ATPase pumps in osteoclasts and osteoblasts

The International Journal of Biochemistry & Cell Biology 34 (2002) 459–476

Review

ATPase pumps in osteoclasts and osteoblasts Martin J.O. Francis a , Rita L. Lees b , Elisa Trujillo c , Pablo Mart´ın-Vasallo c , Johan N.M. Heersche b , Ali Mobasheri d,∗ a

d

Nuffield Department of Orthopaedic Surgery, Nuffield Orthopaedic Centre, University of Oxford, Oxford OX3 7LD, UK b Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5G 1G6 c Departamento de Bioqu´ımica y Biolog´ıa Molecular, Universidad de La Laguna, La Laguna 38206, Tenerife, Spain Department of Veterinary Preclinical Sciences, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ, UK Received 16 March 2000; accepted 4 October 2001

Abstract Osteoblasts, osteocytes and osteoclasts are specialised cells of bone that play crucial roles in the formation, maintenance and resorption of bone matrix. Bone formation and resorption critically depend on optimal intracellular calcium and phosphate homeostasis and on the expression and activity of plasma membrane transport systems in all three cell types. Osteotropic agents, mechanical stimulation and intracellular pH are important parameters that determine the fate of bone matrix and influence the activity, expression, regulation and cell surface abundance of plasma membrane transport systems. In this paper the role of ATPase pumps is reviewed in the context of their expression in bone cells, their contribution to ion homeostasis and their relation to other transport systems regulating bone turnover. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Na,K-ATPase; Ca-ATPase; Gastric H,K-ATPase; Vacuolar H-ATPase; Osteoclast; Osteoblast; Osteocyte; Bone

1. Introduction The transport of ions and molecules across cell membranes is a fundamental property of all cells [1,2]. Here we review recent research on ATPase ion pumps in bone forming (osteoblast) and bone resorbing (osteoclast) cells and compare the transport machinery these cells utilise to achieve their physiological and functional objectives with that of cell types, such as epithelial cells, for which more complete data is available. Transport proteins embedded in the plasma ∗ Corresponding author. Tel.: +44-151-794-4284; fax: +44-151-794-4243; URL: http://www.pcweb.liv.ac.uk/vets/ research/connective/staff.htm E-mail address: [email protected] (A. Mobasheri).

membrane regulate cell shape [3], volume [4,5], intracellular pH [6,7], the membrane potential [8–10], [11] and transepithelial transport [12]. The last 20 years have seen many advances in the molecular characterisation of ATPase ion pumps from the central and peripheral nervous systems, cardiac and skeletal muscle and several epithelia, all tissues in which ion transport systems dominate cellular activities [2,13–15]. An important reason for these advances has been the relative simplicity with which these tissues can be manipulated. In contrast, calcified tissues are difficult to manipulate in the laboratory and obtaining homogeneous cell populations in sufficiently large quantities has been challenging [16]. Consequently calcified tissues remained relatively unexplored until methods for the isolation and culture of bone cells

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became established [17,18]. Nevertheless, information on ion pumps, transporters and channels in bone cells remains limited compared to other tissues. Plasma membrane transport systems play a significant role in the homeostasis of musculoskeletal cells [19] and are the targets of many synthetic and naturally occurring compounds [20–22]. In structurally or metabolically compromised connective and calcified tissues, as in other tissues, membrane transporters may prove to be appropriate targets for the modulation of the metabolic activities of the resident cells. Transport systems are involved in the provision of essential nutrients, maintenance of intracellular pH and regulation of cell volume and are therefore potential targets for the treatment of multifactorial diseases such as osteoarthritis and osteoporosis. Their study should be part of multidisciplinary approaches to the treatment of these cartilage and bone disorders [23,24]. Metabolic acidosis brought about by a decrease in the concentration of serum bicarbonate leads to increased calcium excretion and a net negative calcium balance [25]. In bone, metabolic acidosis disturbs the equilibrium between bone formation and resorption; acidity inhibits bone mineralisation by osteoblasts and promotes bone resorption by osteoclasts [26,27]. Acidosis combined with age-related hormonal changes or metabolic hormonal imbalance brought about by

genetic, dietary or environmental factors results in the loss of bone mineral and consequent osteoporosis. Although acid regulating transport systems have already been selected for preliminary evaluation as therapeutic targets, these are not ideal targets as such systems are generally expressed in vital organs [28]. Knowledge of other related transport systems in polarised bone cells has therefore become essential for the discovery of realistic therapeutic strategies for the treatment of bone disease.

