WO1993001280A1

WO1993001280A1 - Inhibition of proton pump in osteoclast cells

Info

Publication number: WO1993001280A1
Application number: PCT/US1992/005498
Authority: WO
Inventors: Roland Baron
Original assignee: Roland Baron
Priority date: 1991-07-08
Filing date: 1992-07-07
Publication date: 1993-01-21

Abstract

The present invention relates to a uniquely identified ATP dependent proton (H+)-pump in osteoclast membranes which is sensitive to both vanadate and NEM and which contains one or more immunologically distinct subunit(s) not found in other proton pumps, and which is highly sensitive to nitrate. The invention also teaches a method for the introduction of suitable therapeutic agents into the aforesaid uniquely identified proton (H+)-pump in osteoclast membranes in order to treat the effect of osteopenia and other bone and joint diseases with increased osteoclastic activity (Arthritis, Paget's disease, Hypercalcemia).

Description

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation-In-Part of applicant's co-pending parent application Serial No. 726,480 filed on July 8, 1991.

FIELD OF THE INVENTION

This invention relates to an ATP dependent proton (H⁺)-pump in osteoclast membranes which is sensitive to both vanadate and NEM, is inhibited by low concentrations of nitrate and contains an immunologically distinct subunit(s) not found in other proton pumps. This unique pump is a potential target for the introduction of various drugs that will inhibit osteoclast bone resorption without affecting functions of other cell types.

BACKGROUND OF THE INVENTION

Osteoclasts and osteoblasts are classes of bone cells responsible for bone resorption and bone formation, respectively. During normal bone remodeling, osteoclastic bone resorption is followed faithfully by osteoblastic bone formation at the previous resorption sites. For bone formation to occur at the site of previous resorption, osteoclastic bone resorption must cease. Osteopenia results from an imbalance of the opposing activities of osteoclasts and ostoblasts such that the rate of bone resorption exceeds the rate of accretion. When the osteopenia has progressed sufficiently, the bone strength is decreased below the critical fracture point, and a fracture results. Formally, the term osteoporosis denotes the occurrence of fractures in osteopenic patients. It is thought that osteopenia can be treated by manipulating the opposing activities of the osteoclasts and osteoblasts. This reasoning has led to the development of factors that can inhibit the formation and/or activities of osteoclasts and stimulate the formation and/or activities of osteoblasts.

Osteoclasts are tightly attached to the bone surfaces that they resorb, and the extracellular compartment between the bone and the ruffled border membrane of the osteoclast is actively acidified by the cell. This

acidification is thought to be essential for osteoclastic bone resorption. The mechanisms responsible for the

acidification are most likely comparable to those found in many intracellular organelles, namely synaptic vesicles, chromaffin granules, endσsomes, lysosomes, plant vacuoles, the stomach and the kidney tubule plasma membrane. In all these cases, ATP-dependent II -pumps play a vital role in acidification, i.e., in the establishment and maintenance of a pH gradient across the membrane. The H+-ATPases of several different organelles have been recently purified and characterized and several of their subunits cloned and sequenced. On the basis of the available information, it may be concluded that there are three types of H-+-ATPase: 1) The mitochondrial F₀-F₁-type ATPase which is composed of several subunits and in inhibited by oligomycin, azide, and efrapeptin; 2) the H+/K+-ATPase, a phosphoenzyme ATPase, composed of two subunits, α and β , which is inhibited by vanadate; and 3) vacuolar and secretory granule ATPase, also of a F0-F1 type v/ith multiple subunits, which is inhibited by MEM but not vanadate or mitochondrial ATPase inhibitors. The F0-F1 ATPases are all electrogenic while the

H+/K+-ATPase is not. In addition, the plasma membrane of yeast and Neurospora crassa contain another type of H+-ATPase, which consists of a single subunit, is

electrogenic, and is inhibited by NEM and vanadate.

With reference to Figure 5, which shows the structure of a classical vacuolar pump, the vacuolar-type proton pump is a multisubunit structure formed by the assembly of several different polypeptides, some in one copy and some others in multiple copies. These macro-molecular assemblies are organized in three portions, each with a specific functional role:

The catalytic portion of the pump (top) faces the cvtoplasmic side of the plasma membrane and is formed by the assembly of 3 copies of a 70kD subunit (67-72kD), which binds ATP, and 3 copies of a 60kD subunit (57-60kD). - The proton transporting portion of the pump (proton channel) is burried in the plasma membrane that is traverses and is composed of several copies of a 16kD, DCCD binding, proteolipid and, probably of one or more copies of a 20kD hydrophobic polypeptide.

The two functional portions mentioned above are liked by a group of 30-42kD polypeptides forming the "stalk" of the pump and whose function is not yet established

(regulatory?)

Not shown here are several "accessory"

proteins, probably involved in the regulation of the pump, in the 100-115kD range. It is, therefore, an object of the present

invention to provide for a uniquely identified ATP dependent proton (H⁺)-pump in osteoclast membranes.

It is also an object of the present invention to provide for an ATP dependent proton (H⁺)-pump in osteoclast membranes which is sensitive to both vanadate and NEM.

It is another object of the present invention to provide for an ATP dependent proton pump which is highly sensitive to nitrate ions.

It is a further object of the present invention to provide for an ATP dependent proton (H⁺)-pump in osteoclast membranes which is sensitive to both vanadate and NEM and contains an immunologically distinct subunit (s) not found in other similar proton pumps.

Lastly, it is an object of the present invention to provide for a method for treating osteopenia

hypercalcemia of malignancy, Paget's disease, arthritis and all other bone and joint diseases which involve excessive bone resorption as a pathogenic mechanism, which comprises introducing a suitable therapeutic agent into the unique osteoclast membranes containing the ATP dependent proton (H⁺)-pump identified herein.

