Characteristic localization of carbohydrates in osteoclasts by lectin cytochemistry

Bone, 13, 41 l-416, (1992) Printed in the USA. All rights reserved.

8756-3282192 $5.00 + .OO Copyright 0 1992 Pergamon Press Ltd.

Characteristic Localization of Carbohydrates in Osteoclasts by Lectin Cytochemistry H. NAKAMURA

and H. OZAWA

Department of Oral Anatomy, Niigata University School of Dentistry, Niigata, Japan correspondence and reprints: Gakkocho-dori-2, Niigata 95 1, Japan.

Address.for

H. Nakamura D.D.S., Ph.D., Department of Oral Anatomy, Niigata University School of Dentistry,

Abstract

toradiographic (Owen 1968) and histocytochemical techniques (Takagi et al. 1982). Lectin histocytochemistry is a method commonly used for their detection, because lectins specifically bind to sugar residues (Damjanov 1987). Despite many lectin histocytochemical studies on osteoclasts, very little is known about the process of glycosylation and the localization of carbohydrates on cement lines (Vgllnlnen et al. 1986; Takagi et al. 1988, Illes & Fischer 1989; Nakamura et al. 1989). We therefore assessed the degree of localization of osteoclasts’ glycocali and carbohydrate complexes in cement-line-like structures, as well as the ability of osteoclasts to metabolize sugar. Our experiments resulted in the localization of sugar residues in osteoclasts at the electron-microscopic level with HRPconjugated lectins.

Lectin cytochemistry was performed to clarify the process of glycosylation and the localization of glycocalyx in osteoclasts. Microslicer sections of decalcified rat tibiae were incubated in the presence of HRP-conjugated lectins (Con A, PNA, MPA, WGA, UEA-1). Lectin reactions in cell organelles revealed that glucose (Glc) and mannose (Man) are transferred to carbohydrate chains in nuclear envelopes, rough endoplasmic reticuli, and the cis and medial sides of the Golgi apparatus. N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and/or N-acetyhreuraminic acic (NANA) residues are transferred, in turn, in the Golgi apparatus. Lectin reactions detected in lysosomal structures suggest that some sugar residues are incorporated into carbohydrate chains of hydrolytic enzymes, such as acid phosphatase and arylsulfatase. Others would be transported to plasma membranes as glycocalyx. PNA and MPA reactions were most evident on ruffled borders of osteoclasts. On the other hand, cement-line-like structures on bone surfaces displayed Con A, MPA, and WGA positive reactions. The following factors suggest that osteoclasts actively metabolize sugar: characteristic localization of glycocalyx in osteoclasts reflect the polarity of osteoclasts, and carbohydrate complexes in cementline-like structures seem to play an important role in the coupling phenomenon in bone tissue.

Materials and Methods Tibiae of male Wistar rats (100 g) were used for cytochemical observation. The rats were anaesthetized with nembutal and perfused through the left ventricle, fist with Ringer’s solution, then with 2% paraformaldehyde, 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.3) for 10 min. The tibiae were dissected, immersed in the same fixative for two hours, and decalcified in 4.13% EDTA for two weeks at 4” C, then Microslicer and Cryostat sections of approximately 50 pm thickness were obtained. The sections were rinsed overnight with PBS containing 0.02% saponin to allow lectins to penetrate cell organelles thoroughly, and incubated in HRP-conjugated lectin solution for 24-48 h at 4” C: 10 pgiml Con A (Concanavalin A); 10 pg/ml UEA-1 (V1e.r europeus agglutinin I); 20 pg/rnl PNA (Peanut agglutinin); 5 p&ml WGA (Wheat germ agglutinin) (Seikagaku Kogyo Co., Ltd. Tokyo, Japan); 10 Kg/ml MPA (Maclura pomifera agglutinin) (E.Y. Laboratories, Inc. San Mateo, CA, USA). The sections were washed with PBS and then refixed with 2.5% glutaraldehyde. Once rinsed with PBS and 0.05 M Tris-HCI buffer (pH 7.6), they were then immersed in DAB solution (0.05% Diaminobenzidine in 0.05 M Tris-HCl buffer, pH 7.6), and again in DAB-H,02 solution for 10 minutes at room temperature. They were postfixed with 1% 0~0, in 0.1 M phosphate buffer (pH 7.3) for one hour at 4” C. After dehydration in a graded acetone series, the sections were embedded in Epok 812. Ultrathin sections obtained by a Porter-Blum MT-l were stained with lead citrate. These samples were observed under a Hitachi H-500 electron microscope at an accelerating voltage of 75 kV.