2. ATPase pumps in osteoclast biology Osteoclasts are large, multinucleated, highly motile and specialised cells of hemopoietic origin that resorb bone (Figs. 1 and 2; [29,30]). They are usually found in contact with the bone surface and within a lacuna that is the product of their resorptive activity [31]. When active, they are polarised cells with several distinct plasma membrane domains: an area of extensive membrane folding in apposition to the bone where bone resorption takes place (the ruffled border) [32,33], a microfilament-rich, organelle-free area that surrounds the ruffled border and serves as the point of attachment of the osteoclast to the underlying bone matrix (clear zone) [32,34,35], and a basolateral

Fig. 1. The osteoclast is a progeny of the hemopoietic stem cell capable of giving rise to erythrocytes, platelets and various leucocytes.

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Fig. 2. Transport mechanisms involved in bone matrix acidification and ion homeostasis in osteoclasts. Several transport systems in the osteoclast are polarised. These include the proton ATPase, chloride channels in the ruffled (apical) membrane and chloride bicarbonate band-3 like anion exchangers in the basolateral membrane. Voltage activated calcium channels are present in these cells in addition to potassium channels. Glucose transporters in the basolateral membrane are responsible for glucose uptake, which provides the substrate for glycolysis and structural components required for glycosylation reactions in the Golgi apparatus. Na/H exchanger, Cl/HCO3 exchanger and H,K-ATPase are responsible for maintenance of intracellular pH. The Cl/HCO3 exchanger is also responsible for uptake of chloride, which together with protons are secreted by chloride channels and vacuolar H-ATPase in the apical membrane. The Ca-ATPase has a polar distribution in osteoclasts and is present only on the basolateral membrane and not the ruffled border or clear zone. Na,K-ATPase is polarised to the basolateral domain of osteoclasts with nominal presence also on the ruffled border and clear zone membranes.

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membrane opposite the ruffled border with a central part where the degraded bone matrix components are released extracellularly after being transcytosed through the cell [36,37]. In order to decalcify bone and degrade the organic extracellular matrix, osteoclasts must secrete 1–2 protons for every calcium ion liberated [38]. This acid secretion and subsequent transport of free calcium requires the functional, biochemical and morphological polarisation of a large number of plasma membrane and intracellular transport systems (Fig. 2). These include the electrogenic H-ATPase, a highly conductive chloride channel, chloride bicarbonate exchangers, carbonic anhydrase and accessory pumps; these serve to maintain the overall intra and extracellular ionic milieu [31]. In this context the osteoclast may be considered a unicellular proton and calcium transporting epithelium [39]. The presence of numerous mitochondria, lysosomal vesicles and Golgi complexes suggest high biosynthetic and metabolic activities in osteoclasts [40]. Since these processes require energy in the form of ATP, early studies concentrated on the investigation of ATPase activity of osteoclasts in this context. Severson et al. [41,42] histochemically defined a Mg-dependent

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and N-ethylmaleimide (NEM)-sensitive ATPase associated with mitochondrial-like structures in mouse osteoclasts, but in these early studies there was no evidence for a plasma membrane ATPase. In contrast, Gothlin and Ericsson [43] demonstrated ATPase activity in the ruffled border region of osteoclasts, which they hypothesised, might be the same type as within lysosomes. Currently, a variety of ATPases have been located in distinct plasma membrane domains of the osteoclast. These include Na,K-ATPase, gastric H,K-ATPase, vacuolar V-ATPase and CaATPase. There is no information on the non-gastric H,K-ATPase in bone cells. The information presented in Table 1 summarises our current understanding of four ATPase systems in osteoclasts and osteoblasts and includes information about their cellular localisation, isoform composition, pharmacology and physiological significance.

3. ATPase pumps in osteoblast biology ATPase pumps are hypothesised to have physiological functions in osteoblasts and in particular in the control of mineralisation. However, little distinction

Fig. 3. (A) The osteoblast is derived from multipotential mesenchymal stem cells capable of differentiating into fibroblasts, chondroblasts, adipocytes, myoblasts and osteoblasts and hence give rise to cartilage and bone (B); the osteoblast is responsible for depositing a matrix of collagen type I which is subsequently calcified to form bone (C).

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has been made as to whether the cells studied were of osteoblastic or osteocytic phenotype. Hence, much of the osteoblast literature reviewed here may refer to the osteocyte. Osteocytes are the resident cells within bone and contribute to its physiological maintenance. Osteoblasts synthesise bone (Figs. 3 and 4; [44]). They

are specialised cells responsible for the formation of bone extracellular matrix, a combination of collagenous (collagen type I) and non-collagenous proteins (i.e. osteopontin, osteocalcin, osteonectin) [45]. Osteoblasts control the subsequent mineralisation of bone matrix and also participate in bone remodelling [46].

Fig. 4. Transport systems involved in ion homeostasis in osteoblasts. Transport systems in the osteoblast are also polarised but not to the same extent as osteoclasts. There are no proton ATPases in osteoblasts and recent studies suggest that band-3 like anion exchangers (AE2) are almost exclusively present in ER and Golgi compartments [24]. In osteoblasts Ca-ATPase is abundant in the lateral and basolateral (plasma membrane facing away from bone) membranes. Na,K-ATPase expression does not appear to be polarised in osteoblasts unlike the plasma membrane Ca-ATPase, which is polarised.

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Osteoblasts and osteocytes are derived from multipotential mesenchymal stem cells capable of differentiating into cartilage, bone, and fat cells (Fig. 3; [47]). In culture, bone-derived osteoblast-like cells exhibit a characteristic mesenchymal morphology [17] and the expression of the enzyme alkaline phosphatase is often used as a bone specific marker for the osteoblastic phenotype. In comparison with osteoclasts, significantly less attention has been paid to ion pumps and ATPases in osteoblasts (Table 1). 4. Na,K-ATPase 4.1. Background Na,K-ATPase is expressed in almost all animal cells and serves as the principal regulator of intracellular ion homeostasis, coupling the hydrolysis of a molecule of ATP to the inward transport of two potassium ions and the outward movement of three sodium ions (for reviews see [48,49]). It is responsible for the generation and maintenance of the transmembrane ion gradients vital for essential cellular activities: nutrient uptake, volume regulation, pH maintenance, and generation of action potentials and secondary active transport. Na,K-ATPase is composed of two polypeptide subunits (␣ and ␤) and belongs to the P-type superfamily of ATPases [49]. In terms of three dimensional structure and mechanistic function Na,K-ATPase closely resembles the gastric H,K-ATPase [50]. The ␣ subunit (110 kDa) is a multipass transmembrane protein containing the binding sites for sodium, potassium, ATP and cardiac glycosides, a class of naturally existing steroid inhibitors that includes ouabain and digoxin. The ␤ subunit (55 kDa) is a glycoprotein with a single transmembrane domain and is understood to be essential for the biogenesis and activity of the enzyme complex. The ␣ subunit contains the phosphorylation, nucleotide binding and cation binding sites and is often referred to as the catalytic subunit. Thus far, four ␣ and three ␤ isoforms of Na,K-ATPase have been identified each of which exhibit tissue specific and developmental expression patterns [49]. 4.2. Na,K-ATPase in osteoclasts Akisaka and Gay [51] and Baron et al. [52] have demonstrated Na,K-ATPase activity in avian

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osteoblasts. Ouabain inhibitable potassium-dependent p-nitrophenylphosphatase activity was detected on the ruffled border of many (but not all) osteoclasts attached to bone, suggesting the presence of functional Na,K-ATPase complexes [51]. These authors detected no such activity on the basolateral membranes or the clear zones of the cells, and neither on osteoclasts unattached to bone or those whose resorption activities were inhibited by calcitonin. In contrast, Baron et al. [52] used the more specific method of antibody localisation of the ␣ and ␤ subunits of Na,K-ATPase and observed the highest concentrations of Na,K-ATPase on the basolateral domains of the osteoclasts with Na,K-ATPase also present in ruffled border and clear zone membranes. However, Baron et al. [52] did not report whether the osteoclasts were attached to bone or not. Further studies with radiolabelled ouabain identified a 40–60-fold increase in the osteoclast compared to bone marrow peripheral blood mononuclear cells [52]. This suggests a highly enriched Na,K-ATPase expression in osteoclasts. The precise role of such large quantities of Na,K-ATPase in the basolateral and/or ruffled border membranes of osteoclasts remains unknown. However, Na,K-ATPase pumps are associated with secondary active transport mechanisms such as Na/Ca exchange and Na/H exchange. These and other Na+ -dependent transport systems exploit the steep Na+ concentration gradient already established by Na,K-ATPase function (see Fig. 2) [53–55]. Such ion fluxes are considered important in driving osteoclast mediated bone resorption [51,52]. 4.3. Na,K-ATPase in osteoblasts Na,K-ATPase is important in the regulation of ion homeostasis and cell function in many cell types [49] but little is known about its expression, regulation and molecular properties in osteoblasts. The function of Na,K-ATPase is likely to be most critical for sodium dependent transport systems that rely on the inward sodium gradient. These systems are likely to include sodium dependent amino acid transport systems, Na/Ca exchange and osteoblast specific sodium dependent phosphate transport (Nad Pi ) [56,57]. The transfer of inorganic phosphate (Pi ), important in bone formation, into osteoblastic cells is a carrier-mediated Nad Pi co-transport process driven by the transmembrane

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electrochemical gradient of sodium maintained by Na,K-ATPase [58]. Bone mineralisation critically depends upon optimal calcium homeostasis in osteoblasts. To establish optimal intracellular [Ca2+ ] and to maintain this in face of calcium influx through various channels and exchangers, osteoblasts utilise Ca-ATPase and Na/Ca exchange systems for calcium extrusion (see below) whose activities depend on the expression and activity of Na,K-ATPase. Pharmacological studies on the effects of diphenylhydantoin (a major antiepileptic drug whose long-term use leads to clinical symptoms of osteomalacia and rickets) on cultured osteoblast-like cells first suggested that Na,K-ATPase is active in such cells and is inhibited by diphenylhydantoin [59]. Later Stark et al. [60] demonstrated histochemically abundant Na,K-ATPase expression in the plasma membrane and lysosomes of cells derived from a human osteosarcoma [60]. More recent studies on human bone-derived osteoblasts suggest that several isozymes of Na,K-ATPase are present in plasma membranes and internal membranes [61]. Immunofluorescence techniques have revealed abundant expression of Na,K-ATPase ␣1, ␤1 and ␤2 isoforms [61,62], while RT-PCR has also been used to demonstrate the presence of transcripts encoding Na,K-ATPase ␣1 and ␣3 isoforms in human osteoblast-like cells but not those of ␣2 [62]. However, the catalytic ␣1 isoform and the ␤1 and ␤2 isoforms dominate. Confocal microscopy has confirmed that Na,K-ATPase ␣1 isoforms are present in the plasma membrane, intracellularly, in the endoplasmic reticulum and in the Golgi apparatus, the site of synthesis of the ␣-␤ heterodimer (Fig. 5) [63]. The high intracellular abundance of the ␣1, ␤1 and ␤2 isoforms is indirect evidence for high turnover of sodium pumps in osteoblasts. The presence of the ␤2 isoform of Na,K-ATPase in musculoskeletal cells [49,62] suggests that its expression is not confined to neuronal and cardiac cells but is more widespread. Whether ␤2 performs adhesive functions in the musculoskeletal

cells including osteoblasts as it does in the central nervous system remains to be seen. Articular chondrocytes, also originate from multipotential stromal stem cells (Fig. 3; [47,64]) and express two catalytic (␣1 and ␣3) and two regulatory (␤1 and ␤2) isoforms of Na,K-ATPase [23,65]. While both osteoblasts and chondrocytes express several Na,K-ATPase isoforms, the pattern of isoform expression in these cell types is clearly distinct and may relate to their physiological and cell-specific roles. Thus, the expression of ␤2 appears to be a more universal phenomenon and not specific to the central nervous system. It remains to be seen if ␤2 performs any adhesive functions in musculoskeletal cells as it does in the central nervous system. The expression and subcellular localisation of Na,K-ATPase isoforms with different affinities for Na+ and K+ may be physiologically important in controlling the intracellular concentrations of these cations and confer unique cell-type specific physiological properties [49]. In view of the importance of Na,K-ATPase in the regulation of inorganic phosphate (Pi ) uptake and calcium homeostasis by the Ca-ATPase [66] it is perhaps not surprising that different isozymes of Na,K-ATPase may be present in osteoblasts since several isoforms of Ca-ATPase are also present (see Table 1; [67,68]). Why therefore is the ␣1 isoform predominant? The rat ␣1 isoform appears to have a relatively high affinity for intracellular sodium [69] and a low affinity for ouabain. Thus, Na,K-ATPase units containing the ␣1 isoform are likely to play a housekeeping role in osteoblasts facing large inward sodium gradients. Sodium ions may enter the osteoblast via other membrane proteins such as the Na/Ca exchanger [70,71], the Na/H exchanger [24,72] and Nad Pi symport [58] all of which are expressed in osteoblasts. Na,K-ATPase would be needed to extrude the sodium that has gained access to the cytosol via these transport systems and indeed it is found in the intracellular membranes of osteoblasts and chondrocytes [60,62,65]. Further treatment of

䉴 Fig. 5. Confocal slices of primary human bone-derived osteoblasts stained with monoclonal antibodies to Na,K-ATPase (␣1 isoform), plasma membrane Ca-ATPase (PMCA1), Na/H exchanger (NHE1), anion exchanger (AE2) and secondary rhodamine conjugated antibody. Plot profiles shown under each panel demonstrate the intensity of the fluorescent signal along the dotted arbitrary horizontal lines drawn on each panel. Na,K-ATPase expression is not polarised whereas the plasma membrane Ca-ATPase is polarised. Expression of the NHE isoform of the Na/H exchanger is mainly intracellular and vesicular whereas AE2 expression is found in intracellular Golgi compartments [24,62]. The control panel shows staining of cells incubated with rhodamine conjugated secondary antibody. The grey scale look-up table (LUT) on the left of the figure gives an indication of staining intensity.

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chondrocytes with the anterograde vesicular transport inhibitor Brefeldin A resulted in accumulation of large quantities of ␣ subunits in juxtanuclear pools [65]. This abundance of Na,K-ATPase isoforms in intracellular membrane of chondrocytes and osteoblasts may be explained in several ways. The Na,K-ATPase isoforms that are abundant in the endoplasmic reticulum or Golgi apparatus may be en route to the plasma membrane. Alternatively, the intracellular Na,K-ATPase isoforms may be held in membrane bound intracellular stores as “reserve pumps” which can be rapidly inserted into the plasma membrane in response to hormonal stimulation or functional demands. Na,K-ATPase in osteoblasts appears to be regulated by insulin as in skeletal muscle and adipose tissue (for a review see [73]). Rat osteoblast-like cells contain specific and abundant high affinity binding sites for insulin [74]. Physiological concentrations of insulin (1–10 ng ml−1 ) stimulate active K+ transport into osteoblasts that is mediated by Na,K-ATPase. Thus, insulin can directly affect the metabolism of osteoblasts and the hormone’s action on transmembrane ion transport may be linked to insulin’s interaction with its receptor in osteoblasts. This could explain the reduction in bone mass observed in human type I (insulin-dependent) diabetes mellitus [75,76]. Insulin exerts direct anabolic effects on bone metabolism and can, for example, stimulate collagen synthesis [77]. Acute effects on insulin on cation transport rate are likely to be pre-requisite for subsequent DNA synthesis and cell proliferation. In type I diabetes cation transport will not be stimulated by insulin deficiency with the possible loss of equilibrium between bone deposition and resorption, leading to the observed bone loss. In conclusion the osteoblastic Na,K-ATPase plays an important role in the osteoblast by maintaining the transmembrane gradient of Na+ that drives intracellular calcium homeostasis and extracellular matrix calcification.

5. H,K-ATPase 5.1. Background There are two types of H,K-ATPase; the gastric H,K-ATPase and the non-gastric, renal or colonic

H,K-ATPase [78,79]. H,K-ATPases are structurally and functionally related to Na,K-ATPase and are responsible for acid secretion in the stomach [80] and potassium homeostasis in the colon and the nephron [79,81]. Proton pumping by gastric H,K-ATPase is coupled to the electroneutral uptake of potassium in gastric parietal cells [82]. The gastric H,K-ATPase is mainly expressed in the stomach epithelium in the apical membrane plasma membrane domain of gastric parietal cells and is inhibited by omeprazole and orthovanadate [83]. In terms of pharmacology, the renal/colonic H,K-ATPase is highly homologous to Na,K-ATPase and is sensitive to ouabain [81]. 5.2. H,K-ATPase in osteoclasts Osteoclast acidity is dependent largely or partially on maintenance of potassium and sodium gradients, sodium channels, chloride-bicarbonate exchange, and H,K-ATPase [84]. The fact that the resorption lacuna underneath the ruffled border membrane is acidic and this low pH favours dissolution of bone mineral raised the possibility that osteoclasts might secrete acids during bone resorption [85,86]. Baron et al. [85] were the first to show the presence of a proton ATPase on the ruffled border membrane. They found a 100 kDa lysosomal membrane polypeptide that showed immunological similarity to the gastric H,K-ATPase. The demonstration by Tuukkanen and Vaananen [87], Mizunashi et al. [88], and Sarges et al. [89] that omeprazole and other H,K-ATPase inhibitors also inhibit bone resorption suggest that there is an active H,K-ATPase dependent proton pump on the ruffled border responsible for bone resorption. However, the inhibition by omeprazole was not very large (15–30%). Omeprazole only inhibited ATP-dependent proton transport in bone-derived membrane vesicles at concentrations 1000-fold higher than that required for inhibition of H,K-ATPase activity in kidney membrane vesicles [90]. Other studies have also failed to show either inhibition of acidification of osteoclast-derived membrane vesicles by orthovanadate [91,92] or to detect any H,K-ATPase activity or protein on osteoclast ruffled borders [51,93]. Thus, the gastric H,K-ATPase pump may be present on the osteoclast plasma membrane in relatively low quantities but its expression (and that of the non-gastric H,K-ATPase) has not been

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satisfactorily investigated using molecular biology (RT-PCR, Northern blotting), biochemical (subcellular fractionation, purification and mass spectrometry), immunological (Western blotting and immunohistochemistry using monoclonal and polyclonal antibodies) or pharmacological tools. Moreover, though the gastric H,K-ATPase may be involved to a certain extent in the resorption process, there is stronger evidence to suggest that a vacuolar (V-type) proton pump, similar to the kidney H-ATPase is involved in acidification of the extracellular resorption zone (see below and Fig. 2). 5.3. H,K-ATPase in osteoblasts Expression of the gastric or non-gastric H,K-ATPase in osteoblasts and osteocytes has not been studied.

6. Vacuolar (V-type)H-ATPase 6.1. Background The vacuolar or V-type H-ATPases are a family of ATP-driven proton pumps present in a variety of intracellular compartments and on plasma membranes of eukaryotic cells that couple the energy released from ATP hydrolysis to drive the formation of a linear proton gradient across the membrane [93]. They are composed of two functional domains: the V1 domain which is a 570 kDa peripheral complex responsible for hydrolysis of ATP and the V0 domain which is a 260 kDa integral complex that is responsible for proton translocation across the membrane [94]. V-type H-ATPases are inhibited by the macrolide antibiotic bafilomycin A1 , a potent and highly specific inhibitor [95]. 6.2. Vacuolar H-ATPase in osteoclasts Blair et al. [91] and Vaananen et al. [92] were the first to show the existence of a V-type H-ATPase in the ruffled border of avian and rat osteoclasts. This V-type H-ATPase could also be demonstrated in the microsomes derived from chick medullary bone [96]. V-type H-ATPases are polarised to the ruffled border membrane in actively resorbing osteoclasts

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with the function of acidification of the extracellular resorption zone or lacuna necessary for bone resorption [97–100]. Messenger RNA for the V-type H-ATPase subunits is among the most abundantly expressed in osteoclasts [101]. As in kidney cells, V-type H-ATPase activity on the osteoclast ruffled border is charge coupled to passive chloride permeability [102]. Inhibitors of the V-type H-ATPase, such as bafilomycin A1 and antisense RNA and DNA molecules targeted against V-type H-ATPase also inhibit bone resorption [103,104]. The bisphosphonate tiludronate also inhibits osteoclast V-type H-ATPase function although this may not have been the major mechanism of osteoclast inhibition as the dose required was very high and other bisphosphonates had no effect [20]. Chatterjee et al. [105,106] also found that vanadate, a classic inhibitor of P-ATPases, inhibited the osteoclast proton pump at very high concentrations, suggesting a unique pharmacological profile for the osteoclast V-type H-ATPase. Recent work on osteoclast V-type H-ATPase has concentrated on potential subunits that may be distinct from those of V-type H-ATPase present in other cell types. This subunit could then be targeted specifically when bone resorption is to be inhibited without affecting essential V-type H-ATPase in other tissues. Li et al. [107] cloned and characterised a putative novel human osteoclast-specific 116 kDa V-type H-ATPase subunit. However, Scott and Chapman [108] found by PCR amplification that this particular subunit was also expressed in many other tissues. However, the relative amounts of this subunit in other tissues is unknown and could be considerably less than in osteoclasts. Li et al. [109] have reported that targeted disruption of the gene in question in mice impaired only extracellular acidification by osteoclasts and neither intracellular acidification of lysosomes in these cells nor kidney or liver microsomal proton transport, suggesting a structurally and functionally unique V-type H-ATPase in the osteoclast ruffled border. Although early studies indicated that V-type H-ATPase were first localised on the ruffled border, or diffusely throughout the cytoplasm in non-resorbing osteoclasts, it has become apparent that V-ATPases are also located on the basolateral membrane [110]. Ravesloot et al. [111] and Lehenkari et al. [112] have suggested that some osteoclasts, distinguishable on

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the basis of cell shape or resorptive activity, regulated intracellular pH (pHi ) by V-type H-ATPase activity. Nordstrom et al. [113,114] found that in bicarbonate-free medium, pHi regulation by V-type H-ATPase activity was negligible in cells cultured at normal pH but became apparent in 40% of the cells following prolonged acidosis, a condition which stimulates osteoclast resorptive activity [115,116]. Larger osteoclasts (those with ≥10 nuclei) cultured on glass coverslips in bicarbonate-free medium regulate pHi mainly by V-type H-ATPase activity while smaller cells (those with 2–5 nuclei) regulate pHi by Na/H exchange activity [117]. When cultured on a resorbable surface, all resorbing osteoclasts regulated pHi by V-type H-ATPase activity and it was apparent that a greater percentage of large osteoclasts were activated for resorption as compared to small ones. Clearly V-type H-ATPase activity and acid extrusion induced by osteoclast attachment to bone are essential for resorptive activity in all osteoclasts as well as for pHi regulation in certain osteoclasts [118]. Further evidence for the importance of V-type H-ATPase in osteoclastic mediated bone resorption comes from recent work [119] on infantile malignant osteopetrosis, a rare autosomal recessive genetic disease that manifests itself during the first months of life (osteopetrosis is caused by a failure of osteoclasts to resorb bone). These investigators demonstrate that mutations in the gene (OC116) coding for the a3 subunit of human V-type H-ATPase caused osteopetrosis. It has also been shown, by the same group, that mutations in the CLC 7 chloride channel (CLCN7 gene) also result in osteopetrosis in mice and humans [120]. Chloride channels provide the chloride conductance required for an efficient proton pumping by the V-ATPase of the osteoclast ruffled membrane. Mutations in either system result in osteopetrosis. It remains to be seen if other transport systems or ATPases are mutated in other skeletal disorders. 6.3. Vacuolar H-ATPase in osteoblasts There is only indirect evidence for V-type H-ATPase in osteoblasts in that bafilomycin A1 treatment inhibits osteoblast growth with cytotoxicity and morphological changes [121].

7. Ca-ATPase 7.1. Background Calcium acts as a second messenger capable of initiating a large number of intracellular events [122]. In bone cells there is a specific, and additional physiological requirement for controlled intraand extracellular calcium homeostasis as both result in large fluxes of calcium [123,124]. To establish very low intracellular [Ca2+ ] and to maintain this against calcium influx through voltage and stretch activated calcium channels and non-specific leakage, a bone cell may possess two systems for calcium extrusion, the active Ca-ATPase and a coupled transporter, the Na/Ca exchanger. In most cells, including cells derived from bone, Ca-ATPase performs the vital physiological function of active calcium extrusion [66,125–127]. Ca-ATPase is expressed in a variety of calcium transporting tissues and its level of expression has been shown to be relatively high in bone [128]. Ca-ATPase is a high affinity, low-capacity calcium extrusion mechanism that is ATP and Mg2+ dependent and is present in the plasma membrane of all eukaryotic cells [129]. Ca-ATPase is highly dependent on calmodulin [66], has a high affinity for calcium, and is inhibited by vanadate. In contrast, the Na/Ca exchanger is a sodium dependent calcium efflux mechanism that catalyses a reversible, electrogenic exchange, with a stoichiometry of three sodium ions to one calcium ion, which is inhibited by La3+ and other divalent cations. It is regulated by intracellular calcium, sodium and ATP concentrations. Na/Ca exchanger is energised by the gradient of sodium established by the Na,K-ATPase and hence depends on its optimal activity. Any pharmacological or humoral agents that interfere with the sodium gradient can inhibit Na/Ca exchange, thus modulating bone formation and resorption by osteoblasts and osteoclasts, respectively [130]. Long-term administrations of calcaemic agents such as PTH, PGE2 or 1,25-dihydroxyvitamin D3 all inhibit Na/Ca exchange as do certain ionophores such as monensin. Thus, plasma membranes Ca-ATPase, along with Na/Ca exchange, are the main lines of defence of bone cells against rapid changes in intracellular calcium concentration.

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7.2. Ca-ATPase in osteoclasts Akisaka et al. [131] demonstrated that avian osteoclast Ca-ATPase had a polar distribution as it was present only on the basolateral membrane and not the ruffled border or clear zone (Table 1). Calcitonin treatment did not affect the distribution pattern or pump density at any site of the osteoclast. Biochemical characterisation of the Ca-ATPase showed that trace Mg2+ was required for activity, it was calmodulin-sensitive and that it had other characteristics similar to Ca-ATPase in other cells [132,133]. Arg-Gly-Asp (RGD)-containing peptides, which are recognised by ␣v ␤3 -integrin and thought to play a key role in osteoclast attachment to bone, stimulated calcium efflux from avian osteoclasts via Ca-ATPase activity [134]. This decrease in intracellular calcium may be functionally important for the stimulation of bone resorption. Although the precise role of the Ca-ATPase in osteoclasts is not known, it is also possible that it is responsible for maintenance of low levels of intracellular calcium as in other cells. Although immunologically and biochemically distinct from plasma membrane Ca-ATPases, inhibition of sarco(endo)plasmic reticulum Ca-ATPases (SERCA) have recently been shown to induce osteoclast-like cell formation, probably via 1,25(OH)2 -Vitamin D3 activity [135,136]. 7.3. Ca-ATPase in osteoblasts Ca-ATPase is abundant in the lateral and basolateral (plasma membrane facing away from bone) membranes of osteoblasts (see Table 1 and Fig. 5). The polar distribution of Ca-ATPase in osteoblasts suggests a unidirectional and vectorial calcium flux in these cells, which may be important for bone-forming activities [131,137]. Fluorescence studies have indicated a direct role for Ca-ATPase in calcium translocation across the osteoblast plasma membrane [138]. In osteoblasts Ca-ATPase has been found to be expressed as three isoforms; PMCA1, PMCA2 and PMCA1b [67,68]. However, the subcellular distribution of these isoforms has not been determined. Lower quantities of mitochondrial Ca-ATPase are present in osteoblasts and osteocytes compared with osteoclasts which indicates that transcellular calcium transport may be more important for bone-resorption than for bone forma-

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tion [131]. Multiple alternatively spliced forms of the Na/Ca exchanger are likely to be expressed by osteoblasts and have been found to be localised in membranes adjacent to bone matrix substrate [139]. On the other hand the Ca-ATPase is restricted to osteoblast membranes opposite the bone matrix [131]. The location of these two transport systems suggests that the Na/Ca exchanger plays a major role in calcium homeostasis by participating directly in bone formation, possibly by delivering free calcium directly to the site of mineralisation. Ca-ATPase plays an indirect role in this process by maintaining intracellular calcium at physiological levels. However, since Ca-ATPase activity is significantly lower than that found in epithelia (i.e. intestine, a tissue involved in massive calcium transport) the calcium pump probably does not support calcium translocation to sites of mineralisation [140].

8. Conclusions and future directions The pumping of ions across the various osteoclast plasma membrane domains by ATPases is crucial for osteoclastic resorption of bone. The V-ATPase is clearly responsible for the extrusion of protons across the ruffled border membrane that acidifies the extracellular resorption lacuna and the Ca-ATPase maintains low levels of intracellular calcium in the osteoclast. The role of the other ATPases (Na,K-ATPase, H,K-ATPase) may simply be to maintain electrochemical balance of the osteoclast or to generate an ion gradient, which could energise other passive mechanisms of ion transport. In the osteoblast the most important pumps involved in ion homeostasis are Na,K-ATPase, H,K-ATPase and Ca-ATPase. Ca-ATPase is important for vectorial calcium flux in these cells, which may be important for bio-mineralisation. Na,K-ATPase is likely to maintain the intracellular sodium and potassium homeostasis that is required for functional activity of intracellular enzymes [141]. The V-ATPase is not expressed in osteoblasts. The expression of gastric H,K-ATPase and non-gastric (renal or colonic) H,K-ATPase needs to be evaluated in bone cells. The research on ATPases in bone cells has revealed that osteoclasts and osteoblasts express a different and unique array of pumps suited to their biological functions. We speculate that future research in this area of bone cell physiology is likely to concentrate less on

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