These and other objects of the present invention will become apparent to those skilled in the art from the following discussion of the invention. STATEMENT OF THE INVENTION

The present invention provides for an ATP dependent proton (H⁺)-pump in osteoclast membranes that is sensitive to vanadate and NEM, is inhibited by low concentrations of nitrate and contains an immunologically distinct subunit(s) not found in other proton pumps. The pump is a potential target for the introduction of various drugs that will inhibit osteoclast bone resorption without affecting functions of other cell types.

DISCUSSION OF THE INVENTION

It has now been found that the ATP-dependent acidification of inside-out membrane vesicles prepared from highly purified chicken osteoclasts can be completely inhibited by both NEM and vanadate. This finding contrasts with that of others who have found that the acidification associated with osteoclast membrane vesicles is inhibited by NEM but not vanadate (Bekker and Gay, 1990; Blair et al., 1989; Vaananen et al., 1990). The unique finding by the applicants in the instant invention also indicates the possible participation of a vanadate sensitive component in the acidification of the bone osteoclast compartment during resorption.

Of particular interest is the fact that both NEM (1mM) and vanadate (1 mM) could completely block the acidification of osteoclast microsomes. NEM inhibited acidification by 60-70% at μM concentrations, but higher concentrations were required to inhibit the remaining 30%. In contrast, a higher dose of vanadate was needed to inhibit acidification by 50% (100μM) after which there was a sharp drop of acidification with a small increase in vanadate concentration. The Ic₅₀ values for NEM and vanadate were 200 nM and 100 ^ιM respectively. These data established that osteoclast derived inside-out vesicles contain a proton transport system that is sensitive to both inhibitors. The fact that each could inhibit 100% of the transport makes the possibility of 2 pumps in the same vesicles or 2 vesicles, each with one type of pump, extremely unlikely.

In addition to NEM sensitivity, the vacuolar nature of the osteoclast proton pump was demonstrated by the fact that there is a high density of "ball and stalk" structures on the purified membranes in negatively stained samples of microsomal preparations, as reported for the vacuolar pumps of N. crassa and kidney.

Finally, and most importantly, the immunological properties of the osteoclast proton pump also appear to be unique. Antibodies raised against the vanadate and NEM sensitive 100 kD plasma membrane proton-ATPase of yeast and N. crassa (Mandala and Slayman, 1989) do not cross react with the osteoclast membrane fractions used by the applicant in the present invention. Moriyama and Nelson (1988) have reported the presence of a vanadate and NEM sensitive ATPase in chromaffin granule membranes. However, the ATPase was not associated with the acidification of the chromaffin granules, indicating the absence of an associated proton pump. The results shown in the instant invention are of major importance because no other cell type or organelle preparation has been reported to have proton pumps sensitive to both vanadate and NEM (both below mM range). In

addition, the identification of a unique subunit(s) that copurifies with the osteoclast vacuolar proton pump presents a potential target for therapeutic intervention in the prevention and treatment of osteopenia and other diseases involving an increased bone resorption.

The review by Forgac et al (1989) Phsiol. Rev. 69:765-796, lists the drug sensitivities exhibited by the various phosphorylated, F1-F0, and vacuolar ATPases. None of the ATPase pumps listed show dual sensitivity to vanadate and NEM as described for the vacuolar H+-ATPase described in the present invention.

Aaronson, L.R. et al (1988) J. Biol. Chem.

263:14552-14558, describe an H+-ATPase in the plasma membranes of yeast and N. crassa which consists of a single 100 kD subunit, is electrogenic, and is inhibited by NEM and Vo₄. Immunoblot analysis of osteoclast microsomal fractions with an antibody against the N. crassa 100 kD protein failed to detect any cross-reacting protein and studies with F₀-F₁. V type antibodies have demonstrated the multi subunit nature of the OC H⁺ATPase. Moriyama, Y., and N. Nelson (1987) J. Biol. Chem. 262:9175-9180, report the presence of a H+-ATPase proton transport in chromaffin granules that is sensitive to NEM. The ATPase activity of the proton pump is not sensitive to 0.1 mM vanadate. In addition, the ATP dependent proton uptake activity was dependent on the presence of Cl- or Br- outside the vesicles, whereas sulfate, acetate, formate, nitrate, and thiocyanate were inhibitory. The pump

described in the instant application shares many of the same properties as the pump described by Moriyama and Nelson, i.e., NEM and Bafilomycin Al sensitivity, and the

requirement for Cl- and Br-. The transport in osteoclast membrane vesicles of the pump described here, however, differ significantly since it is only slightly sensitive to sulfate and acetate (80% of control in the presence of sulfate), and nitrate in the μM concentrations could block the osteoclast pump whereas mM concentrations (100 to 1000 fold higher) are necessary for inhibiting other known proton pumps. However, the overriding difference between the pump described here and Moriyama and Nelson pumps is the vanadate sensitivity exhibited by the unique pump of the instant application.

Moriyama, Y., and N. Nelson (1988) J. Biol. Chem. 263:8521-8527, reported the presence of an NEM and vanadate sensitive 115 kD ATPase in chromaffin granule membranes.

The ATPase is not inhibited by DCCD or nitrate and only slightly inhibited by sulfate. Although, the pump which is the subject of the present invention is only slightly inhibited by sulfate it is very sensitive to DCCD and nitrate. In addition, the 115 kD ATPase is not involved in proton uptake into chromaffin granules, whereas in the present case, the vanadate sensitive ATPase is involved in proton transport.