Key Words: Osteoclast-Lectin cytochemistry-Glycosylation-Glycocalyx--Carbohydrate-Cement line. Introduction Osteoclasts are multinucleated cells that resorb bone by their proton production and hydrolytic enzymes (Vaes 1968, 1988; Gothlin & Ericsson 1971; Luft 1971; Bonucci 1974; Dorey & Bick 1977; Ejiri 1983; Baron 1989; Irie & Ozawa 1990). Osteoclasts also take part in the coupling phenomenon by forming, on the bone surfaces, cement lines that contain acid phosphatase and polysaccharides (Owen 1968; Tran Van et al. 1982a,b; Baron et al. 1984; Oguro & Ozawa 1988, 1989). Glycocalyx, originating in glycolipids and membrane-bound glycoproteins, is known to change character according to the cytodifferentiation and the acquisition of cell polarity (Damjanov 1987; Nakamura & Ozawa 1990). Carbohydrates in osteoclasts have so far been studied by au411

H. Nakamura and H. Ozawa: Lectin cytochemistry

412

controls Parts of the sections were immersed in DAB and DAB-H20, solution without incubation in HRP-conjugated lectin. Nonspecific HRP binding was examined by incubating the sections with 1 purpurogallin/ml of native HRP. Binding specificity was tested by incubating the sections with inhibitory sugars: 0.1 M a-methyl-D-mannoside for Con A; 0.1 M L-fucose (Fuc) for UEA- 1; 0.1 M galactose (Gal) for PNA; 0.1 or 0.5 M a-D-melibiose for MPA; 0.1 or 0.5 M N-acetyl-D-glucosamine for WGA.

Results 1. Lectin-reaction

in cell organelles

of osteoclasts

Osteoclasts had widely distributed rough endoplasmic reticuli in cytoplasms and well-developed Golgi apparatus surrounding nuclei. In these cell organelles, Con A, which binds to Glc and Man residues, reacted in the nuclear envelopes, rough endoplasmic reticuli, and the cis and medial sides of the Golgi apparatus resi(Fig. la). MPA bound to GalNAc and Gal(l-+3)GalNAc

in osteoclasts

dues reacted in the cis and medial sides of the Golgi apparatus (Fig. lb). WGA bound to GlcNAc and NANA residues reacted in the Golgi apparatus (Fig. lc). WGA reaction was also positive in nuclear pores (Fig. lc). However, the PNA reaction which shows the existence of Gal and/or Gal( 1-+3)GalNAc residues was not detected, either in the rough endoplasmic reticulum or in the Golgi apparatus (Fig. Id). Tubular lysosomes and lysosomelike structures displayed positive Con A, PNA, MPA, and WGA reactions (Fig. 1). The four lectins all showed similar and strong staining characteristics in lysosomal structures. UEA-1, however, which binds to Fuc residue, was negative in the cell organelles of osteoclasts. 2. Lectin reaction on the plasma membrane We observed lectin reactions at three distinct regions of the plasma membranes of osteoclasts. First, the plasma membrane of the ruffled border reacted positively to Con A, PNA, MPA, and WGA (Figs. 2a,b and 3). Similar reactions were seen in the membranes of vacuoles located near the ruffled border. As for

Fig. 1. Electron micrographs of cell organelks in osteoclasts stained by HRP-conjugated lectins. (a) Con A reactions detected in the nuclear envelope (arrowheads), endoplasmic reticuli (rER), cis and medial sides of Golgi apparatus (Go) and lysosomal structure (Ly). X23,ooO. (b) MPA reactions in cis and medial sides of Golgi apparatus (Go) and lysosomal structures (Ly). X 15,000. (c) WGA reactions detected in Golgi apparatus (Go) and lysosomal structures (Ly). WGA reactions are also seen in the nuclear pores (arrowheads). X20,000. (d) Positive PNA reactions in tublar lysosomes (arrowhead) and lysosomal structures (Ly) but negative in Golgi apparatus (Go). X 17,000. N: nucleus.

