in the termination of the resorptive episode. In the following paragraphs, we describe the resorptive event with specific reference to recently discovered potential control mechanisms of the termination and recovery stages (Fig. 1). The relationships described are admittedly hypothetical.
The Pre-Resorptive Stage This immediate pre-resorptive stage results in the formation of a highly specialized microenvironment. A sealing zone of close adhesion forms between the bone-apposed surface of osteoclast and the bone substrate. This stage is dependent critically upon the adhesion of an osteoclast to bone and subsequently, upon its cytoplasmic spreading. Once such a resorptive microenvironment has formed, the osteoclast plasma membrane within it is thrown into complex folds, the 'ruffled border'. The later specialization is the site where most activities essential for bone resorption appear to reside.
The Resorption Stage In this phase, enzymes, protons and free radicals are poured into the resorptive microenvironment. Within this highly acidic microenvironment (pH-3 to 4), hydrolases and cysteine-proteinases digest protein matrix, whilst bone
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Pre-resorptive
- -
.~o~ Ho~
, ~ f ~
Fig. 1. The bone resorptive episode and its local control: a hypothetical scheme based upon new data. Abbreviations: H202hydrogen peroxide; O2---superoxide radicals; HO~--reactive intermediate species.
mineral or hydroxyapatite is dissolved by the action of the secreted acid. In addition, free radicals, mainly 0 2 , are thought to aid the digestion of bone collagen. Factors leading to osteoclast detachment or retraction will inhibit resorption by interfering with the maintenance of the highly acidic resorptive' microenvironment. It is notable that during this stage, the osteoclast generates, and is consequently exposed to high concentrations of ambient Ca 2+. In the resorptive hemivacuole, these levels are estimated to be in the region of between 8 and 40 mM (9).
Tile Termination Stage Completion of the resorptive episode appears to be closely followed by osteoclast detachment (or de-adhesion) and retraction. We suggest that the latter responses are triggered by a change in the ambient [Ca 2+1 that developes locally during resorption. There is compelling evidence that this ambient [Ca 2+] change is monitored by a surface membrane Ca receptor on the osteoclast (see above) (10-20). In vitro adhesion assays (54) and time-lapse studies (14) have revealed that activation of the osteoclast Ca 2+ receptor by elevated ambient [Ca 2+] leads
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to a failure of adhesion and to dramatic pseudopodial and podosomal retraction in attached cells. The cell spread area can reduce by up to 60% at 15raM extracellular [Ca a+] (13). Cell retraction and de-adhesion may thus provide the means whereby ambient [Ca 2+] acutely regulates bone resorption by interfering with the cell-bone seal and by diminshing the overall area of cell contact with the bone. By preventing further bone resorption, this will minimize the build-up of Ca 2+ in the resorptive hemivacuole. Whilst such a mechanism may explain what is likely to be a subtle, minute-to-minute dynamic control, the observed inhibition of secretory activity in response to elevated ambient [Ca 2+] (15) may influence resorption in the longer term and in instances where the local concentration of Ca 2+ becomes very high. Osteoclastic retraction may also result from an increased exposure to NO produced locally (Fig. 2). It is not known however, as to whether such an effect can be exerted from within the resorptive hemivacuole. If NO were to be synthesized in the osteoclast and secreted together with O~-, then an interesting set of reactions could take place. O~- may react with NO to yield the peroxynitrite anion, O N O O - , which upon further protonation would form peroxynitrous acid, O N O O H . Upon homolysis, peroxynitrous acid would produce further radicals (55, 56). Whether such reactive oxygen species have a role in osteoclast regulation is presently unknown.
~12" ~
..~-N0 F'-(
OONO'-I~OONOH-I~NO2+HO']
~*
L02- H202..,,,~
Fig. 2. A detailed hypothetical scheme of local osteoclast control. The reactive oxygen species, HaO 2 is likely to provide an excitatory signal following the resorptive episode, and by inducing increased osteoclast motility, would allow the cell to move away and resorb at a different location. Abbreviations: N O - n i t r i c oxide; H202--hydrogen peroxide; O2---superoxide radicals; O N O O --peroxynitrite radical; ONOOH-peroxynitrous acid; HO--hydroxyl radical; HO~--reactive intermediate species; CT---calcitonin; ET--endothelin; P T H - parathyroid hormone; Vit D--vitamin D3; PG--prostag|adin; GF--growth factors; Cyto--cytokines; PGI2--prostacyclin; oc-osteoclast; ob--osteoblast; ec--endothelial cell.
Local OsteoclastControl
377 The Recovery Stage
It is notable that whilst osteoclasts undergo actue retraction in response to elevated extracellular [Ca2+], their pseudopodial motile activities appear not to cease (57). The cells therefore retain the ability to move away from the resorption site and to resorb at a different location. This process could be aided by the local production of the stimulatory reactive oxygen species, H202 (Fig. 2). This may ensue from the ambient pH change caused by the disruption of the osteoclastbone seal in response to an elevated extracellular [Ca2+]. A pH change will allow the O2- secreted locally by the osteoclast (58-60) (and by other cells) to undergo initial protonation to form the reactive intermediate radical species, HO2, and upon further protonation, to generate H202. Production of the longer-lived membrane-permeant species, H202 may at this point, provide an appropriate excitatory signal that could reverse the acute osteoclastic inhibition resulting from an exposure to elevated ambient [Ca 2§ (10, 11, 15). Enhanced motility induced by H202 would thus allow an osteoclast to move away from the lacuna and resorb elsewhere. This hypothesis would be consistent with our in vitro studies showing marked effects of H202 on the motility of isolated osteoclasts (34). Relevance to Bone and Joint Disease The potent stimulatory effects of H202 on bone resorption appear to have direct implications in bone and joint pathology. The generation of rective oxygen species may be particularly important in the bone resorption that occurs in association with inflammatory diseases, such as in rheumatoid arthritis. For example, H202 is released into chronically inflamed joint cavities by activated synovial neutrophils or as a result of hypoxic-reperfusion injury to the endothelium, generated by intermittent pressure changes in the mobile and inflamed joint. Calculated H202 production from activated neutrophils may often be at rates in excess of 500 nmol/min (61, 62); these concentrations clearly would be in line with those required for bone destruction. An efficient source of reactive oxygen species during episodes of hypoxicreperfusion injury appears to be the enzyme, xanthine oxidase. The latter enzyme has been localized to both normal and rheumatoid synovia (63) and to the endothelium (64). The generation of reactive oxygen species from synovial tissue has also been confirmed by electron spin resonance spectroscopy. In addition, free radical generation from synovial tissue is reduced by inhibitors of xanthine oxidase, implicating the enzyme as being their major source (65). Clearly, the inhibition of osteoclastic activity is a major therapeutic aim in osteolytic diseases of the bone and joints. Two major areas of drug development could follow, one using a strategy to inhibit the production and action of reactive species, and another using agents that could accomplish the release of NO at sites of excessive bone resorption. Drugs that inhibit reactive oxygen species and their production have been shown to be active inhibitors of bone resorption in model systems (58). It may be difficult to succeed with the second strategy, as marked hypotension is known to result from the systemic administration of osteoclast-
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inhibitory doses of drugs, such as SIN-1 (32). However, a promising approach that clearly deserves study, is the development of novel NO-generating agents that could be targetted to sites of potential bone destruction. Finally, the generation of reactive oxygen species has also been implicated in the ageing process, particularly in relation to neurological, vascular and dermal changes (66). It would be interesting to determine whether radical-induced changes could also relate to senescent bone cells and hence, for example, to the development of osteoporosis.
ACKNOWLEDGEMENTS The work described above was supported, in part, by grants from The Arthritis and Rheumatism Council, U.K. (Project Grant Z1 and Z2 to M.Z. and Programme and Project Grants to D.R.B.), The Medical Res. Council, U.K. (to M.Z.), The Leverhulme Trust, U.K. (to M.Z.), Sandoz Foundation for Gerontological Res., Basel, Switzerland (to M.Z.) and The Royal Society (to C.L.-H.). A.S.M.T.A. is a recipient of the Overseas Res. Scholars Award from the Council of Vice Chancellors and Principals of the Universities of the United Kingdom. The authors are grateful to Mr. W. Smith (Cambridge) for skilled assistance. REFERENCES 1. Zaidi, M., Breimer, L. H. and MacIntyre, I. (1987) Quart. J. Exp. Physiol. 72:371-408. 2. Breimer, L. H., MacIntyre, I. and Zaidi, M. (1988) Biochem. J. 255:377-390. 3. Zaidi, M., Moonga, B. S., Bevis, P. J. R., Bascal, Z. A. and Breimer, L. H. (1990) Crit. Rev. Clin. Lab. Sci. 28:109-174. 4. Zaidi, M., Moonga, B. S., Bevis, P. J. R., Alam, A. S. M. T., Legon, S., Wimalawansa, S. J., MacIntyre, I., Breimer, L. H. (1991) In: Vitamins and Hormones (Volume 46) (Ed. G. D. Aurbach) New York, Academic Press. pp. 87-164. 5. Chambers, T. J. (1988) Ciba Foundation Symposium 136:92-107. 6. Vaes, G. (1988) Clin. Orthop. 231:239-271. 7. Roodman, G. D. (1991) Crit. Rev. Oral Biol. and Med. 2:389-409. 8. Zaidi, M., Alam, A. S. M. T., Shankar, V. S., Bax, B. E., Bax, C. M. R., Moonga, B. S., Bevis, P. J. R., Stevens, C., Blake, D. R. and Huang, C. L.-H. (1992) Biol. Rev. (ln press). 9. Silver, I. A., Murrills, R. J. and Etherington, D. J. (1988) Exp. Cell Res. 175:266-276. 10. Zaidi, M., Datta, H. K., Patchell, A., Moonga, B. S. and MacIntyre, I. (1989) Biochem. Biophys. Res. Commun. 163:1461-1465. 11. Malgaroli, A., Meldolesi, J., Zambonin-Zallone, A. and Teti, A. (1989) J. Biol. Chem. 264:14342-14347. 12. Donahue, H. J., Ijima, K., Goligorsky, M. S., Rubin, C. T. and Rifkin, B. R. (1992) J. Bone Min. Res. (In press). 13. Zaidi, M. (1990)Biosci. Rep. 10:493-507. 14. Datta, H. K., MacIntyre, I. and Zaidi, M. (1989) Biosci. Rep. 9:247-251. 15. Moonga, B. S., Moss, D. W., Patchell, A. and Zaidi, M. (1990) J. Physiol. 429:29-45. 16. Miyauchi, A., Hruska, K. A., Greenfield, E. M., Duncan, R., Alvarez, J., Barattolo, R., Colucci, S., Zambonin-Zallone, A., Teitelbaum, A. L. and Teti, A. (1990) J. Cell Biol. 111: 2543-2552. 17. Zaidi, M., Kerby, J., Huang, C. L.-H., Alam, A. S. M. T., Rathod, H., Chambers, T. J. and Moonga, B. S. (1991) J. Cell. Physiol. 149:422-427.
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