Three other groups have described vacuolar ATP dependent proton pumps in osteoclast membranes isolated fro calcium deprived egg laying hens, that are inhibited by NEM, but not vanadate (Blair et al (1989) Science 245:855-857, Bekker, P.J., and C. V. Gay (1990) J. Bone Min. Res.

5:569-579 and Vaananen, H.K. et al (1990) J. Cell Biol.

111:1305-1311). The major difference between the

observations of these three groups and the pump of the present invention is the significantly increased vanadate sensitivity observed here for the first time. Blair et al. report that proton transport is not inhibited by 1 mM vanadate and completely inhibited by 05 mM NEM. In the pump of the present application it has been found that 1 mM vanadate inhibits 100% and it requires 1 mM NEM for 100% inhibition. Bekker and Gay report that the osteoclast proton pump is not inhibited by vanadate, although their figures suggest that 1 mM vanadate causes 30% inhibition. In contrast, the invention here observes 50% inhibition at 100 μM vanadate and 100% inhibition at 1 mM vanadate. All groups are using the same source for preparation of

osteoclast membrane vesicles. Moreover, all groups claim that their osteoclasts membrane preparations are about 70% pure. The inventor in the instant application has

determined that the other groups investigating this area of interest were inaccurate and his preparations were truly enriched for osteoclast membranes while those of the other workers in this field were not. When testing osteoclast membrane fractions of varying degrees of purity, it has been found that the ability of the vesicles to acidify (Δ pH/μg protein) and the sensitivity to vanadate co-purified with the osteoclasts. In addition, lysosomal proton pump

contamination was not significant since the vesicles could use only ATP as a substrate when lysosomal pumps can also use GTP. It has, therefore, been concluded that the

vanadate sensitive component of proton transport is

osteoclast associated.

The osteoclast membrane preparations that have now been isolated can be immunologically distinguished from the preparations used by the other groups working in this field. The other groups observe a protein of 70 kD in their

osteoclast membrane preparations that cross reacts with antibodies against a N. crassa 67 kD proton pump subunit. Whereas the invention here observes a 70 kD band in his impure fractions that decreases with osteoclast membrane purity. Furthermore, all other groups observe a 60 kD protein in their osteoclast membrane preparations that cross reacts with antibodies raised against an N. crassa 57 kD proton pump subunit. The invention here observed this protein in his impure osteoclast preparations, but the band decreased with purity.

In summary, there are definite immunological differences between the osteoclast proton pump isolated by the present inventor as compared to other workers in this area of interest. Instead of the classical and previously reported 70kD catalytic subunit found in vacuolar proton pumps and in osteoclasts, as reported by the other groups, the present inventor observed a 63kD subunit. This subunit, when immunoprecipitated after incubation in the presence or radiolabeled phosphate, forms a phosphorylated intermediate which is sensitive to vanadate. This explains the

sensitivity of the osteoclast proton pump to vanadate, an observation that none of the others groups have reported. Furthermore, the other subunit of the catalytic portion of the osteoclast proton pump, 60kD in molecular weight, differs immunologically from that reported by others and is recognized by antibodies to the bovine chromaffin granule but not by antibodies to the N. crassa vacuolar pump, which sees the 60kD subunit in classical vacuolar pumps. These two characteristics, as well as the extreme sensitivity to nitrate, distinguish the osteoclast proton pump from other vacuolar proton pumps and indicate that the pump of the present invention may be a target for therapeutic

intervention.

DESCRIPTION OF THE FIGURES

Fig. 1.A-1 and 1.A-2 Illustrate highly purified osteoclasts.

Fig. 1.B-1 and 1.B-2 Show that the ability of the vesicles to transport protons increases with osteoclast purity.

Fig. 1.C Demonstrates the presence of a high density of characteristic F0-F1 ball-and-stalk multisubunit proton pump structures by electron microscopy of negatively stained microsomal vesicles.

Fig. 2.A-E Illustrate that the osteoclast proton pump is inhibited by all the classical inhibitors of vacuolar ATPas but also by vanadate, defining it as a new class of proton ATPase, and by low concentrations of nitrate.

Fig. 3.A-B Show the immunoblot analysis of the subunit composition of the osteoclast proton pump reveals a new and specific p63 isoform of the vacuolar catalytic 70KD subunit

Fig. 4. Shows in Lane A and F: 20 sec phosphorylation in th absence of vanadate; lane B: 20 sec phosphorylation in the presence of 1 mM vanadate; lanes C, D and E: 20 sec

phosphorylation in the absence of vanadate followed by chas for 15, 30 and 120 sec respectively with cold ATP and Pi.

Fig. 5. Shows the structure of a classical vacuolar proton pump.

DETAILED DESCRIPTION OF THE INVENTION

Experimental designs and methods:

1) Purification of Osteoclasts: Osteoclasts were isolated from medullary bone of calcium deprived laying hens and enriched by unit gravity centrifugation - a procedure routinely used in the laboratory to yield 5-15 × 10 cells (osteoclasts) per hen of particle purity ranging from 1:4 to 1:7. Though the particle purity of osteoclasts is

relatively low, the purity is 70-90% on the basis of total protein (or membrane) as osteoclasts are ~ 30 times larger than the contaminating cells. Furthermore, this level of purity is at least equivalent to that of kidney homogenates previously used by other investigators to purify proton pumps. Other methods of osteoclast isolation, based on percoll or ficoll gradients, have been attempted but they did not significantly improve the purity of the

preparations. Therefore, all experiments have been carried out with crude cell preparations as a control as well as with preparations of various known increasing degree of osteoclast purity in order to determine which components or properties co-purify with these cells.