H. Nakamura

and H. Ozawa: Lectin cytochemistry

in osteoclasts

413

Fig. 2. Electron micrographs of osteoclast stained with HRP-PNA. (a) Positive PNA reactions on ruffled border (RB) and vacuoles (V). x4,C0CJ blocId capillary, Oc: osteoclast. (b) Highly magnified ruffled border. PNA reactions are detected on the biological membranes of ruffled border X20,000. and vacuoles (V). ~7,000. (c)Highly magnified clear zone. PNA reaction is positive only on the coated pit (arrowhead).

stainability among these lectins, reactions to PNA and MPA were stronger than the others. Second, the plasma membrane of the clear zone showed Con A, MPA, and WGA reactions (Fig. 3a). However, no reaction to PNA was detectable, except for the coated pits (Fig. 2~). Third, the basolateral plasma membrane of osteoclasts showed moderate reaction to Con A, PNA, MPA, and WGA (Fig. 2a). No UEA-I reaction was observed on the plasma membrane of osteoclasts.

borders of osteoclasts (Fig. 3) and on cement-line-like structures. WGA reaction was especially strong compared with other lectins. On occasion, we observed mononuclear cells or excavated osteocytes with moderately developed rough endoplasmic reticuli lined on these structures (Fig. 4a). Lectin-reaction-positive coated pits were observed on the plasma membranes of mononuclear cells that came in contact with the cement-line-like structure (Fig. 4b). PNA and UEA-1 reactions were negative on bone surfaces and matrices.

3. Lectin reaction on cement-line-like

4. Control

structure

In this report, laminar structures on bone surfaces that lead to resorbed bone surface and later become cement line will be referred to as “cement-line-like structures.” Con A, MPA, and WGA reactions were positive on bone surfaces under the ruffled

Sections incubated with native HRP or without lectin displayed no reaction whatsoever. In the sections incubated with inhibitory sugar, the lectin reaction was either eliminated or evidently diminished.

H. Nakamura

414

and H. Ozawa: Lectin cytochemistry

in osteoclasts

0a

Fig. 3. Electron micrographs of ruffled borders stained with HRP-conjugated lectins. (RB) and vacuoles (V). Bone surface (arrowheads) under the ruffled border and clear detected on the biological membrane of ruffled border (RB) and vacuoles (V). Bone HRP-MPA. X 10,000. (c) WGA reactions seen on the plasma membrane of ruffled on the bone matrix under the ruffled border (arrowheads). X7.000.

Discussion I. The localization of carbohydrates

in osteoclasts

The lectin-staining patterns in cell organelles of osteoclasts suggest that Glc and Man are transferred to carbohydrate chains in the nuclear envelopes, rough endoplasmic reticuli, and the cis

(a) Con A reactions on the biological membranes of ruffled border zone are also stained by HRP-Con A. X7,600. (b) MPA reactions surface (arrowheads) under the ruffled border are also stained by border (RB) and vacuoles (V). WGA reactions are also positive

and medial sides of the Golgi apparatus. GalNAc is, in its turn, transferred in the cis and medial sides of the Golgi apparatus. GlcNAc and/or NANA are transferred in the Golgi apparatus. This would seem to imply that osteoclasts synthesize N-linked, O-linked carbohydrate chains and/or glycosaminoglycans of proteoglycans, as well as other cells. Some sugar residues would be incorporated as carbohydrate chains of enzymatic glycoproteins, such as acid phosphatase (Wergedal & Baylink 1969; Luft

H. Nakamura and H. Ozawa: LRctincytochemistry in osteoclasts

415

Fig. 4. Electron micrographs of cement-line-like structure stained with HRP-WGA. (a) WGA reactions detected on cement-line-like structure (arrowheads) which continue to Howship’s lacuna. Note mononuclear cell (MNC) on the cement-line-like structure. Mitosis is also observed neighboring it. ~4,900. (b) Highly magnified cement-line-like structure and mononuclear cell. Coated pit (arrow) of mononuclear cell shows WGA positive reaction, as well as cement-lin&like structure. x 16,000.

