MICROSCOPY RESEARCH AND TECHNIQUE 33:165-170 (1996) Role of Microscopy in Elucidating the Mechanism and Regulation of the Osteoclast Resorptive Apparatus CAROL V . GAY Department of Biochemistry and Molecular Biology and Department of Poultry Science, The Pennsylvania State University, University Park, Pennsylvania 16802 KEY WORDS Osteoclasts, Bone resorption, Carbonic anhydrase, Proton-translocating ATPase INTRODUCTION Microscopy has spearheaded investigations into understanding cell function in bone, perhaps more than for any other tissue. The reason is that bone cells lie within or on the extracellular substance of bone, a material not unlike reinforced concrete. It is the purpose of this paper' to survey the role microscopy has played in elucidating osteoclast function. Osteoclasts were identified nearly a century and a half ago by microscopists (Kolliker, 1873; Robin, 1849). These early morphologists correctly interpreted that osteoclasts are bone resorbing cells. This conclusion was based on the crumbling appearance of bone beneath osteoclasts and the fact that pieces of bone appeared to be ingested by the cells. Modern scanning electron microscope studies have made it imminently clear that osteoclasts degrade bone by making resorption pits, whereas other cells such as monocytes, macrophages, and certain tumor cells that resorb devitalized bone do not make resorption pits in vivo (Jones et al., 1985). As microscopic techniques such as immunocytochemistry, histochemical reaction capture methods, autoradiography, and in situ hybridization methods were developed, insight into the cellular basis of bone resorption has progressed steadily. The task of an osteoclast is defined by the composition of extracellular bone matrix, which is mainly hydroxyapatite crystals packed into parallel arrays of a fibrous protein, type I collagen. Two eroding systems, then, are needed: a way to dissolve mineral and a means of degrading protein. The former requirement is now well established as an acid-secreting mechanism that involves carbonic anhydrase and a vacuolar-type H+-ATPase; the latter is due to proteolytic enzymes. The carbonic anhydrases are central to COOmetabolism, and the range of tissues served by this family of enzymes is consequently widespread. That many investigators have become intrigued by carbonic anhydrase is evident from the recent exhaustive publication of Dodgson et al. (1991). Not only is carbonic anhydrase notable for its rapid turnover rate, but it also subserves an array of physiological processes by epithelia. These processes include gas exchange a t the blood lung interface, elaboration of a bicarbonate rich fluid by tissues such as pancreas and salivary glands, and acidification of luminal fluids by the proximal renal tubule and the gastric mucosa. A number of isoforms have been identified, such as one for muscle cells and another for mitochondria. The important carbonic anhydrase isoform in osteoclasts is CAII. This has been shown by immunocytochemistry for chicken (Gay et al., 1974), rat (Vaananen and Parvinen, 1983), and human (Vaananen, 1984) osteoclasts. A CAI, deficiency syndrome in humans is associated with a mild form of osteopetrosis (Sly et al., 19831, a finding that suggests a role for carbonic anhydrase in osteoclastic bone resorption. Messenger RNA for CAI1 has been localized by in situ hybridization, a molecular tool that exploits the power of microscopy to capture space relationships, in osteoclast-like cells derived from an osteosarcoma (Zheng et al., 1993) as well as in neonate rat osteoclasts (Laitala and Vaananen, 1993). Both studies found good corroboration of in situ hybridization with Northern analysis. The latter study also showed that active osteoclasts expressed substantial levels of CAI1 mRNA, whereas nonresorbing osteoclasts expressed very little mRNA. Transcription of CAI1 mRNA has been shown to be regulated by 1,25-dihydroxyvitamin D, in osteoclast precursor cells (Billecocq et al., 1990; Lomri and Baron, MECHANISM OF MINERAL DISSOLUTION 1992). Localization and Role of Carbonic Anhydrase The carbonic anhydrase isoform CAVhas been localCarbonic anhydrase reversibly hydrates COz to form ized in the matrix of osteoclast, renal, and gastric micarbonic acid. Definitive identification of carbonic an- tochondria by ultrastructural immunocytochemistry, hydrase in osteoclasts came from imrnunocytochemi- but the role of CAv is not currently understood (Karcal, autoradiographic, and histochemical microscopic hukorpi et al., 1992). Much of CAI1 is distributed throughout the cytoanalyses. Using these methods, carbonic anhydrase has been localized in osteoclast cytoplasm of chickens plasm in osteoclasts. The role of cytosolic carbonic an(Anderson et al., 1982; Gay and Mueller, 1974), rats (Marie and Hott, 1987; Simasaki and Yagi, 1960; Sundquist et al., 1987; Vaananen and Parvinen, 1983)), mice (Jilka et al., 19851, and humans (VaanaReceived Januarv 3. 