2) Cell fractionation: Purified osteoclasts were homogenized gently in buffer and centrifuged at 100 x g for 5 min to remove nuclei and cell debris. The supernatants were centrifuged at 10,000 × g for 20 min to remove

mitochondrial fractions. The supernatant from this step was spun at 100,000 × g for 30 min to get the microsomal

fraction. This fraction, which is highly enriched in the vanadate-sensitive proton transport system, was further fractionated in percoll gradients or by free-flow

electrophoresis. A total of ~5 × 10° cells is washed twice in TEA-sucrose (10 mM triethanol araine, 10mM acetic acid; pH 7.4; 1 mM EDTA and 250 mM sucrose) and resuspended in 5 volume of TEA-sucrose. Cells are homogenized with 1-3 passes of a ball bearing homogenizer to obtain > 80% lysis, as monitored by phase-contrast microscopy, leaving nuclei intact and allowing for large sheets of membranes.

Microsomes are prepared from a postnuclear supernatant and resuspended to 1 mg protein/ml as previously described. To allow better separation of membranes, microsomes might have to be incubated for 5 min 37°C in 20-25 μM/ml TPCK-trypsin (Worthington). The digestion is stopped by adding 100 μM/ml soybean trypsin inhibitor (Sigma) and cooling on ice.

Control experiments indicate that trypsin treatment had no major effect on the protein composition of endosomes or on the functional integrity of their membranes, as assayed by ATP-dependent acidification in vitro and we were able to verify this is osteoclast membrane preparation. FFE is performed using a Bender and Hobein Elphor Vap 21 instrument at 130 mA and 1700-1800 V.

The different subcellular fractions are characterized by assaying marker enzymes such as Na⁺,

K+-ATPase, 5'-nucleotidase and alkaline phosphatase for plasma membrane, monoamino oxidase and succinate

dehydrogenase for mitochondria, beta hexosaminidase for lysosomes, galactosyl transferase for Golgi and glucose-6- phosphatase for endoplasmic reticulum fractions. All the purified fractions are tested for the vanadate-sensitive acidification using the vesicular acidification assay.

3) Vesicular Acidification Assay: ATP-dependent acidification is assayed as previously described and as routinely used in previous work by the applicant, using vesicles isolated from osteoclasts instead of intracellular organelles. The vesicles are diluted in buffer containing

100mM NaCl, 50 mM KCl, 20/mM HEPES-TMA, and 5 mM MgSO₄ (pH

7.4). Preloading is accomplished by incubating the vesicles in a solution of 0.5 uM valinomycin and 1.5 μM Acridine

Orange in the same buffer for 20 min. Proton transport is initiated by the addition of 2.5 mM K₂ATP and acidification will be estimated from the characteristic quenching of flouresence emission (on a Hitachi 2000 Fluorimeter) with excitation at 460 nm and emission at 520nm, which occurs as a function of decreasing pH. Verification of the integrity of the vesicles is accomplished by adding the proton ionophore Nigericin at 1.25 nM, which should rapidly dissipate the proton gradient. The fraction enriched in that activity is used for purification of the osteoclast proton pump.

DISCUSSION OF THE EXPERIMENTAL RESULTS

Given the high degree of contamination of

osteoclast preparations with vesicles derived from other cell-types or from acidified intracellular orgenelles, the possibility that indeed the pharmacology and structure of the ruf fled-border osteoclast proton-pump (OC- H+ATPase) could differ from that of other proton pumps but remain undetectable under the experimental conditions used in previous studies, was considered.

This possibility was raised mostly because it was found that H+-transport by inside-out vesicles derived from oεteoclast-enriched cell preparations was, unlike other known proton-transport systems [with the exception of the N crassa plasma membrane P-ATPase (Aaronson et al 1988;

Al-Awaqati, 1986] not only sensitive to the V-ATPase inhibitors NEM and Bafilomycin Al but also to the P-ATPase inhibitor vanadate, and this despite the absence of P-ATPase subunits in the preparations used in carrying out the present invention.

Osteoclast membranes contain a high number of FO-Fl-like proton pumps

To monitor the contamination of preparations by proton pumps derived from other cells and organelles (Fig. 1Λ) , microsomal fractions were obtained from osteoclast suspensions at several steps in the purification procedure, ensuring that the pharmacological and structural properties of the proton transport system(s) present in these membranes co-purified with the osteoclasts. Using our procedures, osteoclasts were purified 400-fold and plasma membrane enriched another 10-fold after fractionation (Table 1). The ability of the inside-out vesicles present in these membrane fractions to transport protons upon addition of ATP in the acidification assay, expressed as the Δ pH/mg protein (Fig. 1B-2) increased proportionally to the purity of the cell preparation, thereby demonstrating that most of the proton transport systems present in the microsomal fraction are derived from osteoclasts.

Despite the fact that only partially purified microsomes from the osteoclast fractions were used the ability of these vesicles to transport protons was about 50-fold higher than that of microsomes from other cells TABLE - 1

ENRICHMENT OF DIFFERENT ORGANELLE MARKER ENZYMES IN PURIFIED MEMBRANE FRACTION OF OSTEOCLAST

Enzyme Specific Activity

Total Homogenate P1 P2

Na+ K+-ATPase 1.5±0.4 12.2±1.3 16.8±2.1

5'-Nucl eoridase 21.2±2.8 1 19.3±12.4 180.8±16.3

Succinic dehydrogenase 12.2±2.1 1.1±0.4 0.6±0.2

Glucosc-6-phosphatase 6.8±0.7 0.6+0.3 0.5±0.2

Galactosyl transferase 8.1±0.9 0.4±0.2 0.2±0.1

(Fig. 1B) and at least 2-fold greater than that of highly purified endosomes (Schmid et al 1989; Fuchs et al 1989). This very efficient H+ transport suggested that a high concentration of pumps was present in these membranes. In order to directly determine whether this was the case, high magnification electron microscopy on negatively stained osteoclast microsomes was performed (Fig. 1C). 30-40% of the vesicles were found to contain in their limiting membranes high densities of characteristic ball-and-stalk structures, compatible with the presence of high number of copies of F0-F1-ATPases and reminiscent of kidney tubule apical membranes (Brown et al 1987).

These microsomal preparations v/ere then used to study the properties of proton transport and for

immunochemical analysis of the OC H+-ATPase structure. As demonstrated below, the osteoclast membrane F0-F1 proton pump is of a novel type, albeit closely related to the V-ATPases. The main differences found between the OC-ATPase and V-ATPases are 1) Its complete inhibition by both NEM and vanadate; 2) Its complete inhibition by low

concentrations of nitrate and 3) Its unique expression of a 63kd subunit, related to but distinct from the 67-70kd catalytic subunit of the other V-ATPases. The OC-H+ ATPase is sensitive to both NEM and vanadate, defining it as a novel class of proton pump

Pharmacologically (Fig. 2), all the inhibitors of V-ATPases inhibited 100% of the acidification by osteoclast membrane vesicles. The k1/2 for NEM, DCCD, quercetin and Bafilomycin Al were 0.1 μM, 35 uM, 3 μM and 6 nM respectively

(Fig. 2A-1 through 2A-4). The mitochondrial proton ATPase

inhibitors, oligomycin, azide and fluoride has no effect. This data confirmed that, as reported by others (Blair et al 1989; Bekker, and Gay, 1990; Vaananen et al 1990), the osteoclast proton pump exhibits properties of the vacuolar type, a finding in agreement with the high density of

F0-F1-like ball and stalk structures we found on the

σsteoclast-derived microsomal vesicles used in this assay (Fig. 1B), as described on purified kidney membranes (Brown et al 1987).

However, and unlike any other V-ATPase, the

OC-ATPase could also be inhibited by vanadate, which blocked

100% of this acidification at a concentration of 1mM and with an k1/2 of 100 uM (Fig. 2A-6 and B). Most importantly, the sensitivity of proton transport to vanadate co-purified with the osteoclasts (Fig. 2C) and was not detected in kidney microsomes derived from the same animals (Fig. 2D), eliminating the possibility of an artefact or of a species specificity. Since the 70kD subunit forms part of the catalytic portion of the proton pump, the hypothesis that the unique p63 subunit may represent the molecular counterpart for the unique sensitivity of the OC-H+ATPase to vanadate was tested. The membranes were incubated with 32P-ATP in the presence or absence of vanadate (28), solubized and p63 immunoprecipitated with the antibodies to the N. crassa 70kD subunit. The results (Fig. 4) unequivocally demonstrated that the 63kD subunit of the OC-H+ ATPase forms a

phosphorylated intermediate, a property not previously reported in V-type ATPases, and that this is inhibited by vanadate.

Given the importance and novelty of this observation, several alternate possibilities were

considered. First, these membranes could contain two type of proton pumps, a V-ATPase conferring the NEM and

Bafilomycin sensitivities and a P-ATPase conferring the vanadate sensitivity. This possibility was ruled out on the basis of several experimental results. First, it is

possible to inhibit 100% of proton transport with each inhibitor used separately (Fig. 2), which strongly argues against the presence of two types of pumps or two types of vesicles in our microsomal preparations. Second, kinetic analysis of H+ transport by inside out vesicles in the presence of various concentrations of ATP demonstrated the presence of only one Km for ATP in these preparations (Fig. 2E), a very unlikely result if indeed two H+ ATPases were present in these membranes. Third, immunoblot analysis with antibodies raised against the two P-ATPases that are

sensitive to vanadate, the N crassa plasma membrane

electrogenic pump (Aaronson et al 1988; Mandala, and

Slayman, 1989) and the gastric H+/K+ ATPase [HK9 and HKB, (Caplan, 1991)], did not recognize any protein in our microsomal preparations or in situ. Fourth, both the morphological and the pharmacological data demonstrate a high concentration of F0-F1, V-like proton pumps in these membranes.

The possibility that vanadate could inhibit other ATPases, indirectly affecting proton transport, was also considered. This is, however, unlikely because inhibition of the Na,K ATPase or a Ca-ATPase (even though Ca is not present in the acidification buffer used in the present invention) would favor (and not inhibit) proton accumulation in the vesicles (Fuchs et al 1990). Furthermore, although an NEM- and VO4-sensitive ATPase has been reported in chromaffin granule and coated vesicle membranes, the eκzyme was not a H+ ATPase and its activity did not affect H+ transport under similar experimental conditions (Moriyama, and Nelson, 1988). Finally, the fact that three other studies, using the same starting material, concluded that the osteoclast proton pump was not sensitive to vanadate (Blair et al 1989; Bekker, and Gay, 1990; Vaananen et al 1990) can be explained on the basis of purity of the osteoclast preparations. In fact, all three studies show a slight effect of vanadate, but one too small to be interpretable. As shown in Fig. 2C, vanadate sensitivity becomes appreciably visible only at purities higher than 1:50 (osteoclasts:other cells) and is still of only 50% even at purities of 1:10. In one study (Vaananen et al 1990), experiments were performed using total bone homogenates, conditions under which osteoclasts are not purified. In another (Blair et al 1989), a purity of 98% is claimed, based on a cross-reference paper, but careful examination of the methods indicates that only the first step of the cited purification procedure was performed in the second study. In applicant's hands, this step gave purities of 1:150±25, thereby explaining the lack of

vanadate sensitivity of proton transport in these studies. The results obtained here show that, while under the

conditions of these other studies vanadate sensitivity is not detectable (Fig. 2C), it becomes very clear at higher osteoclast purities and with the fractionation procedures we used. These observations, therefore, strongly suggested that the OC-H+ATPase represents a new type of vacuolar proton pump, which, in addition to being sensitive to classical inhibitors, is also blocked by vanadate.

The PC- H+ ATPase is highly sensitive to nitrate and insensitive to sulfate and acetate

The novelty of the OC-ATPase is also demonstrated by its sensitivity to various ions (Table 2).

Addition of valinomycin to the proton transport assay's buffer allows the membranes of the vesicles to become permeable to K+, thereby allowing an equilibration of + charges across the membrane. This permits H+ transport independent of anion transport. In the absence of

valinomycin, the assay allows the identification of the anions whose transport is required, and possible, across the vesicular membranes (here Cl- and Br-, both through chloride conductances).

The most striking observation was that nitrate was inhibitory at concentrations 100 to 1000-fold lower than required to inhibit other V-ATPases (Moriyama et al 1986; Wang, and Gluck, 1990): 1 mM NO3- could completely inhibit proton transport by osteoclast membrane vesicles, with an Table 2

Effects o f Different Ions on Acidification of Osteoclast-

Microso mes

Salt (150 mM) Acidification

(% of Control)

with valinomycin without valinomycin

KCl 100 83±4

K3r 79±3 70±6

KNO₃ 0 0

KH₂PO₄ 32±2 20±2

K-acetate 77±2 6S±6

K₂SO₄ 75±3 7 l±ό

KF coagulation of proteins coagulation of protein

NaCl 90+6 70±4

NaBr 74±5 68iβ

NaNO₃ 0 0

NaH₂PO₄ 30±5 31±4

Na-acetatc 23±4 22±6

Na₂SO₄ 26±2 20±4

NaF 0 0

CsCl 80±3 78+6

CaCl₂ 0 0

MgSO₄ 0 0 Choline chloride 60±4 62±6 Choline bromide 46±3 42±8

Sucrose 16±4 14±6 K₂SO₄ (75 mM)+KCl (75 mM) 80±5 77±5 K-acccate (75 mM)+KCl (75 mM) 76±4 70±6

k1/2 of 100 μM (Fig. 2A-5).This inhibition was not, however, due to oxyanion chaotripic effects since we did not observe, as reported for the N Crassa V-ATPase (Moriyama, and Nelson, 1989), a dissociation of the F0 and F1 components of the OC-ATPase, even at higher concentrations of nitrate (data not shown). Like other V-ATPases, the OC-ATPase was

electrogenic and probably associated with a parallel

chloride conductance since it specifically required Cl- or Br- outside the vesicles or the K⁺/valinomycin system to neutralize the charges developed during transport of H inside the vesicles. However, and differing again from other pumps, up to 75% of the acidification could still occur in the presence of SO₄╌ or acetate without

valinomycin (Table 2). Hence, the OC-ATPase is vacuolar in nature but has a unique pharmacological and ionic

sensitivity profile.

The OC- H+ ATPase differs from other H+ ATPases in its subunit composition

Sensitivity to vanadate is a pharmacological property previously found only in P-ATPases (Al-Awqati, 1986). Since antibodies to P-ATPases failed to detect the presence of known P-ATPases in applicant's preparations or in situ., the possibility that the catalytic subunits (67-70 and 57-60kd) of the OC- H+ ATPase, sites where vanadate is known to be competing with phosphate for binding (Arai et al 1987; Macara 1980), could differ from other V-ATPases was tested.

Immunoblot analysis of the membrane preparations from osteoclasts at different stages of purification were carried out with antibodies against several subunits of vacuolar proton pumps of various sources (Fig. 3 A and 3B). Antibodies against the 115, 57 and 39 kd subunits purified from chromaffin granule membrane (Wang et al 1988) or antibodies against the 16kd DCCD-binding proteolipid from plant (Lai et al 1988) revealed that these 4 subunits co-purified with the osteoclasts (Fig. 3A), further

confirming the vacuolar nature of the OC-ATPase.

Different results were, however, obtained with antibodies to the two catalytic subunits of the N crassa V-ATPase. First, the 60kd subunit decreased with osteoclast purity (Fig. 3A) when blotted with the N Crassa antibodies (Bowman et al 1988). This is in contrast with the results obtained with the anti 57kd chromaffin granule antibody and suggests that the 60kd subunit associated with the

OC-ATPase is more closely related to the chromaffin granule 57kd than to the N crassa 60kd subunit, despite their highly conserved sequences (Nelson, and Taiz, 1989). Second, the 70 kd subunit, whether detected by antibodies raised against the 70 kd subunits of chromaffin granules (Moriyama, and Nelson, 1988), coated vesicles (Sudhof et al 1989) or N crassa (Bowman et al 1988)

decreased with osteoclast purity (Fig. 3A), suggesting that this subunit was mostly present in contaminating cells.

Most interestingly, the antibodies again N crassa V-ATPase 70kα subunit demonstrated that while the epitope(s)

recognized on the corresponding subunit in chicken V-ATPase decreased with osteoclast purity, a new band of approximate mol. wt. 63 kd progressively appeard and increased with osteoclast purity (Fig. 3A and B). These results suggested that the nature of the catalytic subunits of the OC-ATPase differ from that of the classical V-ATPase and that a 63kd isoform (p63) of the 70kd subunit might be specifically expressed in this cell.

The specificity and nature of the 63kd polypeptide (p63) was further confirmed by the following experiments (Fig. 3B). First, antibodies raised against the N crassa 70kd subunit expressed in E coli as a recombinant fusion protein (Bowman et al, unpublished results) gave similar results (Fig. 3B, lane B), thereby eliminating the

possibility that the original antibodies recognized other proteins contaminating the gel preparation. Second, the 63kd band was absent from kidney, bone marrow or macrophages (Fig. 3B) but could specifically be induced in bone marrow macrophage cultures in the presence of 1,25-dihydroxyvitamin D3 (data not shown), conditions which induce the expression of several osteoclast gene products (Billecocq et al 1990) . Third, the 70kd, 63kd and 60kd subunits could be

distinguished by their electrophoretical mobility when blotting with the antibodies to N crassa 70 and 60kd

subunits simultaneously (Fig. 3B, lane A). The possibility that this 63kd band resulted from proteolytic degradation in the osteoclast preparations was also ruled out by the observations that processing the unpurified fraction

(1/1000) for up to 6 hours under the conditions of

osteoclast purification did not induce any decrease in the amount or apparent Mr of the 70kd subunit, nor did it induce the appearance of a 63kd band, and also that the p63 band could be hormonally induced. The OC-H+ATPase, therefore, specifically contains a 63kd subunit immunologically related to but distinct from the 70kd subunits of other vacuolar proton pumps. Taken together, these results confirm that nature and specificity of the 63kd as an isoform of the 70kd catalytic subunit of the proton pump which is specifically expressed in the OC-H+ATPase. The fact that the ability of these membranes to transport protons and their sensitivity to vanadate

co-purify with the osteoclasts clearly demonstrate that the proton pump which is the subject of this invention is indeed associated with these cells. Although these pumps could be derived from intracellular membranes, a number of

observations indicate that they are present as the apical plasma membrane of this cell (the ruffled-bσrder). First, the enzyme assays and the nucleotide specificity of H+ transport show a marked enrichment in plasma membrane and little contamination with endocytic V-ATPases (Table 1). Second, examination of the ruffled border membrane in situ showed the presence of the same F0-F1 structures found in the inside-out vesicles present in our microsomal

preparations (Fig. 1C). Although the nature of these structures was not known, this coat has been previously described in rat osteoclasts (Kallio et al 1971). Third, the 70kd, 60kd (Vaananen et al 1990) and 31kd (Blair et al 1989) subunits have been immunolocalized at the ruffled border membrane, an observation that was reproduced in the laboratory. The OC-H+ATPase is, therefore, present at the apical ruffled-border plasma membrane of the osteoclast. The OC- H+ ATPase constitutes a novel class of plasma membrane H+ ATPase

It has, therefore, been concluded that the

PC-ATPase represents a new class of F0-F1 H+-ATPase, formed by the assembly of several subunits common to other

V-ATPases and as least one specific and inducible isoform (p63) of the 70kd catalytic subunit. It is speculated that, since this unique subunit forms part of the catalytic component of the proton pump, where vanadate inhibition would occur, it may represent the molecular counterpart for the unique sensitivity of the PC-ATPase to vanadate.

The 63kD subunit of the PC-H+ATPase forms a vanadate- sensitive phosphorylated intermediate.

With reference to Fig. 4, 50 μg of microsomal membrane protein was resuspended in 100 μl of acidification buffer (150 mM KCl, 20 mM HEPES, pH 5.5 with tetramethyl ammonium hydroxide and 5 mM MgSO4) and incubated in the absence (Lanes A and F) or in the presence (Lane C) of 1 mM sodium orthovanadate for 5 min (30). The reaction was started by addition of 200 μCi of [ɣ³²P] ATP (50 ul) and stopped after 20 sec by addition of 50 ul of 20% TCA. For the chase experiment, the reaction was stopped with TCA and the reaction mixture was diluted to 2 ml with 1 mM cold ATP and 1 mM sodium phosphate for 15, 30 or 120 sec. The membranes were pelleted down by centrifugation at 12,000 rpm for 2 min, washed twice and the final pellet was solubilized in 0.5 ml of 10 mM MOPS (ph 7.0), 0.3 M sucrose, 25% glycerol, 2 ug/ml pepstatin A, 5 ug/ml leupeptin and 1% C₁₂E₉. The suspension was vortexed, centrifuged at 12000 rpm for 4 min and the supernatants were incubated 2 hrs at room temperature with the anti N. crassa 70 kD antibody (final dilution 1:100) and protein A-agarose beads were added for 1 hr at room temperature. The suspension was centrifuged at 12000 rpm for 4 min, pellets were washed in the same buffer once, solubilized in sample buffer and separated in acid gels as described (31).

These results demonstrate that the 63kD subunit forms a phosphorylated intermediate.

The novel 63kD subunit also applies to humans.

In order to determine whether the expression of the novel 63kD subunit was unique to avian species or whether it would also apply to humans, we have analyzed by Western blot a Giant Cell Tumor of bone (osteoclastoma). The results clearly demonstrated that, in this tissue, the antibodies to the N. crassa 70kD subunit also recognized a 63kD band, as in chicken osteoclast preparations.

These results demonstrate that the unique 63kD subunit is also present in human osteoclasts. While the invention has been described with respect to various specific examples and embodiments, it is to be understood that the invention is not limited thereto and that it can be variously practiced within the scope of the following claims.

Claims

C LAIMS :

1. An ATP dependent proton (H⁺)-pump in osteoclast membranes which is sensitive to both vanadate and NEM.

2. An ATP dependent proton (H⁺)-pump according to claim 1 which is characterized by a sensitivity to both vanadate and NEM in amounts below the ml. range.

3. An ATP dependent proton (H⁺)-pump according to claim 1 which is characterized by a sensitivity to both vandate and NEM and which is immunologically distinguished from other proton (H⁺)-pumps by the presence of a 63 kD subunit.

4. An ATP dependent proton (H⁺)-pump according to claim 1 which is characterized by a sensitivity to vanadate resulting in 50% inhibition of activity at a level of 100 μM of vanadate.

5. An ATP dependent proton (H⁺)-pump according to claim 1 which is characterized by a sensitivity to vanadate resulting in 100% inhibition of activity at a level of 1 mM of vanadate.

6. An ATP dependent proton (H⁺)-pump according to claim 1 which is 100 to a 1000 fold more sensitive to nitrate ions than known proton pumps.

7. A method of treating the effects of osteopenia comprising introducing an effective amount of a suitable therapeutic agent into the osteoclast membrane containing an ATP dependent proton (H⁺)-pump which is sensitive to both vanadate and NEM.

8. A method of treating the effects of osteopenia according to claim 7 wherein the ATP dependent proton

(H⁺)-pump is characterized by a sensitivity to both vanadate and NEM in amounts below the mM range.

9. A method of treating the effect of osteopenia according to claim 7 wherein the ATP dependent proton

(H⁺)-pump is characterized by a sensitivity to both vanadate and NEM and which is immunologically distinguished from other proton (H⁺)-pumps by the presence of a 63 kD subunit.

10. A method of treating the effects of osteopenia according to claim 7 wherein the ATP dependent proton (H ) -pump is characterized by a sensitivity to vanadate resulting in 100% inhibition of activity at a level of 100 μM of vanadate.

11. A method of treating the effects of osteopenia according to claim 7 wherein the ATP dependent proton (H⁺)-pump is characterized by a sensitivity to vanadate resulting in 100% inhibition of activity at a level of 1 mM of vanadate.

12. A method of treating the effects of

osteopenia based upon targeting the unique 63 kD subunit.

13. A method of treating the effects of osteopenia using the unique 63 kD subunit as a basis for one or more specific drugs or antibodies to inhibit proton transport by osteoclasts.

14. A method of treating the effects of osteopenia using the unique 63 kD subunit to target to the osteoclast compounds that would inhibit bone resorption.

15. A method of treating the effects of osteopenia wherein the ATP dependent proton (H⁺)-pump is characterized by a sensitivity to nitrate resulting in 100% inhibition of activity at a level of 1 mM nitrate.

AMENDED CLAIMS

[received by the International Bureau on 2 November 1992 (02.11.92);

original claims 1-15 replaced by amended claims 1-14

(3 pages)]

CLAIMS :

1. A purified ATP dependent proton (H+)

-pump in osteoclast membranes which is characterized by a high sensitivity to each of vanadate and NEM and which has a 63kD subunit.

2. The ATP dependent proton (H+) -pump according to claim 1 which is characterized by a high sensitivity to each of vanadate and NEM in amounts less than lmM.

3. The ATP dependent proton (H+) -pump according to claim 4 which is characterized by a sensitivity to vanadate resulting in 50% inhibition of activity at a level of 100 uM of vanadate.

4. The ATP dependent proton (H+) -pump according to claim 17 which is characterized by a sensitivity to vanadate resulting in 100% inhibition of activity at a level of lmM of vanadate.

5. An ATP dependent proton (H+) -pump in osteoclast membranes which is characterized by a high sensitivity to each of vanadate and NEM in amounts less than lmM and which has a 63kD subunit, wherein said proton (H+)-pump also exhibits 100 to 1000 fold higher sensitivity to nitrate ions than other known proton pumps, when such sensitivity is measured by known assay techniques used to measure the characteristics of proton transport into inside-out vesicles and proton ATPase activity, and which is

characterized by a sensitivity to nitrate resulting in a 100% inhibition of activity at a level of lmM nitrate.

6. A method of treating the effects of osteopenia comprising introducing an effective amount of a suitable therapeutic agent into the osteoclast membrane containing an ATP dependent proton (H+) -pump which is sensitive to both vanadate and NEM.

7. A method of treating the effects of osteopenia according to claim 6 wherein the ATP dependent proton (H+)-pump is characterized by a sensitivity to both vanadate and NEM in amounts below the mM range.

8. A method of treating the effects of osteopenia according to claim 6 wherein the ATP dependent proton (H+)-pump is characterized by a sensitivity to both vanadate and NEM and which is immunologically distinguished from other proton (H+) -pumps by the presence of 63kD subunit.

9. A method of treating the effects of osteopenia according to claim 6 wherein the ATP dependent proton (H+)-pump is characterized by a sensitivity to vanadate resulting in 100% inhibition of activity at a level of 100 uM of vanadate.

10. A method of treating the effects of osteopenia according to claim 6 wherein the ATP dependent proton (H+) -pump is characterized by a sensitivity to vanadate resulting in 100% inhibition of activity at a level of lmM of vanadate.

11. A method of treating the effects of osteopenia based upon targeting the unique 63kD subunit.

12. A method of treating the effects of osteopenia using the unique 63kD subunit as a basis for one or more specific drugs or antibodies to inhibit proton transport by osteoclasts.

13. A method of treating the effects of osteopenia using the unique 63kD subunit to target to the osteoclast compounds that would inhibit bone resorption.

14. A method of treating the effects of osteopenia wherein the ATP dependent proton (H+) -pump is characterized by a sensitivity to nitrate resulting in 100% inhibition of activity at a level of lmM nitrate.