1971), tar&ate-resistant acid phosphatase (HammarstrGm et al. 1971; Minkin 1982; Oguro & Ozawa 1988), and arylsulfatase (Dorey and Bick 1977, Baron et al. 1985, 1986a); this hypothesis is supported by the tubular lysosomes and lysosomal structures that show positive lectin reactions. Other sugar residues would be transported to the plasma membranes as carbohydrate moieties of glycolipids and/or membrane-bound glycoproteins, due to the positive reaction on the plasma membranes of osteoclasts. Many reports note that the plasma membrane of a ruffled border contains H+-ATPase (Baron et al. 1985; Akisaka & Gay 1986; Blair et al. 1989; V%&%nen et al. 1990) and basolateral plasma membrane possesses Na+-K+-ATPase (Baron et al. 1986b). In our study, lectin reactions were strong on the plasma membranes of ruffled borders and moderate on the basolateral plasma membranes. These reflect the localization of membrane-bound glycoproteins, such as H + -ATPase and Na + K+-ATPase, and therefore correlate to the polarity of osteoclasts. In addition, the plasma membranes of ruffled borders and coated pits of clear zones would contain much Gal and GalNAc by PNA and MPA reaction. These osteoclast structures are related to the endocytosis of bone matrix and seem to be receiving signals from bone matrix. Strong lectin reactions on these portions suggest that carbohydrate chains recognize bone matrix by such mechanisms as the endocytosis of asialoproteins present in hepatocytes (Quintart et al. 1983). Furthermore, the intense PNA and MPA reactions on the biological membranes of vacuoles, as well as the plasma membranes of ruffled borders, suggest that both membranes contain similar carbohydrate chains. Hence, vacuoles in osteoclasts may result from the withdrawal of ruffled border, as suggested in the study of calcitonin administration (Baron et al. 1990).

On the other hand, WGA reaction on nuclear pores of osteoclasts would reflect GlcNAc residues of nuclear pore glycoproteins, elsewhere reported to contain O-linked GlcNAc (Holt et al. 1987). No UEA-1 reaction in cell organelles of osteoclasts suggests that osteoclasts hardly metabolize Fuc. 2. Lectin reactions in cement-line-like structures In bone remodeling, osteoclastic bone resorption is always followed by osteoblastic bone formation (Takahashi et al. 1964). Howard et al. (1981) reported that coupling factors that stimulate bone formation are released during the process of bone resorption. Furthermore, Baron et al. (1984) suggested that coupling factors exist on a part of the cement line that contains polysaccharide and acid phosphatase (Owen 1968; Tran Van et al. 1982a,b; Oguro & Ozawa 1988, 1989). Our study demonstrates that cement-line-like structures contain Glc, Man, GlcNAc, GalNAc, and/or NANA. The cement line would consist of three probable components: (a) those secreted by osteoclasts, such as acid phosphatase, (b) those from serum and adsorbed to bone matrix, such as a,HS-glycoprotein, and (c) those synthesized by osteoblasts and present in the bone matrix. IGF-I,11 (Canalis et al. 1988; Frolik et al. 1988; McCarthy et al. 1989), TGF-P (Seyedin et al. 1986; Pfeilschifter & Mundy 1987; Noda & Camilliere 1989), and BMP (Wozney et al. 1988), which belong to third group, have been especially suggested to localize on the cement line at the activated forms by acids and/or hydrolytic enzymes of osteoclasts. Hence, lectin-reactions on cement-linelike structures would reflect the localization of such glycoproteins and/or proteoglycans. Since some proteoglycans in extracellular matrix can bind some growth factors (Gordon et al. 1987; Yamaguchi et al. 1990), carbohydrate complexes on ce-

H. Nakamura and H. Ozawa: Lectin cytochemistry in osteoclasts

416

ment-line-like structures could adsorb and reserve coupling factors to promote the proliferation and/or the cytodifferentiation of osteoprogenitor cells. In conclusion, characteristic localization of glycocalyx in osteoclasts would reflect the functional polarity of osteoclasts, and carbohydrate complexes in cement-line-like structures may play an important role in the coupling phenomenon.

We wish to thank Dr. S. Ejiri, Associate Professor, Department of Oral Anatomy, Niigata University School of Dentistry. Thanks are also due to the staff of the Department of Oral Anatomy for their assistance. We are also grateful to Dr. Takashi Suzuki for the

Acknowledgmenrs:

helpful discussions

in the preparation

of this manuscript.