1994: accepted August 18. 1994. nen, 1984).Its role in osteoclasts has been reviewed by Address reprint requests to Carol V. Gay, 468A N. Frear Building, University Gay (1991, 1992). Park. PA 16802. 0 1996 WILEY-LISS, INC. 166 C.V.GAY hydrase is not completely understood; however, a potential role is to facilitate diffusion of CO, from the cell interior to the cell surface, as described in model systems by Enns (1967) and Shultz (1980).Given the enormity of osteoclasts, which can have a volume 50 times or more than other cells, facilitated diffusion is likely to be involved. It was observed by ultrastructural immunocytochemistry that carbonic anhydrase in osteoclasts is, under some circumstances, associated with the inner surface of the ruffled border (Anderson et al., 1982; Cao and Gay, 1985). Membrane-associated carbonic anhydrase has also been reported in a giant cell tumor of bone (Kuwahara et al., 1986). The observations of membrane-associated carbonic anhydrase led us to postulate that one role of carbonic anhydrase is to provide hydrogen ions to a proton-pumping ATPase in the ruffled border of osteoclasts. Localization of the Proton-Translocating ATPase One way to localize an enzyme is by capturing histochemical reaction products. In 1985, a number of methods existed for localizing Na+,K+-ATPases and Mf+ +,Ca++-ATPases, however, none existed for an H -ATPase at the ultrastructural level. Using the existing histochemical methods for ATPases as models, we devised a way to identify sites of H+-ATPase activity (Akisaka and Gay, 1986). The reaction depended on the presence of ATP and Mg++ a t high pH. Released phosphates were trapped in the conventional manner by lead ions. Staining, which was observed along the ruffled border and in cytoplasmic vesicles, was blocked by proton-pumping ATPase inhibitors p-chloromercuribenzoate, tri-n-butyltin, and duramycin. Meanwhile, by use of an antibody to gastric H+,K+-ATPase immunostaining of the ruff led border was demonstrated (Baron et al., 1985). In 1989, antibodies to bovine kidney vacuolar H+-ATPase were found t o bind specifically to the ruffled border (Blair et al., 1989). In addition, Vaananen et al. (1990) used antibodies to Neurospora crassa vacuolar ATPase and found ruffled borders of both rat and chicken osteoclasts to be immunostained. It is now known that the osteoclast H+-ATPase is not of the gastric type. Presumably, epitopes in common to both types of ATPase antibodies (gastric and vacuolar) recognized the antigen. Thus, microscopic methods, both histochemical and immunochemical, have revealed sites of a proton ATPase in the ruffled border where it was hypothesized to be. An intense investigation to demonstrate function of the proton ATPase has followed. These studies have made it clear that pumping of protons to the exterior of osteoclasts is driven by a vacuolar type pump (Bekker and Gay, 1990; Blair et al., 1989, 1991; Chatterjee et al., 1992; Vaananen et al., 1990). Regulation of expression of the H+-ATPase was the next logical quest, and here microscopic methods also played a role. In situ hybridization has allowed localization of mRNA transcripts for the vacuolar H+-ATPase in isolated osteoclasts from neonate rats (Laitala and Vaananen, 1993). Additionally, substantial levels of mRNA were present in the cytoplasm. Northern analysis revealed high levels of transcript in active osteoclasts and markedly re- Fig. 1. Diagram summarizing the mechanisms of osteoclast acidification and their potential regulation by parathyroid hormone (PTH), calcitonin (CT), and 17P-estradiol (EJ. Acidification depends on carbonic anhydrase (CA) activity and a proton-translocating ATPase and is enhanced ( + ) by ETH and diminished (-) by CT and E,. At least one signaling pathway utilized by PTH appears to involve CAMP.See text for discussion and references. duced levels in inactive cells. Amplified expression of the H+-ATPase has been shown in late stage osteoclasts precursors (Kurihara et al., 1990; Wang et al., 1993). Insights Into Osteoclast Acidification Using Fluorescence Microscopy A microscope photometer and a pH-sensitive dye, acridine orange, have been used to monitor changes in acid levels in single living osteoclasts. Acridine orange behaves as a weak base that diffuses through intact cell membranes but is sequestered when protonated in acidic compartments (Moriyama et al., 1982). Thus, increased levels of fluorescence are proportional to trapped protons. Cells were first neutralized with 20 mM ammonium chloride added to culture medium; then, by removal of the neutralizing solution, acidification was reinitiated (Hunter et al., 1988, 1991). The sites of trapped acid appeared to be within intracellular vesicles. It is believed that the vesicles fuse with the ruffled border membrane, releasing both acid and proteolytic enzymes into the resorption lacuna. This view is supported by electron microscopic observations that have revealed many vesicles in inactive osteoclasts but few in active cells (for reviews see Gay, 1992; Holtrop, 1991). Further, a unique histochemical marker, an alkaline p-nitrophenyl phosphatase, has been found to stain both vesicles and ruff led border membrane but not other membranes of the osteoclast (Fukushima et al., 1991). The fusion of vesicles with the ruffled border membrane is believed to be the means of positioning the proton pump in the ruffled border (Fig. 1). Table 1 summarizes and Figure 1 diagrams the ef- MICROSCOPY AND OSTEOCLAST FUNCTION TABLE 1 . Detection of changes in protonated acridine omnge by fluorescence microscopy of isolated osteoclasts treated with regulatory substances' Treatment A. Calcitonin (7 x lo-" M) Acetazolamide M) HTS (10-9 MI B. PTH (lo-' M) DbcAMP M) PTH (lo-' M) PTH + alloxan (lo-' M) Cholera toxin (0.01 &g/ml) C. Estradiol (2 x lo-'' M) Diethylstilbestrol ( x 10-lOM) Estradiol (2 x lo-' M) + PTH M) Estradiol (2 x lo-* M) + DbcAMP M) Estradiol (2 x lo-' M) + alloxan (lo-' M) Effects on acidification' (% change t s.d.) 1 (44 f 4) 1 (30 f 3) 1 (48 t 6) t (24 f 4) t (45 ? 4) t (27 f 5) 1 (20 f 5) t (38 * 6) .1 (41 f 3) .1 (39 f 2) Reference Gay et al. (1993) Hunter et al. (1988) Hunter et al. (1991) Gay et al. (1993) May et al. (1993) Gay et al. (1993) 1 (27 f 3) 1 (45 * 2) 1 (47 t 3) 'Abbreviations: DbcAMP, dibutyryl cyclic adenosine monophosphate;HTS, 543hydroxybenzoyllthiophene-2-sulfonamide;FTH, parathyroid hormone. 'Based on single cell values averaged for 20 cells per data point at 1 h treatment times. fects of some regulatory agents on osteoclast acid production. Both calcitonin and the carbonic anhydrase inhibitors, acetazolamide and hydroxythiophene sulfonamide, blocked reacidification of acridine orange stained osteoclasts (Table 1A). These results substantiate that carbonic anhydrase plays an integral role in the acidification mechanism. Furthermore, since calcitonin when injected into chickens reduced the association of carbonic anhydrase along the inner face of the ruffled border (Anderson et al., 19821,it is possible that a role of calcitonin may be to control the supply of hydrogen ions to the proton pump by altering the position of carbonic anhydrase. However, the physiological role of calcitonin is unclear. Its effects in humans appear to be transient and of little use in controlling trabecular bone volume or calcium homeostasis (Mundy, 1990). Parathyroid hormone (PTH) was found to enhance acidification (Table 1B). Dibutyryl cAMP mimicked the PTH effect, implying that the PTH response is CAMP-dependent. Inhibition of CAMP generation by alloxan, an inhibitor of adenylate cyclase (Cohen and Bitensky, 19691, blocked the PTH response. The involvement of a G-protein of the Gs type, whose major role is adenylate cyclase stimulation, was shown by the use of cholera toxin, a substance which permanently activates the a-subunit of Gs-proteins. In addition, pertussis toxin which prevents receptor interaction with G-proteins other than Gs-protein blocked the PTH stimulatory effect on acid production (May and Gay, 1993). The implication of this is that PTH interacts with more than one type of G-protein. These results suggest that PTH regulates osteoclast acidification through at least two signaling pathways. However, these inhibitor studies require confirmation by other approaches due to the potential pitfalls associated with 167 the use of inhibitors on living cells. In an earlier study, PTH was found to stimulate CAMP levels in isolated osteoclasts (It0 et al., 1985). Carbonic anhydrase has been identified as an effector molecule for the PTHcAMP signaling pathway (Dietsch, 1987; Silverton et al., 1987). It is possible that these actions of PTH occur as a result of direct activation on osteoclasts. The isolated osteoclasts are relatively pure preparations and typically include less than 10% of other cell types, only 2% of which are alkaline phosphatase positive (Gay et al., 1983,1993). Secondly, PTH has been shown to bind specifically to osteoclasts, as will be discussed in the next section. However, proof of direct activation of osteoclasts by FTH awaits the demonstration of receptors at protein and mRNA levels. Also, as shown in Table lC, 17P-estradiol and diethylstilbestrol were found to slow reacidification of osteoclasts. The stimulatory effects of PTH and its second messenger cAMP were overridden by treatment with estrogen. This implies that the action of the estrogens is to inhibit a step in the signal transduction pathway at a later point than cAMP production. By the use of culture methods, a fluorescent probe, and a photometer integrated into a microscope it has been possible to gain insight into one function of individual living osteoclasts: the process of acidification. It should be pointed out that the use of photometry lacks the precision that can be obtained by the use of instrumentation capable of processing ratiometric images. The use of acridine orange to show acidification in lysosomes, as well as its several other uses, is reviewed by Zelenin (1993). Detection of Specific Binding of Calciotrophic Hormones to Osteoclast Surfaces Parathyroid Hormone. Results obtained from studying the effects of parathyroid hormone on acidification by osteoclasts in relatively pure cultures of osteoclasts (Table 1A) led us to devise a way to test whether or not parathyroid hormone could bind specifically to osteoclasts (Agarwala and Gay, 1992). Osteoclasts were isolated from 2-3-week-old chick tibia and maintained in culture 4-6 days. A technique which had been devised to show specific PTH binding to kidney cell surfaces (Niendorf et al., 1988) was employed. Biotinylated bovine PTH was applied to the cultured osteoclasts for 2-20 min. After rinsing away unbound biotinylated PTH, avidin labeled with fluorescein isothiocyanate was applied and the excess removed by rinsing. Bright spots of fluorescence were observed on the osteoclast surfaces. The surface binding displayed the properties of receptor-dependent binding, namely saturability, time dependence, temperature dependence, and competition with unlabeled PTH. In the same study, osteoblasts were found to be intensely stained, in accordance with other studies. PTH binding to osteoclasts has also been shown by well-controlled immunocytochemistry in sections of rat bone (Rao et al., 1983) and 1251-PTHlabeling of isolated avian osteoclasts by autoradiography (Teti et al., 1991). The amount of PTH that binds to osteoclasts is substantially less than that which binds to osteoblasts (Agarwala and Gay, 1992). Further, osteoclast surfaces 168 C.V. GAY are rapidly cleared of bound PTH (within 20 min). These characteristics may contribute to the difficulty in showing the direct involvement of PTH with osteoclasts. The demonstration of specifially bound PTH to osteoclasts supports the view that osteoclasts are directly regulated by PTH, in addition to indirect regulation through the action of PTH on osteoblasts. However, the magnitude of both direct and indirect effects of PTH on osteoclasts has not been assessed. Calcitonin. The binding of radiolabeled calcitonin to osteoclasts was first demonstrated in rat bone by Warshawsky et al. (1980), who used autoradiography at the ultrastructural level. Rao and colleagues (1981) have shown binding of endogenous calcitonin to osteoclasts in sections of rat bone using antibodies to calcitonin. Application of radiolabeled calcitonin to isolated osteoclasts followed by autoradiography has shown substantial binding of calcitonin to rat osteoclasts, while binding to avian osteoclasts was undetectable (Nicholson et al., 1986, 1987). This has led to the view that avian and mammalian osteoclasts are regulated by substantially different processes. However, it is well documented that avian osteoclasts respond to calcitonin in low doses (for a review, see Gay, 19881, providing evidence for the presence of calcitonin receptors in avian osteoclasts. Apparently, rat osteoclasts have substantially more calcitonin receptors than the avian cells. By using the biotinylated hormone technique of Niendorf et al., 1988 and Agarwala and Gay, 1992, specific binding of calcitonin to avian osteoclasts has now been shown (Hall et al., 1994). Living osteoclasts were exposed to biotinylated calcitonin at room temperature. The cells were then chilled to disrupt microtubules and prevent possible uptake of the labeled hormone. Bound biotinylated hormone was made visible by using fluorescein-tagged avidin. In order to amplify the signal, a biotinylated antibody to avidin was applied, followed by a second application of fluorescein tagged avidin. Characteristics of receptor binding were shown: saturability, time dependence, competition with unlabeled calcitonin and lack of competition by unrelated peptides. Clearance from the cell surface occurred in 10 min, even more rapidly than found for PTH. Because avian osteoclasts resorb bone more aggressively than osteoclasts derived from other species (Jones et al., 1986), it is reasonable to speculate that the avian cell is more tightly regulated and utilizes fewer receptors. Estradiol. Nuclear estrogen receptors have been described in osteoblasts (Turner et al., 19931, osteoblast-like cells (Eriksen et al., 1988; Komm et al., 19881, and osteoclasts (Oursler et al., 1991). However, the response of osteoclasts to 17P-estradiol in the acidine orange assay used to monitor acidification (Table 1C) was detectable within 10-15 min. The rapidity of this response suggested that estrogen may also be acting at a nonnuclear site. Further, proton pumping by a vesicle preparation of osteoclast plasma membranes was inhibited by 17p-estradiol at 2 x lop8 M (Gay et al., 1993). In order to further evaluate possible interaction of estrogen a t plasma membrane, a complex of 17P-estradiol conjugated to bovine serum albumin-fluorescein isothiocyanate has been applied t o osteoclast surfaces (Brubaker and Gay, 1994). Binding was observed with fluorescence microscopy; it was dose- and time-dependent and could be blocked with 17P-estradiol or tamoxifen, an estrogen antagonist. Both 17P-estradiol and the 17p-estradiol-BSA-FITC complex stimulated cell shape changes within minutes of application, further documenting rapid responses that point to the existence of nonnuclear receptors. How the estrogens affect osteoclasts in vivo is unclear. For example, they have failed to directly influence bone in organ cultures (Caputo et al., 1975). On the other hand, the importance of estrogens in the reduction of bone loss in the immediate postmenopausal period is well known (Lindsay et al., 1976, 1980). MECHANISM OF ORGANIC MATRIX DEGRADATION Lysosomal proteinases are secreted into the resorption pit in a manner that is regulated by parathyroid hormone, calcitonin, and other hormones (see review by Delaissb and Vaes [19921).The most important proteinases appear to be cathepsin B and L, members of the cysteine proteinase family, which can ef€iciently degrade collagen a t an acidic pH. Most studies on matrix degradation have involved biochemical analyses. However, the spatial precision afforded by microscopy has shown that osteoclasts synthesize B, L and D cathepsins on the basis of immunolocalization and that the B and L forms are secreted, whereas D cathepsin remains as an intracellular enzyme (Goto et al., 1992,1993).An ultrastructural study has shown that matrix is not destroyed when bone explants have been cultured in the presence of the cysteine proteinase inhibitor, leupeptin (Everts et al., 1988). The question of the presence and secretion of procollagenase by osteoclasts is not completely resolved. The major source of collagenase in bone is osteoblasts (Vaes et al., 19921, although immunoreactive collagenase in osteoclasts has not been reported (Delaisse and Vaes, 1992), suggesting that collagenase found in resorption lacunae may be secreted by osteoclasts. Further support for this view is provided by the detection of collagenase mRNA by in situ hybridization in odontoclasts (Okamura et al., 1993). The role of collagenase in resorption pits provides an intriguing problem since collagenase is inactive at a low pH. An attractive hypothesis presented by Delaisse and Vaes (1992) poses the possibility that collagenase is activated in regions of the pit that have been neutralized by the HPO,' released as hydroxyapatite is dissolved. SUMMARY Microscopic studies have assisted in revealing some of the components of the resorptive apparatus of osteoclasts, specifically carbonic anhydrase and the protontranslocating ATPase. Further, microscopy has helped substantiate the types of proteolytic enzymes secreted into the resorption lacuna. Regulatory agents affecting the resorptive process in vitro include parathyroid hormone, 17P-estradio1,calcitonin, and 1,25-dihydroxyvitamin D,. Studies showing the specific binding of parathyroid hormone, estradiol, and calcitonin to osteoclast MICROSCOPY AND OSTEOCLAST FUNCTION plasma membrane are discussed. While specific binding suggests that direct effects may occur, further investigation is needed to substantiate this possibility. ACKNOWLEDGMENTS The osteoclast acidification studies were supported by NIH grant DE 04345. REFERENCES Aganvala, N., and Gay, C.V. (1992) Specific binding of parathyroid hormone to living osteoclasts. J. Bone Miner. Res., 7:531-539. Akisaka, T., and Gay, C.V. (1986) Ultracytochemical evidence for a proton-pump adenosine triphosphatase in chick osteoclasts. Cell Tissue Res., 245507-512, Anderson, R.E., Schraer, H., and Gay, C.V. (1982) Ultrastructural immunocytochemical localization of carbonic anhydrase in normal and calcitonin-treated chick osteoclasts. Anat. Rec., 204:9-20. Baron, R., Neff, L., Louvard, D., and Courtoy, P.J. (1985) Cell-mediated extracellular acidification and bone resorption: Evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border. J. Cell Biol., 101:2210-2222. 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