References Akisaka, T.; Gay, C. V. Ultracytochemtcal evidence for a proton-pump adenosine triphosphatase in chick osteoclasts. Cell Tissue Res. 245: 507-512; 1986. Baron, R.; Vignery, A.; Horowitz, M. Lymphocytes, macrophages and the regulation of bone remodeling. Peck, W. A., ed. Bone and Mineral Research, Annual 2. Amsterdam: Elsevier; 1984; 175-243. Baron, R.; Neff. L.; Louvard, D.; Courtoy, P. J. Cell-mediated extracellular acidification and bone resorption: Evidence for a low pH in resorbing lacunae and localization of a 100.kD lysosomal membrane protem at the osteoclast tuffled border. J. Celf Biol. 101:221&2222; 1985. Baron, R.: Neff, L : Tran Van. P.: Nefussi, I. R.: Vignery. A. Kmettc and cytochemical idennfication of osteoclast precursors and then differentiation mto multinucleated osteoclasts. Am. J. Pnthol. 122: 3633378: 1986a Baron, R.; Neff, L.; Roy, C.: Boisvert, A.; Caplan M. Evidence for a high and specific concentration of (Na+ ,K * )ATPase in the plasma membrane of the osteoclast. Cell 46: 31 l-320: 1986b. Baron, R. Molecular mechanisms of bone resorption by the osteoclabt. Anur. Ret 224: 317-324: 1989. Baron, R.; Neff. L.; Brown, W.; Louvard, D.: Cowtoy, P. J. Selecttve mtemalization of the apical plasma membrane and raptd redistnbutmn of lysosomal enzymes and mannose 6-phosphate receptors during osteoclast mactivation by calcitonin. J. Cell Sci. 97:439-447; 1990. Blair, H. C.; Teitelbaum, S. L.; Ghiselli, R.; Cluck, S. Osteoclasttc bone resorp tion by a polarized vacuolar proton pump. Science 245:855-857; 1989. Bonucci, E. The organic-inorganic relationships in bone matrix undergoing osteoelastic resorption. C&if Tissue Res. 16~13-36: 1974. Canalis, E.; McCarthy, T.; Centrella. M. Isolation and charactertzatton of insuhnlike growth factor 1 (somatomedin-C) from cultures of fetal rat calvariae Endocrinology 122:22-27; 1988. Damjanov, 1. Biology of disease. Lectm cytochemistry and htstochemtstry. Lob. Invesf. 57:5-20; 1987. Dorey, C. K.; Bick, K. L. Ultrahistochemical analysis of glycosammoglycan hydrolysis in the rat periodontal lig&ment. C&if. Tissue Res. 24: 143-149: 1977 Ejiri, S.; The preosteoclast and its cytodifferentiation into the osteocla$t: Ultrasttuctural and histochemical studies of rat fetal pa&al bone. Arch. histo/. ./up 46~533-557; 1983. Frolik. C. A.; Elhs, L. F.; Williams, D. C. Isolatmn and charactertzatton of tosulin-like growth factor-11 from human bone. Eiochem. Biophw Res. Comnun. 151:1011-1018; 1988. Gordon, M. Y.: Riley. G. P.; Watt, S. M.; Greaves, M. F. Compattmentalizauon of a haematopoietic growth factor (GM-fSF) by glycosaminoglycans in the bone marrow microenvironment. Nature 326~403405; 1987. Giithlin, G.; Ericsson, J. L E. Fine structural localization of actd phosphomonoesterase in the brush border region of osteoclasts. Histochemie 28:337344; 1971. Hammarstrom. L. E., Hanker, J. S.; Toverud, S. U. Cellular differences in acid phosphatase isoenzymes in bone and teeth. Clin. Orrhop. 78:151-167: 1971 Halt, G. D.; Snow, C. M.; Senior, A.: Haltiwanger, R. S.; Gerace. L.: Han. G. W. Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine. J. Cell Eiol. 104:1157-I 164; 1987. Howard, G. A.; Bonemiller, B. L.; Turner, R. T.: Rader. J. I. Parathyrotd hermane stimulates bone formation and resorption in organ culture: Evtdence for a coupling mechanism. Proc Narl. Acad. Sci. USA 78:3204-3208; 1981. Illes, T.; Fischer, J. Distribution of lectin binding glycoprotein in osteoclasts Hisrochemisrv 91:5>59: 1989.

Irte. K.. Ozawa, H. Relationships between tooth eruption. occlusion and alveolar bone resorption: Cytological and cytochemical studies of bone resorption on rat incisor alveolar bone facing the enamel. Arch. Hisrol. Cytol. 53:497-509; 1990. Lutt, U Acid phosphatase of osteoclasts demonstrated by electron macroscopic histochemistry. Hisrochemie 28:103-l 17: 1971. McCarthy, T. L.; Centrella. M.; Canalis, E. Regulatory effects of insulm-like growth factors I and II on bone collagen synthesis in rat calvarial cultures. Endocrinology 124:301-309; 1989. Minkin, C. Bone acid phosphatase: Tartrate-resistant acid phosphatase as a marker of osteoclast function. C&if. Tissue Int. 34:285-290; 1982. Nakamura. H.; Ozawa, H. Lectin cytochemistty on the StraNm intermedium and the papillary layer in the rat incisor enamel organ. Arch. Histol. Cyroj. J3:351369; 1990. Nakamura, M.: Akita. H.; Mizoguchi, 1.; Kagayama. M. A histochemical localization on Macluro pomifero lectin during osteogenesis. Hisrochemisrr) 92:225-230; 1989. Noda, M.; Camilliere. J. J In viva sttmulation of bone formation by transforming growth factor-p. Endocrmology 124:2991-2994; 1989. Oguro. 1.; Ozawa, H. The histochemical localization of acid phosphatase activity in BMU. J.B.M.M. 6:19C-195: 1988. Oguro, 1.; Ozawa. H. Cytochemical studies of the cellular events sequence m bone remodeling: Cytological evidence for a coupling mechanism. J.B.M.M. 7:3& 36: 1989. Owen, M. Uptake of 3H~glucosamine by osteoclasts. Nnture 220:1335-1336: 1968 Pfeilschtfter, J : Mundy, G. R. Modulation of type B transfotming growth factor activity in bone cultures by osteotropic hormones. Proc. Nafl. Acad. Sci USA 84:2024-2028; 1987. Qumtart, J . Courtoy, P. J., Limet, 1. N.; Baudhuin, P. Galactose-specrfic endocytosis in rat liver Biochemical and morphological characterization of a lowdensity compartment tsolated from hepatocytes. Eur. J. Biochem. 131: 105112: 1983 Seyedin. S. M.: Thompson. A. Y.; Bentz, H.; Rosen. D. M.; McPherson, J. M.; Corm. A.; Siegel, N. R.: Galluppi. G. R.; Pier. K. A. Cartilage-inducing factor-A. Apparent identity to transforming growth factor-B. J. Biol. Chem. 261:5693-5695: 1986. Takagi. M.: Pan&y. R. T.. Toda, Y.; Denys, F. R. Extracellular and intracellular digestion of complex carbohydrates by osteoclasts. Lab. Invest. 46:288-297; 1982. Takagi. M ; Yagasaki. H.; Baba. T.; Baba, H. Ultrastructural visualizatton of selective peanut agglutinin binding sites in rat osteoclasts. J. Hisfochem. Cyrochem. 36:95-101: 1988 Takahashi. H : Hattner, R., Epker, B N.; Frost, H. M. Evidence that bone resorption precedes formation at the cellular level. Henry Ford Hosp. Med. Bull. 12:35%364; 1964 Tran Van, P.: Vignery, A., Baron, R. An electron-microscopic study of the boneremodeling sequence in the rat. Cell Tissue Res. 225:283-292; 1982a. Tran Van, P.: Vignery. A., Baron, R. Cellular kinetics of the bone remodeling sequence in the rat. Anot. Rec. 202:445-451; 1982b. Vllnlnen, H. K.; Malmi, R.; Tuukkanen. 1.; Sundquist. K.; Harkdnen, P. Identification of osteoclasts by rhodamine-conjugated peanut agglutinin. C&if. Tissue Inr. 39:161-165, 1986. VBLnBnen, H. K.: Karhukorpi, E. K.; Sundquist, K.; Wallmark, B.; Roininen, 1.; Hentunen, T.; Tuukkanen, J.; Lakkakorpi. P. Evidence for the presence of a proton pump of the vacuolar H+-ATPase type in the ruffled borders of osteoclasts. J. Cell Biol. 111:1305-1311; 1990. Vaes. G. On the mechanisms of bone resorption. The action of parathyrotd hormone on the excretion and synthesis of lysosomal enzymes and on the extracellular release of acid by bone cells. 1. Cell Biol. 39~676697: 1968. Vaes. G. Cellular biology and biochemical mechanism of bone resorption. Uin. Orthop. 231:23%271; 1988. Wergedal. J E.; Baylink. D. 1. Distribution of acid and alkaline phosphatase activity in undemineralimd sections of the rat tibia1 diaphysis. J. Hisrochem. Cyrochem. 17:799-806; 1969. Wozney. J. M., Rosen, V.: Celeste. A. J.; Mitsock, L. M.; Whiners, M. I.; Ktitz, R. W ; Hewick, R. M.: Wang, E. A. Novel regulators of bone formation. Molecular clones and activities. Science 242: 15281534; 1988. Yamaguchi, Y.: Mann, D. M.; Ruoslahti, E. Negative regulation of transfomting growth factor-B by the proteoglycan decorin. Nature 346~281-284; 1990.

February 12, I992 April 30, 1992 Dote Accepted: May 19, 1992

Date Received: Date Revised: