ATTACHMENT, POLARITY AND COMMUNICATION CHARACTERISTICS OF BONE CELLS

JO ANNA ILVESA RO
Department of Anatomy and Cell Biology, University of Oulu Biocenter Oulu, University of Oulu

OULU 2001

JOANNA ILVESARO

ATTACHMENT, POLARITY AND COMMUNICATION CHARACTERISTICS OF BONE CELLS

Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium L101 of the Department of Medical Biochemistry, on April 20th, 2001, at 12 noon.

O U L U N YL I O PI STO, O U L U 2 0 0 1

Copyright © 2001 University of Oulu, 2001

Manuscript received 20 March 2001 Manuscript accepted 26 March 2001

Communicated by Docent Antero Salminen Professor Karl Åkerman

ISBN 951-42-5935-1

(URL: http://herkules.oulu.fi/isbn9514259351/)

ALSO AVAILABLE IN PRINTED FORMAT ISBN 951-42-5934-3 ISSN 0355-3221 (URL: http://herkules.oulu.fi/issn03553221/) OULU UNIVERSITY PRESS OULU 2001

These results suggest that cadherin-like molecules may mediate the tight attachment of osteoclasts in the sealing zone area and that the decrease of resorption in AHAVSE-treated osteoclast cultures is due to prevention of sealing zone formation. known gap. We studied the polarity of mesenchymal osteoblasts using osteosarcoma cell line UMR-108 and endosteal osteoblasts in situ in bone tissue cultures. were studied using the well-characterized pit formation assay. and that the blocking of gap junctions decreases both the number and the activity of osteoclasts. Furthermore. Furthermore. suggesting that it is not mediated by cadherins. suggesting a defect in the fusion of mononuclear osteoclast precursors to multinucleated mature osteoclasts. Keywords: cell adhesion. Joanna. Biocenter Oulu.O.Ilvesaro. P. polarity and communication characteristics of bone cells Department of Anatomy and Cell Biology. we demonstrate that rat osteoclasts show connexin-43 staining localizing in the plasma membrane of the cells in cell-cell contacts and over the basolateral membrane of osteoclasts. These findings suggest the bone attaching plasma membrane of osteoblasts is apical. Cadherins are transmembrane glycoproteins usually mediating homophilic calciumdependent cell-cell adhesion. Pan-cadherin antibodies localized cadherin-like molecule in the sealing zone area of osteoclasts. we show rapid inactivation of osteoclasts with AHAVSE. Electron microscopy showed that VSV particles were budding from the culture medium-facing surface. the total resorbed area and the number of resorption pits also decreased in the cultures. osteoblasts. FIN-90014 University of Oulu. cell polarity.junctional inhibitors. In the present work we have studied the effects of the cadherin CAR sequence HAV-containing hexapeptide AHAVSE on osteoclasts. In endosteal osteoblasts. Immunofluorescence confocal microscopy revealed that the vesicular stomatitis virus glycoprotein (VSV G) was targeted to the culture mediumfacing surface. In the present work. The effects of heptanol and Gap 27. osteoclasts. cell communication . Attachment. Influenza virus hemagglutinin (HA) was localized to the bone substrate-facing surface of the UMR-108 cells.B. University of Oulu. and the circulation or bone marrow facing plasma membrane is basolateral in nature. 5000. VSV G protein was found in the surface facing the bone marrow and circulation. On the contrary. which is seen as a decrease in the percentage of osteoclasts with actin rings. These results suggest that gap-junctional connexin-43 plays a functional role in osteoclasts. whereas Influenza viruses budded from the bone substrate-facing plasma membrane. The primary attachment of osteoclasts to bone surface is not affected by the peptide. Finland 2001 Oulu. Finland (Manuscript received 20 March 2001) Abstract Bone resorbing osteoclasts require tight attachment of their plasma membrane to the bone surface in order to retain the specific microenvironment and thus to be able to dissolve the bone matrix underneath. The inhibitors decreased the number and activity of osteoclasts. Gap junctions often mediate communication between different cells and cell types. Treatment of osteoclast cultures with AHAVSE decreased the number of resorption pits and the total resorbed area.

To my husband Vesa .

I want to express my heartfelt gratitude to my parents for always encouraging me to aim as high as I possibly wanted and for always telling me that if I wanted to achieve something. during the years 1995-2001. The expertise of my co-authors. Doctors Päivi Lakkakorpi. for introducing me to the research world and especially to the world of bone. His encouragement. Kalervo Väänänen. for his interest towards my research. in particularly the present and former Bone Heads for the stimulating working atmosphere.D. It is my great pleasure to thank the following persons. I thank him for his love . if I just put my heart into it. for carefully reviewing the thesis and for teaching me during my first years in the University of Jyväskylä and for once sending me to Oulu.. M. sense of humour and enthusiastic attitude towards research and life in general have been inspiring and guiding me during these years and have been more than I could have ever asked for. Ph. M. Especially Anita Kapanen. I could do it.there is nobody quite like her. Ph.D. support. Jussi Halleen and Teuvo Hentunen are warmly acknowledged for fun times in and outside laboratory. Also. I owe my loving thanks to my best girlfriend and colleague Anne Tuomisto for the special bond we two share . M. Kalervo Metsikkö and Pasi Tavi is most sincerely acknowledged.Acknowledgments This work was carried out at the Department of Anatomy and Cell Biology and Biocenter Oulu. for kindly providing me the surroundings. Ph.. I thank my first teacher and supervisor Professor H. deserves my warmest thanks.D. I am grateful to Professor Karl-Erik Åkerman. thus making it possible for me to work in the Department of Anatomy and Cell Biology..D. My husband Vesa.D. Ph.. I wish to express my appreciation to Professor Hannu Rajaniemi. I also thank Professor Juha Peltonen. My humble thanks are due to the whole staff of Department of Anatomy and Cell Biology.. I also wish to thank Sari Annunen. I am in debt to Docent Antero Salminen. Eero Oja and Marja Välimäki for their skilful technical assistance..D. DDS. who have taken part of this work and thus made it possible: I owe my deepest thanks to my mentor Professor Juha Tuukkanen.D..D. University of Oulu. and.D. M. Ph. for his critical appraisal of this thesis. Allan Haimakainen. Ph.D.. Minna Orreveteläinen.

Oulu. Oulu University Scholarship Foundation and the Finnish Cultural Foundation. I am priviliged to have him as my husband. Vesa’s 24-hour ITsupport is also warmly acknowledged. March 2001 Joanna Ilvesaro .and his never-ending optimism and encouragement during hard times. This research was financially supported by Biocenter Oulu.

3’ diaminobenzidine tetrahydrochloride Dulbecco’s modified Eagle’s medium Dimethyl sulfoxide Filamentous actin Fetal bovine serum Fetal calf serum Fluorescein isothiocyanate Functional secretory domain Histidine-alanine-valine N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulphonic acid) Kilodalton Macrophage colony-stimulating factor -6 Micromolar. 10 g/mol Non-collagen protein Osteoclastogenesis inhibitory factor Osteoclast differentiation factor Osteoprotegerin Osteoprotegerin ligand Phosphate-buffered saline Paraformaldehyde Parathyroid hormone .Abbreviations ALP BL BMU BRU BSA CAR CA-II CT Cx-43 DAB DMEM DMSO F-actin FBS FCS FITC FSD HAV Hepes KDa M-CSF µM NCP OCIF ODF OPG OPGL PBS PFA PTH Alkaline Phosphatase Basolateral Bone multicellular unit Bone remodeling unit Bovine serum albumin Cell adhesion recognition Carbonic anhydrase II Calcitonin Connexin-43 3.

RANK RANKL RB RGD SZ TRANCE TRANCER TRAP TRITC SDS SDS-PAGE TEM VNR VSV WGA Receptor activator for NF-κB Receptor activator for NF-κB ligand Ruffled border Arginine-glycine-aspartic acid Sealing zone TNF-related activator-induced cytokine TNF-related activator-induced cytokine receptor Tartrate-resistant acid phosphatase Tetramethylrhodamine isothiocyanate Sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis Transmission electron microscopy Vitronectin receptor Vesicular stomatitis virus Wheat germ agglutin .

Tuukkanen J (2000) Bone-Resorbing Osteoclasts Contain Gap-Junctional Connexin-43. which are referred to in the text by their Roman numerals: I Ilvesaro JM. Väänänen K. Ilvesaro J. II III IV . Tuukkanen J Connexin-Mimetic Peptide Gap 27 Decreases Osteoclastic Activity. Ilvesaro J. Lakkakorpi PT. Väänänen HK (1998) Inhibition of Bone Resorption by Cadherin Cell Adhesion Recognition Sequence Containing Peptide AHAVSE Is Due to Prevention of Sealing Zone Formation. Metsikkö K.List of original publications This thesis is based on the following articles. Tavi P. Submitted. Tuukkanen J (1999) Polarity of osteoblasts and osteoblast-like UMR-108 cells. Väänänen K. Ilvesaro J. Journal of Bone and Mineral Research 14 (8): 13381344. Journal of Bone and Mineral Research 15 (5): 919-926. Experimental Cell Research 242:75-83.

....................... 30 2............... 36 4................................................................................................ 35 4..................1................................2............... 27 2......2.............. 38 4........ 24 2...... III-IV) ...................................3 Confocal laser scanning microscopy (I-IV) ....................................................................................1 Bone matrix components....................... Introduction 2.........3.................................................... 36 4...................4 Degradation of the mineral and organic matrix.......1...........................1 Light microscopy (I.............................................................................................................6 Disorders of bone modeling and remodeling ..........2 Cellular mechanisms of bone resorption .................... 37 4....4..........2 Cell culture (I-IV) .. 31 4............................................1 Antibodies (I-IV)...................................................... 37 4...............................................3 Tissue culture (II) ........................................3............ 38 ............................3 Fluorescence and microscope techniques (I-IV) ...............................2 Attachment of osteoclasts to bone matrix....2.................1 Structure and function of bone tissue ...................................... 38 4.................2........................... 16 2.........................................................................................4 Communication between bone cells......................................3 Cell polarity ......... 29 2......................................5 Digital image analysis (I -IV).................. III-IV) ............. III-IV)................................... 15 2..........1 Reagents (I-IV) ........................2 Bone cells.................... 17 2....2..........1 Isolation and culture of osteoclasts (I..2............................2.Contents Abstract Dedication Acknowledgements Abbrevations List of original articles 1..............2 Conventional fluorescence microscopy (I...........................................2..................... 20 2.2 Cell line (II)............................................ 35 4.......................3........................2............1 Gap junctions ....................................................3...................1 Osteoclastic membrane domains ............3 Cadherins ..........1.................................................................4 Immunoelectron microscopy (II) .................. 36 4......5 Regulation of osteoclastic bone resorption ................................................... 31 2...........3.... 25 2..................................................................... 36 4...................................... 23 2........... 37 4...... 28 2........................

.............................................................2 Osteoclastic bone resorption .................. 39 4. 45 6......................... 39 4........4.......................2 Effects of HAV-containing peptide (I)...............2 Cell surface biotinylation (II) .............................. 41 5..........1 Immunofluorescence data .....3 Polarity of osteoblasts (II)....2.............................. 49 ...........................................................................3 Polarity of osteoblasts .................................4 Gap junctions in osteoclasts (III and IV)........................................................ 39 5.. 40 5................................................................4....... 40 5..4 Gap junctions in osteoclasts .......3 Activity of osteoclasts .............................................1 Metabolic labeling (II) ................................................. III-IV)...4..................................................... 39 4...........................4................. 41 5........................ 40 5.......................2 Effects of gap-junctional inhibitors heptanol and Gap 27 ....3 Biochemical data............ 42 5... 43 5.........1 Methodological aspects.................................................................................. 42 5..3.................................2.............................................2 Electron microscopy data .................... 43 5......................................................................3................................ 46 6......................................................................................................................................................................................................2..............5 Statistical analysis (I... 43 6........... 41 5..1 Primary attachment of osteoclasts to bone surface......2 Cadherins and attachment of osteoclasts to bone ..3.................................4 Viral infections (II)............... 41 5..................................................................4.....................................................1 Cadherin localization in osteoclasts (I) .................... 48 6.............1 Connexin-43 localization in osteoclasts (III) .......................................

or excess bone formation. when serum estrogen levels decrease. but they might also appear to be useful targets for therapy of metabolic skeletal disorders. which remodels and repairs itself throughout life. which leads to disturbance in the bone microarchitecture. Bone also provides the environment for hematopoiesis. The clinical complications of osteoporosis. . which takes place in the marrow of long bones in adults. Excessive bone resorption results in net bone loss. femoral neck and at the wrist. Bone is also a dynamic tissue. At the cellular level. whereas decreased bone resorption. it protects major vital organs and it maintains calcium and phosphate homeostasis. Osteoporosis is a metabolic bone disease leading to decreased bone mass and increased susceptibility to fractures.1 Introduction Bone tissue has three major functions in the body: it offers mechanical support to the body. the polarization of the osteoblasts and the communication between osteoclasts and osteoblasts. Special interested was focused on the attachment of osteoclasts to bone surface. results in increased bone mass. Osteoporosis typically affects women after menopause. Solving of the cellular and molecular mechanisms of bone formation and resorption is important because of the increasing rate of osteoporosis in the aging population. In normal bone tissue the formation and resorption of bone are well balanced so that when old bone is resorbed by osteoclasts. which in turn increases the probability of fracturing. osteoporosis is characterized by extensive bone resorption. The two cell types primarily involved in bone turnover are the bone-forming osteoblasts and bone-resorbing osteoclasts. The experimental part of this thesis work was carried out to clarify some cell biological questions concerning different bone cells. fractures occur most often in the spine. All of these are interesting biological questions. similar amount of new bone is formed by osteoblasts.

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long bones at joints. is found inside the cortex and it is responsible for the metabolic function of bone. During the growth period (first 20-30 years of life). which bear the weight of body. either with intramembranous ossification or endochondral ossification. This means that during embryonic development mesenchymal stem cells of connective tissue differentiate straight into bone forming osteoblasts and start to synthesize bone. form via endochondral ossification. bone-lining cells. form by intramembranous ossification. is reached by late adolescence (Matkovic et al. There are two morphologically distinct types of bone. The remaining volume. This remodeling begins with a localized resorption that is succeeded by a precisely equal formation of bone at the same site (Parfitt 1994). such as the long bones of legs and arms. osteocytes and bone-resorbing osteoclasts. An extensive blood circulatory system provides bone nutrients. On the other hand. Mesenchymal stem cells differentiate into chondroblasts. is composed of blood vessels and bone marrow. a sponge-like structure of bone trabeculi. including bone-forming osteoblasts. which constitutes 85% of total bone in the body and trabecular (cancellous) bone that constitutes the rest. Later on. Mundy 1995). During the next three decades (and beyond) the adult skeleton is maintained by removing and replacing a fraction each year. which first form a cartilage model of the skeleton. more bone is formed than is resorbed. a compact bone layer that forms the body of the bone.1 Structure and function of bone tissue Bone consists of mineralized organic matrix and bone cells. Trabecular bone. 1994. the spaces between the bone trabeculi in long bones.2 Review of the literature 2. hormones and growth factors. such as bones of the skull. which is the site of erythrocyte synthesis. cortical (compact) bone. the . The core of each bone consists of cortex. meaning the maximal amount of bone matrix an individual has attained. which is mainly responsible for the mechanical and protective function. resulting in an increase in bone mass. Peak bone mass. the cartilage model is replaced by bone matrix with the help of osteoblasts and osteoclasts working synchronically. Sometime after the fifth decade. Bone growth in embryonic development can occur in two mechanisms. The flat bones of the body.

but matrix gla-protein can also be found in cartilage in addition to bone. 1990). two types of glycosaminoglycan are found: chondroitin sulfate and heparin sulfate. depending on the magnitude by which resorption and formation are uncoupled. XI and XIII (Gehron 1989). This reduces skeletal strength and increases the risk of fracture over time. making it an excellent method to measure bone resorption rate from urine (Uebelhart et al. whereas the others can also be found in many tissues other than bone.16 formative phase of the remodeling sequence fails to keep the pace with resorptive activity and skeletal mass. In bone. Osteocalcin expression is restricted to bone. the primary secretory product of osteoblasts. Type I collagen represents the majority of bone collagen.1. All connective tissue cells interact with their extracellular environment in response to chemical stimuli that direct and coordinate specific cell functions. These actions require cell attachment and spreading by focal adhesions via integrins. osteocalcin (bone gla-protein) and matrix gla-protein (Glowacki et al. which can be broken down into four general groups of proteins: 1) cell attachment proteins 2) proteoglycans 3) γ-carboxylated (gla) and 4) growth-related proteins. These pyridinium cross-links are only released on degradation of the mineralized collagen fibrils during bone resorption. 22% protein and 8% water (Lane 1979).and intermolecular cross-links. 1989). The molecules are packed laterally in a one-quarter stagger array so that each molecule is offset from its neighbor by approximately one-fourth of its length. This three-dimensional arrangement constitutes the fiber structure found in the bone extracellular space. 1986. Somerman et al. Bone cells synthesize four proteins that affect cell attachment: fibronectin. comprises about 90% of the organic bone matrix. Robey et al. Of these. including the connectivity of trabecular bone. The bone collagen fibrils are highly insoluble because of their many covalent intra. migration and differentiation. thereby rendering it completely insoluble (Eyre et al. 2. In the matrix. 1987. γ-Carboxylation occurs in two bone NCPs. but biochemical analysis have also indicated trace amounts of types III. individual collagen molecules are packed end to end with a short space (gap) between them. Collagen. 1988). thrombospondin. 1988). 1989. 1987). Multiple crosslinking sites combine extracellularly to form pyridinium ring structures tying several collagen monomer molecules together within the formed collagen fiber. bone sialoprotein is exclusively found in bone. such as proliferation. decreases. Type I collagen is the most abundant form of collagen found in the body and is widely distributed in connective tissue. 70% of bone is mineral. osteopontin and bone sialoprotein (Oldberg et al. Proteoglycans are macromolecules that contain acidic polysaccharide side chains (glycosaminoglycans) attached to central core protein. Measurements of . Robey et al. that transduce signals to the cytoskeleton (Ruoslahti et al. The remaining 10% of the organic matrix is composed of non-collagenous proteins (NCPs). the type and pattern of which differs from that in soft connective tissues. V.1 Bone matrix components Bone matrix consists of an organic matrix that is strengthened by deposits of inorganic calcium salts.

Osteoblasts produce and secrete the major part of the organic bone matrix. Four maturational stages of osteoblast development can be distinguished in situ – the preosteoblast. 1988. . The most abundant NCP produced by osteoblasts is osteonectin. osteoblast. Indeed. 1988.1. A number of proteins in bone appear to be associated with the life cycle and function of osteoblasts (Ripamonti et al. pliable organic matrix now becomes relatively hard.17 osteocalcin in serum have proved to be valuable as markers of bone turnover in metabolic diseases (Price et al. 1986. cell membrane permeability and blood coagulation. with traces of calcium and magnesium fluorides (Levy 1894). The composite arrangement of bone tissue yields a material that is very strong for its weight and much stronger than either component would be alone. cells of mesenchymal origin lining the new bone matrix. and strains. about 85% phosphorous.1 Osteoblastic cells Bone formation is the responsibility of osteoblasts. Hauschka et al. osteoblast secretion products that can stimulate osteoblast cell growth in an autocrine or paracrine fashion (Canalis et al. which in a well-regulated process becomes calcified to form mineralized bone. 2+ Osteonectin has high affinity for binding Ca and hydroxyapatite and it also binds to collagen (Termine et al. Preosteoblast is considered the immediate precursor of the osteoblast and is identified in part by its localization in the adjacent of one or two cell layers distant from the osteoblasts lining the bone formation surfaces. Osteoblasts arise from the same pluripotent stem cell with chondroblasts. With the mineral phase. nerve impulse transmission. These proteins may be bone morphogenetic factors or growth factors. 1988). a crystalline lattice in which the principal components are calcium and phosphate ions (DeJong 1926). the otherwise soft. a phosphorylated glycoprotein accounting 2% of the total protein of developing bone in most animal species. 1980). Robey et al. 1992).1. stresses. 1991. rigid material which possesses the necessary mechanical properties that permit it to withstand the forces. and from 40-60% of the body sodium and magnesium are associated with the bone crystals.2. Rapid responses to calcium excesses and deficiencies in the body are crucial for a number of processes. 1988. Osteoblasts are postproliferative. Hauschka et al. Yamaguchi et al. The importance of the role of bone tissue as an ion reservoir can be appreciated from the fact that about 99% of the body calcium. 1996). Grigoriadis et al. 1981) and thrombospondin (Clezardin et al.2 Bone cells 2. 1987). such as TGF-β and insulin-like growth factors. osteocyte and bone-lining cell (Aubin et al. 1991). the most abundant inorganic components of the bone matrix are phosphate and carbonate salts of calcium. Other bone cell products may be associated with the growth or differentiation of osteoblasts in an indirect or as yet unidentified fashion. 2. myoblasts and fibroblasts (Bennett et al. The mineral is in the form of hydroxyapatite. These consequently serve as the major source for the transport of these ions to and from the extracellular fluids. adipocytes. including the regulation of muscle contraction.

They share a common stem cell with monocyte-macrophage lineage cells (Ash et al. Menton et al. such as osteoblasts and maybe even osteoclasts (Bonewald 1999. Kahn et al. and cytokines and growth factors. Walker 1973a. 1992). Osteoblasts can also be recognized by their ability to synthesize a number of phenotype-specific or -associated macromolecules.18 cuboidal. but it has been hypothesized to be involved in the mineralization process (Wennberg et al. thin. The canaliculi are small tunnels in the bone. including the bone matrix proteins. which are thought to represent the inactive form of osteoblast in terms of matrix production. It is generally accepted that osteoclasts are formed by the fusion of mononuclear precursors derived from haemopoietic stem cells in the bone marrow. Walker 1975b. 1990). Feldman et al. 1980. 1992. Walker 1975a. M-CSF (WiktorJedrzejczak et al. 1992) through which osteoblastic/stromal cells stimulate osteoclast formation and resorption via . 1989). Osteoblastic or bone marrow stromal cells are also required for osteoclast differentiation. Doty 1981. 1988. 1999. 1990. which are considered the most mature form of osteoblastic lineage are osteocytes. bone sialoprotein and osteopontin. Zheng et al. 1976. The precise physiological role of alkaline phosphatase is unknown. also known as TRANCE. 1980. Bone lining cells detach from the surface of bone when osteoclasts start the resorbing of bone. Yoshida et al. strongly alkaline phosphatase-positive cells lining the bone matrix at sites of active bone production. Suda et al. osteocyte. rather than incomplete cell divisions (Chambers 1989. Göthling et al. measuring about 1-2 µm in diameter. 2. proteoglycans. Kerby et al. 1984). are covered by flat.1. elongated bone lining cells. Walker 1975a). certain hormone receptors.2 Osteoclasts Osteoclasts. some evidence is gathering that osteocytes sense the mechanical stress to bone and therefore can participate in the bone remodeling process (Burger et al. the majority of bone surfaces that are not being remodeled (i. The differentiation of osteoclast precursors into mature multinucleated osteoclasts requires different factors including hormonal and local stimuli (Athanasou et al. are poorly understood. Osteocyte cell bodies are found in lacunae inside the bone matrix and they have multiple long cytoplasmic processes extending through canaliculi by which they communicate with each other and possibly with other surface bone cells. Mikuni-Takagaki 1999). 1975. 2000). Although the functions of this most abundant cell type of bone.e. are multinucleated cells differentiating from haemopoietic cells. One of the factors produced by these cells that support osteoclast formation is macrophage colony-stimulating factor. Receptor activator for NF-κB ligand (RANKL.2. In the adult skeleton. ODF and OPGL) is the vital signal (Suda et al. first named by Kölliker (Kölliker 1873). such as osteocalcin. undergoing resorption or formation). Osteocytes are smaller than osteoblasts and they have lost many of their cytoplasmic organelles. 1991) and living bone and bone cells have been shown to play a critical role in osteoclast development (Hagenaars et al. After the completion of bone formation a small portion (10-20%) of osteoblasts incorporate themselves within the newly formed extracellular matrix. these cells trapped inside the matrix.

1998. OPG Decoy receptor. 1997a. Regulation of osteoclast formation and resorption by TRANCE/OPG. This makes them relatively easy to identify by light microscopy. which acts as a decoy receptor for TRANCE. Wong et al. Yasuda et al. RANKL. 1. 1986). such as tartrate resistant acid phosphatase (TRAP) (Anderson et al. 1997b. osteoblasts TRANCER. by which osteoblastic or stromal cells can stimulate precursor differentiation into mature osteoclasts.25(OH)2D3 Fig. Osteoclast precursors in turn have a receptor for TRANCE. osteoclasts STIMULATION INHIBITION OPG PTH 1. calcitonin receptor (Warshafsky et al. Osteoblasts express in their plasma membrane TRANCE.1). also known as OCIF). 1998a. 1998b). Osteoclasts are responsible for dissolving both the mineral and organic bone matrix (Blair et al. but they may contain up to 100 nuclei being between 10 and 100 µm in diameter (Göthling et al. 1976). soluble TRANCE. carbonic anhydrase II (Väänänen et al. They are highly vacuolated when in active . Yasuda et al. Multinucleated osteoclasts usually contain less than 10 nuclei. 1997. which acts as a decoy receptor for the RANKL thus inhibiting the true connection between osteoclasts and osteoblasts via RANK and RANKL (Fig. 1.25(OH)2D3 M-CSF PGE2 PGE2. Tsuda et al. Osteoclasts represent terminally differentiated cells expressing a unique polarized morphology with specialized membrane areas and several membrane and cytoplasmic markers.19 a receptor RANK (TRANCER) located in osteoclasts (Lacey et al. called TRANCER. 1983). Osteoblasts also secrete protein that strongly inhibits osteoclast formation called osteoprotegerin (OPG. 1979). 1989). Wong et al. Through this receptor osteoblastic cells can also stimulate mature osteoclastic bone resorption. 1985) and vitronectin receptor (Davies et al. Osteoblasts also secrete a soluble protein called OPG. RANK. thus blocking the stimulating effect of TRANCE to osteoclasts or their precursors.

Understanding the mechanisms that control the remodeling process and its regulation will clarify not only the local control of the function of bone cells but also the pathophysiology of accelerated bone loss and osteoporosis. being continuously broken down and reformed by the coordinated actions of osteoclasts and osteoblasts on trabecular surfaces and in Haversian systems. preventing its resorption by osteoclasts. Throughout life old bone is continuously removed by osteoclasts and new bone is formed by osteoblasts in a process called bone remodeling cycle. The non-mineralized osteoid covers the mineralized bone matrix in vivo. Tran et al. Parfitt 1983).20 state. The factors that determine why a particular site of bone is chosen for remodeling are not clear. In the normal adult skeleton. 1991). Frost 1973). In the normal adult skeleton. Thus. The remodeling of each unit takes about 3-4 months. The remodeling cycle is a coordinated series of events. 1986). the rate of which varies considerably both within and among different bones of the skeleton. Activation involves recruitment of osteoclast precursors to the bone and their differentiation and fusion into functional osteoclasts. Proteases released by osteoblastic cells have been shown to be responsible for dissolving the osteoid and thereby inducing osteoclastic resorption and determining its location (Chambers et al. Frost 1986). as described below. 1986). The modeling of each packet takes a finite period of time resulting that approximately 10-15% of bone surface is continuously being remodeled. Bone resorption is recognized as being a multi-step process and the work done by each BRU is divided into four distinct phases: activation.2 Cellular mechanisms of bone resorption The process of bone remodeling is the key phenomenon in bone cell biology. removing both mineral and organic components of bone matrix (Blair et al. indicating a high metabolic rate (Mundy 1990). One of the most attractive of the current hypotheses is that osteocytes may be involved in the activation (Chambers 1985. the osteoid must be dissolved or mineralized. 2. The adult skeleton is in a dynamic state. bone formation occurs only at sites where bone has previously been resorbed. Bone remodeling occurs in focal and particular units. 1982a. after which osteoclasts can attach to the mineralized matrix and initiate bone resorption (Jones et al. before osteoclasts can begin the actual resorption in vivo. Frost 1973. The term “activation” is used to describe the initiating event that converts a resting bone surface into a remodeling surface. It also involves the penetration of the bone lining cells and recognition of the bone sites to be resorbed (Tran et al. a new BRU is activated every 10 s (Parfitt 1983). reversal and formation (Baron 1977. During the resorption phase osteoclasts work in a concerted fashion. This turnover or remodeling of bone occurs in focal and discrete packets throughout the skeleton. and also contain many mitochondria. called basic multicellular units (BMU) or bone remodeling units (BRU) throughout the skeleton (Frost 1964. 1982b). resorption. where the actions of osteoblasts and osteoclasts are tightly coupled to retain the balance between formation and resorption of bone. The hallmark of the .

creating a tunnel about 2. During the reversal phase macrophages may appear on the resorption surface (Raisz 1988). Wuthier 1969). the osteoclasts drill through the matrix in a direction that is parallel with the long axis of the bone. In cancellous bone. Later it was shown that mitochondria of bone-forming cells might produce a local rise in the levels of mineral ions. 1991). not necessarily excluding one another: the nucleation theory and the matrix vesicle theory (Anderson 1984. The roles of these macrophages are not known. 1969). Formation of new bone begins after the short reversal phase. Two general theories exist for how calcification is initiated in skeletal tissue. certain glycosaminoglycans or lipids could trigger the initiation of calcification (Baylink et al. The mitochondria of columnar and hypertrophic zones of growth plate cells were found out to contain more enzymes associated with calcification (alkaline phosphatase. 1964). osteoclasts resorb saucer-shaped cavities some 50 µm in depth towards its center. they might produce factors that help initiate osteoblastic bone formation (Raisz 1988). 1988). If the fate of osteoclasts at the reversal site is apoptosis. The factors could also be released from the bone matrix during the resorption phase (Mundy et al. Irving 1958. Spector et al. This has been shown to occur with other cells (Arends et al. 1991).21 resorbing surface is the appearance of a scalloped erosion. In cortical bone. Osteoclasts are highly motile and actively migrating cells. The mechanism by which osteoblasts are summoned into the resorption lacuna is uncertain. Williams et al. 1972. Shuttleworth et al. an organic matrix consisting primarily of type I collagen and various other components. The nucleation theory holds that the type I collagen fibril is the major site of crystal nucleation where calcium phosphate crystals are deposited in the hole regions of these fibrils. lysozyme and lactate dehydrogenase) than did those of the resting zone (Arsenis 1972). there is a reversal phase. which was capable of being mineralized (Shapiro et al. It is during the reversal phase that the coupling of bone resorption to formation takes place. In normal adult bone. they can move along the bone surface to another site and restart the resorption phase (Kanehisa et al. biochemical process where the presence of collagens. which lasts from 7-14 days and heralds the switchover from destruction to repair. but it has been suggested that they complete the task of bone resorption. alkaline pyrophosphatase. Wuthier 1968. 1984). but it is probable that a number of paracrine factors produced in or around the remodeling site are involved. osteoid is laid down in discrete lamellae about 3 µm thick. Bone formation is a twostage process beginning with the deposition of osteoid.5 mm long and 200 µm in diameter (Eriksen 1986). The second stage in bone formation is mineralization of the organic matrix. so that after completion of one resorption lacuna. The first line of evidence that cellular activity might be needed for the mineralization of bone arose out of the finding that mitochondria accumulate calcium (Engstrom et al. which occurs after a delay of about 20 days called the mineralization lag time. also called an eroded surface or Howship’s or resorption lacuna. and also a matrix. Once the osteoclasts have resorbed most of the mineral and organic matrix. the function of macrophages could be related to destruction of osteoclast corpses. Alternatively. 1972. 1972. The resorption phase takes about two-three weeks (Baron et al. Anderson 1989. Originally it was believed that the initiation of bone mineralization is mainly an extracellular. 1987). These “coupling factors” could be elaborated by cells involved in the activation of resorption (lining cells) or by osteoclasts themselves or some other cell types present in the resorption lacunae. The matrix vesicles .

Calcified cartilage and woven bone seem to mineralize via matrix vesicles (Bonucci 1971). fibronectin and osteonectin). There is no doubt that in mature calcified bone collagen and the inorganic phase are strongly bound (Irving 1973). 1987). matrix vesicles are rarely. and are thus not lysosomal origin (Ali et al. once initiated. The cell buds off organelles capable of mineral accumulation and then synthesizes proteins that can control the rate at which crystallization proceeds. Somewhat more mineral seems to be associated with aligned gap or hole regions of the fibers. which have more room for inorganic ions than rest of the fibril structure. which in solution seem to regulate the kinetics of the mineralization process (Termine et al. The driving force for the mineral cascade. The rate of mineralization in both woven bone and in lamellar bone seems to depend on the presence of inhibitor molecules (e. These spherulites conglomerate until a continuous mineralized mass is achieved throughout the matrix space. However. Either because there simply is insufficient space for them or for developmental reasons. seems to be the mineral crystals themselves that are first associated with the matrix vesicle membrane. the other in lamellar bone (Gorski 1998. The principal component of the mature mineral phase is hydroxyapatite. alkaline pyrophosphatase and ATPase content. After the mineralization process is triggered.g. Schwartz et al. . mineralization proceeds in association with the heteropolymeric matrix fibrils themselves. The lipid-rich inner membrane of these vesicles becomes the nidus for hydroxyapatite crystal formation and. Instead. These vesicles have high alkaline phosphatase. crystals.g. showing that membrane bound vesicles bud from the long processes of osteoblastic cells and that 2+ the mineralization is induced by the release of Ca -containing vesicles (RingbomAnderson et al. but hardly any acid phosphatase. The extracellular matrix in non-fetal and more abundant lamellar bone is tightly packed with well-aligned collagen fibrils that are “decorated” with complexed NCPs (e. seen in lamellar bone. Termine 1993). membrane-bound bodies that exocytose from the plasma membrane and migrate to the loose extracellular matrix space. It is unknown whether the driving force for mineralization is the bone collagen itself or its associated NCPs. often in contact with the initial mineral crystals (Anderson 1984. crystallization proceeds to the point of obliteration of the vesicle membrane producing a spherulite of clustered. Anderson 1995. one is predominant in both calcified cartilage and primitive woven bone. 1952). pyrophosphate and acidic NCPs). tiny.22 theory states that bone-forming cells produce small 100-200 nm in diameter organelles that have been observed in mineralizing tissues and cell cultures. 1970). but it takes from 3 months up to a year for the matrix to reach its maximum mineral content (secondary mineralization) (Amprino et al. Plausible evidence has been demonstrated to support the matrix vesicle theory. 1986). later it has been speculated that the two mechanisms for bone mineralization are not alternative but parallel. eventually. Other loci for the bone mineral appear to be between collagen fibrils in a brick and mortar fashion (Weiner et al. the mineral content of the matrix increases rapidly over the first few days to 75% of the final mineral content (primary mineralization). if ever. 1994). 1980).

2. 1) When an osteoclast is not resorbing bone. basolateral membrane and functional secretory domain (Fig.1 Osteoclastic membrane domains Inactive osteoclasts. are not polarized and fail to display specialized membrane domains (Holtrop et al. the cell membrane forms a tight attachment to the bone surface. Ruffled border (RB) is a membrane domain facing the bone surface. 1 2 BL 3 FSD BL SZ RB SZ SZ RB SZ Fig. In the middle of the basolateral membrane is the fourth membrane domain. Palokangas et al. 1997). The sealing zone is seen as an electro-dense area free of organelles (Schenk et al. 3) When the osteoclast is actively resorbing bone. the functional secretory domain (FSD). sealing the proteolytic enzymes and acid into the forming resorption lacuna. a fourth domain arises into the basolateral membrane. Sealing zone (SZ) forms a tight contact to the bone. Basolateral membrane (BL) faces towards the bone marrow. Lakkakorpi et al. 1997). 1973) consisting of bundles of actin filaments (King et al. 1977. which is called the resorption lacuna. the functional secretory domain (FSD). it quickly polarizes its membrane into distinct domains. 1991a. which appears when matrix degradation is started (Salo et al. Ruffled border (RB) is a highly convoluted plasma membrane domain under which the actual bone resorption takes place. also called as clear zone. Lakkakorpi et al. in turn. The membrane areas of active osteoclasts can be separated into the following domains: sealing zone. Acidic intracellular vesicles fuse to the cell membrane facing the bone matrix. 1989. 1996). Actively resorbing osteoclasts. Holtrop et al. because it has both lysosomal and endosomal features (Akamine et al. Baron et al. ruffled border. 2).23 2. Osteoclastic membrane domains. . In the sealing zone.2. 1975). 1967) and it has a striated appearance in electron microscopy. it shows no signs of polarized membrane domains. 1977). RB does not fit to any type of previously described plasma membrane domains. 1993. which acts as a route of osteoclasts to exocytose the resorbed material. Sealing zone surrounds the RB. although they can still adhere to the bone surface. 2) Once the osteoclast starts the resorbing. 1988. where the actual resorption takes place. 1990. Bone degradation occurs in the extracellular space between the bone matrix and the RB. thereby isolating the resorption lacuna from the extracellular fluid and permitting the maintenance of a specific microenvironment in the lacuna. thus defining the outlines of the resorption lacuna. Salo et al. are highly polarized cells and they reveal distinct membrane domains and cytoskeletal structures that are not seen in inactive or immature osteoclasts (Baron et al. thereby forming RB membrane. with alternating dark and light areas orientated perpendicular to the bone surface (Malkani et al.

are secreted (Baron 1989). This structure has been suggested to be a late endosome-type compartment (Palokangas et al. It has been suggested that vitronectin receptor mediates the tight attachment of osteoclasts to bone matrix (Nakamura et al. the vitronectin receptor αvβ3 (Davies et al. 1991. Although VNR is important in the function of osteoclasts. from which different hydrolytic enzymes. 1987). 1989. Yamamoto et al. . in echistatin-treated mice. 1998). 1990) and that osteopontin. Väänänen et al. 1990. later identified as anti-vitronectin receptor antibodies (Davies et al. Horton 1990) and α2β1 (Horton et al. basolateral membranes and intracellular vesicles of osteoclasts. and the cadherin family. 1986). In order to retain the specific microenvironment created by the cell organelles in the ruffled border membrane. 1995). 1998. the final tight attachment of osteoclasts to the bone surface is mainly not mediated through it. 1991b. the number of osteoclasts on the bone surface was increased rather than decreased and their electron microscopic morphology was found to be normal (Masarachia et al. via a β3 integrin (Helfrich et al. inhibition of integrin function did not cause detachment of osteoclasts from the bone surface or changes in the clear zone or ruffled borders. 1996. and these have been classified into at least three major molecular families. 1994. 1990).2 Attachment of osteoclasts to bone matrix Bone resorption occurs in an extracellular compartment below the osteoclasts. 1992).24 2. Hence. A number of cell surface glycoproteins have been identified as intercellular adhesion molecules. Nevertheless. Sato et al. but missing from the area of the tight sealing zone (Lakkakorpi et al. Osteoclasts express two members of integrin superfamily. 1991b). Reinholt et al. Controversial data has been reported on the precise localization of vitronectin receptor in osteoclasts. Integrins are adhesion molecules that mediate cell attachment to the substrate by binding to an arginine-glycine-aspartic acid (RGD) consensus sequence in their ligands (Ruoslahti et al. the immunoglobulin (Ig) superfamily. 1989). Horton et al. 1989). It has been shown that peptides containing the RGD sequence potentially inhibit bone resorption (Horton et al. The mechanism by which osteoclasts attach to bone is not fully understood (Dresner-Pollak et al. 1986) is the ligand of the osteoclast vitronectin receptor. 1995). Suda et al. according to their known structural features (Horton 1997.2. 1991b. the integrin superfamily. VNR has also been shown to be located only in the ruffled border. inhibited osteoclastic bone resorption in vitro. Lakkakorpi et al. It has also been shown that osteoclasts adhere in vitro to a wide range of RGD peptide containing proteins. where bone resorption is inhibited. 1989. together with acid. Sato et al. a bone matrix component containing the RGD sequence (Oldberg et al. Integrins have been identified as a family of cell surface receptors that recognize extracellular matrices (Mundy et al. including bone sialoproteins. 1997). Interestingly. Lakkakorpi et al. 1986). osteoclasts need to have a tight attachment to bone matrix in the sealing zone. The first indications of the importance of vitronectin receptor came from the experiments in which monoclonal antibodies against osteoclasts (Chambers et al.

and are predominantly localized at adherent junctions of epithelial cells (Boller et al. Takeichi 1991). The demonstrated role of cadherins in mediating cell interactions. suggest that cadherins play a critical role in embryogenesis and morphogenesis (Grunwald 1993. 1988. 1985. along with the more recently found R.2. These classical N-. posses similar structural and functional domains. and it is thought to be mediated by integrins. calcium-dependent cell-cell interactions. Geiger et al. . Kemler 1992) (Fig. Takeichi 1977). cytoskeletal interactions. 1981.and P-cadherins. phosphorylation and proteolysis (Geiger et al. 1992. B and EP-cadherins. The attachment of these non-resorbing osteoclasts is referred as primary attachment. calcium binding.3 Cadherins Cadherins are a family of structurally and functionally related molecules that usually mediate homophilic. This observation suggests that αvβ3 may play a role in osteoclast differentiation in vitro. possibly by interfering with the migration of osteoclast precursors for their fusion (Tanaka et al. such as echistatin. 1992. 3). inhibited the formation of multinucleated TRAP-positive cells. coupled with the complex spatio-temporal patterns of cadherin expression during embryonic development. 1995). and RGD-containing disintegrins. membrane integration. which allow the cells to attach to the matrix by many simultaneous but weak bonds. The classical cadherins are represented by a group of highly conserved integral membrane proteins initially identified biochemically. E. Expression of vitronectin receptor has also been detected in mononucleated tartrate resistant acid phosphatase (TRAP) positive cells during in vitro osteoclastogenesis in culture. 1991). Takeichi 1991).25 Osteoclasts can attach to and migrate on bone surface in vitro without performing bone resorption. Takeichi et al. and post-translational processing such as glycosylation. Grunwald 1993. immunochemically and functionally about 20 years ago. 2. which include sites for adhesive recognition. Cadherins are protected by 2+ 2+ Ca against proteolysis but they are readily degraded if Ca is absent (Grunwald et al. This enables the cells to move along the bone surface (Väänänen et al.

. The HAV sequence is also found in fibroblast growth factor receptors and the deletion of this region completely abolishes fibroblast growth factor function (Byers et al. The most highly conserved region of cadherins is the cytoplasmic domain. Cadherins associate with the cytoskeleton of cells via proteins called catenins. 1992). Cadherins have generally been associated with homotypic cell interactions among solid tissues. which interact with the cadherins (Knudsen et al. The cadherins and catenins both contain phosphorylation sites that regulate their function in intercellular junctions. Structural organization of cadherins. Cadherins contain a common cell adhesion recognition (CAR) sequence HAV (His-Ala-Val) residing in the outermost region of the extracellular domain (Blaschuk et al. 3. This protein family has at least three major classes. Moreover.COOH Adhesive recognition site α γ β Catenins Fig. a recent study has shown that cadherins can interact with collagen type I (Mbalaviele et al. a proteolytic cleavage site near the cell membrane and functionally critical calcium-binding and adhesion recognition sites. α. many mature tissues also continue to express cadherins. Indeed. 1990a). 1990b). The extracellular domains include multiple glycosylation sites. it has been suggested that cadherins may also be involved in heterotypic or even heterophilic interactions (Cepek et al. The cytoplasmic domain includes sites for interaction with catenin cytoskeletal proteins. Cadherins may thus be partly responsible for maintaining tissue integrity. 1996) and hence could act as cell-matrix adhesion molecules. Interestingly. and cytoplasmic (CYTO) domains. Different cadherins are similar in their overall primary structure: they have a single transmembrane domain that divides the molecules into the amino-terminal extracellular and the carboxy-terminal cytoplasmic domain. Ozawa et al. transmembrane (TM).26 Calciumbinding site Glycosylation sites Phosphorylation sites Cytoskeletal binding site NH2 . 1992. 1993). 1994.EC1 EC2 EC3 EC4 TM CYTO . Indicated are the five extracellular (EC1-EC5). β and γ. and the second most conserved region is around the amino-terminal portion. Tang et al. Even though cadherins have essential roles in embryogenesis. HAV motifs have also been identified in Influenza virus hemagglutinins which are integral membrane proteins mediating attachment and fusion of the virus with the epithelia of the mammalian and avian upper respiratory tract (Blaschuk et al. 1990).

1974. Väänänen et al. Acidification of the extracellular resorption lacuna is completed by passive. Salo et al. 1983). 1990). 1994. Sundquist et al. Rifkin et al. 1994. is present in high amounts in the membranes of a population of intracellular vesicles.2. 1990) releasing their proton content to the lacuna. 1972. Inorganic bone matrix is dissolved by the acidic environment in the resorption lacuna revealing the organic collagen network. possible that since the HAV sequence inhibits osteoclast formation. 1990. . Delaisse et al. 1983. Delaisse et al. the fusion of mononuclear osteoclast precursors mediated by E-cadherin may also involve cytoplasmic signaling events. When the osteoclast stops resorption and moves away from the resorption lacuna. It is. 1985. 1987. Carbonic anhydrase II (CA II) is a cytoplasmic enzyme hydrolyzing carbon dioxide into bicarbonate and protons (Gay et al. therefore. 1984. Blair et al. after which various proteolytic enzymes. Osteoclasts resorb bone by generating a pH gradient between the cell and bone surface (Anderson et al. which are then transported and fused to the RB membrane (Bekker et al. 1994). Hydroxyapatite is first solubilized in the acidified lacuna. Väänänen et al. Laitala et al. 1991) and bone-derived collagenases (Chambers et al. 1992). 1988) secreted by osteoclasts through RB. 1986. Blair et al. 1997. which probably continues to function by transporting protons directly from the cytoplasm to the lacuna. phagocytes clean up the remains and make room for osteoblasts to begin bone formation in the newly formed resorption cavity. E-cadherin is expressed in osteoblasts (Babich et al. It has also been shown that the fusion of osteoclast precursors into multinucleated osteoclasts can be inhibited using a peptide containing the HAV sequence (Mbalaviele et al. Only recently it has been shown that osteoclasts remove the degradation products of bone matrix from the resorption lacuna by transcytosis (Nesbitt et al. potential-driven chloride transport (Blair et al. 1990. 1989. An acidic pH favors the dissolving of the bone mineral (hydroxyapatite) and provides optimal conditions for the action of the proteinases secreted by osteoclasts. E-cadherin has been suggested to be expressed in osteoclasts and to take part in the fusion of mononuclear osteoclast precursors to multinucleated mature osteoclasts. It seems that E-cadherin could have tumor suppressor function. V-ATPase. 1993). as recently proposed (Cowin 1994). Väänänen et al. 1985. 1991). CA II is suggested to be the main source of protons for the acidification of the resorption lacuna (Blair et al.4 Degradation of the mineral and organic matrix Both the mineral and organic component of bone matrix are degraded by osteoclasts in resorption lacunae under ruffled border membranes. such as lysosomal enzymes (Baron et al. 2. V-ATPase transports the protons generated by CA II into these vesicles. Baron et al. 1988. RB thus contains large amounts of V-ATPase. Gay et al. 1995). A vacuolar-type proton pump. 1989. Delaisse et al. Laitala et al. digest the exposed organic matrix.27 reduction of E-cadherin expression is positively correlated with progression of various cancers and increased cellular invasiveness (Umbas et al. 1994) and a new cadherin called OB-cadherin has also been cloned from osteoblasts (Okazaki et al. Minkin et al.

This enables that osteoclasts can remove large amounts of degradation products via transcytosis without detaching from the bone surface and loosing the tight attachment of the sealing zone to the bone surface.25(OH)2D3) (Raisz et al. and finally released into the extracellular space through the FSD. gamma interferon (Gowen et al.25dihydroxyvitamin D3 (1. 1991). Gowen et al. 1987). genetic factors play a major role as determinants of variation in BMD (Pocock et al. which fuse to the transcytotic vesicles and participates in destroying the endocytosed material in the transcytotic route (Halleen et al. 1978). 1985). CT.5 Regulation of osteoclastic bone resorption A balance between bone formation and bone resorption is a necessity for the normal function of bone.2. tumor necrosis factor (Abe et al. 1995). 1983. 1987). The transcytosis route provides a possibility for osteoclasts to further process the endocytosed degradation products intracellularly during their passage through the cell. It does that by its effects to inhibit osteoclastic bone resorption (Friedman et al. transforming growth factor α (Takahashi et al. 1986. On the other hand. interleukin-1 (Chambers 1988. a peptide hormone secreted by the thyroid. calcitonin (CT) (Holtrop et al. progestin. 1996) and is also related to the rate of bone loss (Ferrari et al. Tanaka et al. Chenu et al. it has become apparent that the skeleton is a major target tissue for reproductive steroid hormones. 1983b). androgen. and it is the rate of bone formation rather than the rate of bone resorption that is influenced by genes (Kelly et al. It has also been shown that peak bone mass is associated with vitamin D receptor polymorphism (Viitanen et al. It has been estimated that up to 80% of BMD is genetically controlled. 2. Holtrop et al. play a major role in the . transported through the cell in large vesicles. Raisz 1963). The degradation products. Thomson et al. Miller 1978. and to a lesser extent. Systemic stimulators of bone resorption include parathyroid hormone (PTH) (Barnicot 1948. 1974). regulates blood calcium and phosphate levels (Kumar 1963) by causing short-lived falls in the plasma calcium. 1983a. 1990. 1986) and transforming growth factor β (Bonewald et al. Osteoblasts mediate the effects of PTH and 1. PTH and 1.25(OH)2D3 stimulate bone resorption by increasing the activity of existing osteoclasts and by promoting the differentiation of osteoclast precursors into mature multinucleated osteoclasts and 1. testosterone. Taterossian 1973.25(OH)2D3 on the osteoclasts (Narbaitz et al. are endocytosed to the resorbing osteoclast. The sex steroids. This further supports the osteoclastic penetration deeper into the bone. 1994). Weisbrode et al. 1972. Although many acquired or environmental factors are known to affect the adult bone mineral density (BMD) (Välimäki et al. Silve 1982). 1988) are extremely potent in inhibiting osteoclast differentiation and activity. 1986) and to promote renal calcium excretion (Ralston et al. In the past decades.25(OH)2D3 or other vitamin D metabolites seem to play a role in the correction of calcium malabsorption. 1986) and 1. both organic and inorganic. Gowen et al. 1999).28 1997). 1971). estrogen. which is a common feature in osteoporosis (Lamberg-Allardt 1991. 1974. The bone-specific enzyme TRAP is located in cytoplasmic vesicles. especially estrogen and testosterone.

2. 1988. whereas the malignant form is fatal in early childhood. 2000). 1990. or no symptoms. it will accumulate and lead to catastrophic structural failure. Unless microdamage is repaired. Turner et al. On the other hand. Osteopetrosis is a metabolic bone disease characterized by decreased bone resorption due to reduced osteoclastic number and defects in the ruffled border membrane and leading to impaired function of osteoclasts (Marks. With the passage of time. Although having higher bone mass. Sex steroids are essential for maintenance of normal bone volume and estrogen deficiency is an important risk factor for osteoporosis. with sclerosis of the base of the skull (Elster et al. 1992b). Several bone-related disorders have been mentioned in the literature. Ovariectomy leads to a deficit in ash weight and bone mineral density in adult rats and monkeys (Jayo et al. 1988). which lay down new bone and repair the resorption defects caused by the resorbing osteoclasts. are shortly described here. 1991). two of which affecting the bone mass and the mechanical competence. A reduced marrow space results in a decrease in hematopoiesis even to the point of complete bone marrow failure. Immediately after . repeated strain of skeletal tissue that occurs during ordinary mechanical usage results in the development of microdamage and the need to replace or renew bone tissue. namely. This leads to accumulation of excessive amounts bone seen as thickening of cortical region and decrease in the size of medullary space in the long bones. the osteopetrotic bones are easier to fracture (Tuukkanen et al. 1992a.2.6 Disorders of bone modeling and remodeling Normal bone remodeling is initiated by local events that lead to an increase in osteoclastic activity. Tuukkanen et al. 1993. Takano-Yamamoto et al. fracture. and the amount of bone formed is almost always nearly equal to the amount removed. 1990. not at some other location. Lindsay et al. 1991. osteoclastic activity is highly increased leading to decreased bone mass. Walker 1973b). 1992). Wronski et al. Elster et al. Jr. 1987. The remodeling process replaces aged bone tissue with new bone tissue. The causal role of estrogen deficiency in this condition is well established (Odell et al. Estradiol and progesterone are the principal circulating sex steroids in females but also function in males (Landers et al. Nerve compression and vascular compromise can also occur due to decrease in the caliber of cranial nerve and vascular canals. which increases susceptibility to fractures. This is followed eventually by an increased attraction and proliferation of osteoblast precursors followed by the differentiation of these cells into mature osteoblasts. 1970. Kalu et al. 1978) and it is generally believed that treatment with estrogen of ovariectomized rats prevents the increase of bone resorption (Anderson et al. In the mildest form the disease can produce only few.29 sexual dimorphism of the skeleton in mineral homeostasis during reproduction and bone balance in adults. osteoporosis is a bone disease with reduction in bone mass and a change in the microarchitecture. Formation follows resorption at the resorption site. In osteoporosis. Coupling is also manifest at a higher level of organization: global rate changes in resorption are invariably followed by similar global changes in formation.

and the matrix secretion is in the direction of the bone surface making it seem like a polarized event (Cooper et al. No exclusive reports have been presented on the polarity of osteoblastic cells. These plasma membrane domains have different lipid and protein compositions (Simons et al. Knust 1994. Numerous investigations have been performed to study the therapeutic approaches treating osteoporosis (Rodan et al. a feature characteristic of the apical membrane of epithelial cells. 1988. representing a basolateral membrane domain. In epithelium. Most of the plasma membrane proteins in epithelial cells are distributed primarily to either the apical or the basolateral plasma membrane domains.30 menopause. 2000). 2. 1994). Rodriguez-Boulan et al. 1998. the FSD.3 Cell polarity Cellular polarization is best established in epithelium. 1975). neuronal and certain other cells of the immunosystem are maintained by the characteristically polarized phenotype of these cells (Cereijido et al. but very few are distributed to both of these surfaces. Simons et al. 1980. is the target of viral glycoproteins in osteoclasts infected by Influenza A and Sendai viruses. thus identifying it. Jr. Viral particles of VSV-infected epithelial cells bud from the basolateral membrane. it has been shown that during the secretory phase. Smith et al. 1988). . The membrane polarity of epithelial cells has been studied by using glycoproteins of enveloped viruses as markers (Rodriguez-Boulan et al. osteoblasts look highly polarized. 1992. making the cells look highly polarized (Ringbom-Anderson et al. 1997). The plasma membrane of epithelial cells is divided into two domains: an apical domain facing the external milieu and a basolateral domain in contact with the internal milieu and the blood supply. the annual rate being about 2-3% in Caucasian women (Lindsay et al. which separate the apical and basolateral membranes from each other. et al. Rodriguez-Boulan et al. Influenza virus targets its glycoproteins to the apical membrane domain facing the external milieu. Craig et al. Marks. 1992) Polarization is also essential for embryonic development and tissue morphogenesis. The general features defining cell polarity have been described by far in cultured (MDCK. several matrix vesicles can be found to bud from the long processes of U-2 OS osteoblastic cells. 1992). It is probable that only one of the two pathways to the polarized cell surface requires specific sorting signals (Matter et al. van Meer et al. have presented that during matrix formation. In addition. 1989. However. 1994). which is in contact with the internal milieu and the blood supply. Many of the functions of epithelial. Caco-2) and primary epithelial cells (Handler 1989. 1990). suggesting that FSD corresponds to the apical surface of epithelial cells (Salo et al. The special cap structure on the top of an active osteoclast. 1980. Nelson 1993. different plasma membrane domains are clearly separated by tight junctions enabling the maintenance of membrane polarity. the rate of osteoclastic bone loss increases as much as 10-fold. Most of the osteoclast membrane has receptors for VSV. RingbomAnderson et al. 1996). In epithelial cells. Epithelial cells have tight junctions.

and osteocytes. which connect adjacent cells (Kumar et al. osteocytes and osteoclasts communicate with each other resulting in the elaborate balance of bone formation and resorption. 1996. 1996). which connect cells and provide aqueous intercellular channels for bidirectional movement of small molecules and ions between the cytoplasms of adjacent cells (Warner 1988). Each gap junction pore is formed by a juxtaposition of two intercellular hemichannels in neighboring cells. osteoclasts. Each hemichannel is composed of a hexameric array of transmembrane proteins called connexins. and circulating lymphocytes. Thus. Goodenough et al. 1993). and small metabolites. most cells in normal tissues generally communicate via gap junctions. Osteoblasts. but the mechanisms of this communication are not well characterized. such as ions. erythrocytes. It requires wellorganized actions by osteoblasts. 2.31 2. Väänänen 1993). Intercellular communication can occur via secretion of soluble factors. 4 and 5). a highly related multigene family consisting of at least 13 members (Beyer et al. such as skeletal muscle. second messengers.4 Communication between bone cells A series of complex and coordinated events is required for bone remodeling (Marcus 1987. apart from a few terminally differentiated cells. . direct cell-cell contact through cell adhesion molecules or metabolic and electric coupling through gap junctions.4. This coordinated activity between bone cells suggests the existence of intercellular communication. Gap junctions are specialized regions of cell membrane.1 Gap junctions Most cells communicate with their immediate neighbors through exchange of cytosolic molecules. Saez et al. 1990. which interact to span the plasma membranes of two adjacent cells and directly join the cytoplasm of one cell to that of another (Fig. This activity is made possible by clusters of intercellular channels called gap junctions.

Connexin-43 (Cx43) is widely expressed in different tissues. respectively). Although a variety of drugs have been used to block gap-junctional communication. allowing the movement of molecules smaller than 1000 Da. including brain. such as certain morphogens could be directly transmitted between cells via gap junctions. Jones 1974. 2000). 1984). which are found connecting adjacent osteoblasts together. heart. Connexin channels participate in the regulation of signaling between developing and differentiated cell types. 1995a. and some epithelia. ovary. 1990. Bone cells mediate signals to each other through the communicating gap junctions. Cx45 and Cx46 have been identified in bone. The cylinders represent transmembrane domains (M1-M4). Yellowley et al. embryogenesis for regulating the events between cells (Warner et al. The loops between the first and second. . but preventing the movement of proteins and nucleic acids. smooth muscle. but Cx43 is the predominant gap junction protein expressed in bone (Donahue et al. which is important in. and it is actually the most abundant connexin (Goodenough et al. 1995) and glycerrhetinic acid (Davidson et al. 4. the specificity of the blockers remains to be solved.32 Extracellular E1 E2 Gap 27 Membrane M1 M2 M3 M4 Cytoplasm NH2 COOH Fig. the straight-chain alcohols heptanol and octanol (Donahue et al. kidney. as well as third and fourth. Osteocyte nutrition probably takes place across the gap junctions between the cytoplasmic extensions of neighboring osteocytes. A spectrum of drugs has been shown to inhibit gap-junctional communication with varying degrees of efficacy and specificity. 1996). for instance. The gap-junctional channels have an apparent selectivity based principally on molecular size. Weinger et al. Jeansonne et al. 1995b. Musil et al. such as cAMP. anandamide (Venance et al. Stanka 1975. 1995). 1996. transmembrane domains are predicted to be extracellular (E1 and E2. These include for instance volatile anesthetics such as halothane (Nedergaard 1994). between osteoblasts and overlying cells or osteocytes (Akisaka et al. Connexin structure. 1990). Nedergaard 1994). 1974. Vander et al. Small informational molecules. 1979.

Possible arrangements of connexons to form gap junction channels. Connexons consist of six subunits. as has been shown to happen in osteoblasts (Civitelli et al. 5. Gap-junctional communication would thus be an excellent method for bone cells to rapidly propagate locally generated signals throughout the skeletal tissue. connexins. when the two connexons are different. 1990).or one cell type . if connexons are identical or heterotypic. A dependable means of communication is utterly important for correct coupling of osteoclasts and osteoblasts. Connexons associate end to end to form a double membrane gap junction channel. connexons from neighboring cells may form gap junctions in a matter of minutes or even seconds. A connexon may be homomeric.33 On cell contact.to another throughout the skeletal environment may represent an efficient means of coordinating the action of a variety of cells in a highly complex tissue. 1993). suggesting that connexons pre-exist in the plasma membrane (Rook et al. . when it has more than one species of connexins. The channel may be homotypic. Homomeric Homotypic Heteromeric Homotypic Homomeric Heterotypic Heteromeric Heterotypic Fig. when it is composed of six identical connexin subunits or heteromeric. The capability to exchange nutrients and regulatory molecules from one cell .

The bone multicellular unit works synchronically as a cell syncytium. 3. This requires attaching of bone cells to the matrix. The communication between osteoblasts and osteoclasts is essential for proper bone remodeling . We wanted to learn what are the proteins involved in the tight attachment in osteoclasts and what proteins are needed from the bone surface.we wanted to solve how the actual connection between the cells happens. To study if osteoblasts truly are polarized cells. they have to communicate with each other in order to retain the balance between the activities of different bone cells. which is controlled and regulated by hormonal signals and by mechanical forces adjusted to bone. 2. The secretion of bone matrix happens in a polarized manner. The basic mechanism of this coupling has not been previously known. polarizing of the cells’ plasma membranes into specific domains and contacting of bone cells to each other. To study how osteoclasts attach to bone surface. To study the communication mechanisms by which bone cells communicate with each other. .3 Aims of the present study Osteoclastic bone resorption as well as osteoblastic formation of new bone are both required for the correct composition of bone tissue. This is called bone remodeling. The hypothesis of this work was that when the bone cells receive a signal to start the remodeling cycle. The aims of the present study work can be briefly outlined as follows: 1. We wanted to study the mechanisms underlying this feature of osteoblasts.

USA. Heptanol was purchased from Sigma Chemical Co.1 Antibodies (I-IV) Polyclonal rabbit antibody against pan-cadherin (Sigma Chemicals.1. Louis. MO). rabbit anti-mouse or swine anti-rabbit or goat anti-rabbit were also obtained from DAKO. (Eugene. 1992) (II). Secondary antibodies.1 Reagents (I-IV) All cell culture media and reagents were purchased from Gibco BRL (Paisley. Hexapeptides AHAVSE and VASAEH were synthesized in Department of Medical Biochemistry. KY) were used at a dilution 1:100 (III). Yale University School of Medicine . KY) were all used at 1:50 in PBS. IA) and β-catenin (Transduction Laboratories. Lexington. MO). University of Turku. St.4 Materials and Methods 4. 1991). and used at a dilution of 1:100 (I-IV). (St. either tetramethylrhodamine isothiocyanate (TRITC) or fluoresceinisothiocyanate (FITC)-conjugated. Synthetic peptide Gap 27 (SRPTEKTIFII) was synthesized in Keck Foundation Biotechnology Resource Laboratory. IL) and used at a dilution 1:50 (I). DNA-binding fluorochrome Hoechst 33258 (1 . TRITC or FITC-conjugated phalloidin was purchased from Molecular Probes Inc. A monoclonal mouse antibody against chicken gizzard smooth muscle vinculin was purchased from ICN Immuno Biologicals (Lisle. Finland. The VSV G protein and the matrix protein were localized using specific polyclonal antibodies produced in our own laboratory (Metsikkö et al. OR) and was used at 5 units/ml. Lexington. Louis. Iowa City. England). Antibodies against the gel-purified HA band of the WSN strain to Influenza virus hemagglutinin and an antibody against the Influenza virus nucleocapsid protein have been described earlier (Martin et al. 4. Monoclonal antibodies raised against Connexin-43 (Transduction Laboratories. monoclonal antibodies against E-cadherin (Developmental Studies Hybridoma Bank.

and for the experiments the cells were trypsinized and plated on 150 µm thick 1 cm2 bovine bone slices. Lakkakorpi et al. The cells were allowed to attach to sonicated bovine cortical bone slices for 30 minutes. Cells were cultured at +37°C (5% CO2. 1984.36 mg/ml stock.2. 4. 1989). non-attached cells were rinsed away. The cultures were usually stopped by fixing the cells and bone samples with freshly made or refrigerated 3% paraformaldehyde (PFA). Chambers et al. Sigma Chemical Co. heptanol 3 mM or Gap 27 at 500 µM were added to the culture medium of the cells. 100 µg of streptomycin/ml and 7-10% heat-inactivated fetal calf serum (FCS). 100 µg of streptomycin/ml and 7-10% heatinactivated fetal calf serum (FCS). 95% air) for up to 48 hours. 1984. and the bone slices with the remaining cells were transferred into 24-well plates containing fresh medium with appropriate substances to be tested (Boyde et al. 2 mM L-glutamine. 3 to 4 days old rat pup femora. Louis. 4. 100 IU of penicillin/ml. tibiae and humeri were dissected out. 2% sucrose in phosphate-buffered saline (PBS) for 10 minutes at room temperature.84 g of sodium bicarbonate/liter. In further studies some of the following changes were made to the culture medium: hexapeptides AHAVSE or VASAEH at concentration 1 mg/ml. St. III-IV). After the attachment period.. 4.2 Cell culture (I-IV) 4.2. MO) was used to visualize nuclei (I. diluted 1:800.2 Cell line (II) UMR-108 osteoblastic rat osteosarcoma cells (ATCC CRL-1663 similar to UMR-106 CRL-1661) were cultured in MEM (Minimum Essential medium) containing 2 mM Lglutamine.1 Isolation and culture of osteoclasts (I.3 Tissue culture (II) For studying endosteal osteoblasts. 95% air) in small cell culture bottles in 5 ml of medium. which will be later referred to as MEM-FCS. III-IV) Osteoclasts from 1 or 2-day-old Sprague-Dawley rat pup long bones were scraped mechanically into DMEM (Dulbecco’s modified Eagle’s medium) buffered with 20 mM Hepes and containing 0. placed in MEM (Minimum Essential medium) containing 2 mM L- .2. The cells were grown at +37°C (5% CO2. 100 IU of penicillin/ml.

St.or FITC-labeled secondary antibodies for 30-60 min at +37°C. The bones were cultured overnight at +37°C (5% CO2. 100 IU of penicillin/ml. The samples were viewed with a Leitz Aristoplan microscope with appropriate filters. The loose bone marrow was gently removed by injecting culture media over it with a pipette. bones were bisected with a scalpel blade. For immunostaining. Frankfurt) in PBS containing 2. After removing the epiphyseal cartilages the diaphyses. cells were fixed with 10 min methanol at room temperature. 386-A.3. the cells were fixed and permeabilized with various methods for the immunofluorescence staining. The samples were rinsed thoroughly several times with PBS.2 Conventional fluorescence microscopy (I. and further incubated with TRITC. the bone slices or bone samples were incubated with primary antibody one to two hours at +37°C. and the VSV G protein and the matrix protein and Connexin-43 antibodies.1 Light microscopy (I. Then the samples were rinsed in PBS and permeabilized with 0. 1991a).5% 1. III-IV) Osteoclasts were cultured in DMEM-FCS growth medium. the cells were incubated with the DNA-binding fluorochrome Hoechst 33258 (1 mg/ml stock diluted 1:800 in PBS) for 10 minutes at room temperature. Finally all the samples were mounted in either Mowiol (Hoechst. using a Sigma TRAP kit (No.2)-octane or 50% PBS-glycerol. F-actin was stained using either TRITC or FITC-conjugated phalloidin for 20 minutes at +37°C. Sigma Chemicals.1% Triton X-100 in PBS for 10 minutes on ice.3. After growing the cells for 48 hours. . Louis.3 Fluorescence and microscope techniques (I-IV) 4. With pan-cadherin. 4. MO) according to manufacturer's instruction.. vinculin. Influenza virus nucleocapsid protein. 100 µg of streptomycin/ml and 7-10% heatinactivated fetal calf serum (FCS). When using βcatenin. Non-specific binding of antibodies was blocked with swine serum. With E-cadherin antibodies the cells were first fixed with methanol for 5 min at -20°C and quickly rinsed in acetone and let air dry.4-diazobicyclo(2. To visualize the nuclei. a commonly accepted marker of osteoclasts (Minkin 1982). the cells were fixed in 3 % paraformaldehyde in PBS containing 2 % sucrose for 10 min at room temperature. 95% air).37 glutamine. 4.2. phalloidin or Hoechst 33258. The procedure for staining F-actin has previously been published in detail (Lakkakorpi et al. with either hexapeptides AHAVSE or VASAEH (I). heptanol (III) or Gap 27 (IV). the Influenza virus hemagglutinin. III-IV) Osteoclasts were identified by staining them for tartrate-resistant acid phosphatase (TRAP).

05-software (Leica Lasertechnik Gmbh.4.5 Digital image analysis (I -IV) MCID image analyzers utilizing M1 or M2 software (Imaging Research Inc. Chino.3 and 50x1.0 oil immersion objectives (Leitz. The bone slices were decalcified with 7% EDTA in phosphate buffer for 4 days at +4°C with daily changes of the decalcifying medium. Germany) with suitable zoom factors (1x or 2x) of the scanner. were used to the quantification of areas of the resorption lacunae. The Netherlands). a Leitz Aristoplan microscope. The samples were then postfixed with 4% OsO4.1 M phosphate buffer.3.3. St. pH 7. Brock University. 1994) and quantitated by combining the 10 X light microscopy image (Nikon TMD Diaphot) to a color-CCD camera (Sony XC 711P vision camera module).5% glutaraldehyde in 0. In the bone tissue cultures the metaphyseal ends of the long bones were investigated with and the association of labeled cells on bone surface was confirmed by simultaneous observation with Nomarski differential interference contrast optics in transmitted light. Resorption pits were visualized as brown-colored areas after staining with peroxidase-conjugated wheat germ agglutininlectin (WGA) (Sigma Chemical Co. 4. Laser power and offset were adjusted according to the fluorescence label intensity. MO) (Selander et al. The confocal laser scanning microscope consisted of a 750 mW air-cooled argon-crypton laser (Omnichrome. Ultrathin sections (100 nm) were made after removal of mineral from the bone slice.38 4. and 1. Louis.4) or Fluor 40x1. . Canada). The samples were examined with a Philips 410 electron microscope (Philips. Tokyo Japan).3. Heidelberg. dehydrated and embedded in Epon. 4. CA).. Quebec.4 Immunoelectron microscopy (II) The cell cultures were fixed with 2. Scanning was performed in a XY-plane using either a 40 x 0.6 water immersion objective (Nikon.. a PLAPO 63x objective (NA 1. Ontario. Canada) consisting of an Intel 403 E microcomputer linked to an Image 1280 image processor (Matrox Dorral. The localization of immunostaining was acquired by scanning serial optical xy and xz sections. Germany).3 Confocal laser scanning microscopy (I-IV) Samples for confocal laser scanning microscope were prepared in the same way as for conventional epifluorescence microscopy. for 1 to 2 days at 4°C.

(Lisanti et al. the gels were dried and the intensities of radioactivity of both the streptavidin bound fraction and cell lysates were quantified and calculated using a phosphoimager (Molecular Dynamics.5 Statistical analysis (I. 4.. 4.1 Metabolic labeling (II) Cells cultured on bone slices were labeled with S-methionine (Amersham Corp. . Canada).1 rotor. biotinylation was performed. 1988). MA). Sunnyvale. IL) at 0°C as described by Lisanti et al.5 mg/ml for 30 minutes at +0°C were extracted from lysed cells and centrifuged at 30 000 x g for 1 h with a SW50. Adsorption was for 1 h at 37°C with 10 plaque-forming units in MEM containing 0. St. and the cells were infected with wild type vesicular stomatitis virus (Indiana serotype) or Influenza virus of A/WSN strain..4 Viral infections (II) UMR-108 cells were cultured for two days to near confluency.1% BSA. membrane proteins labeled with sulfo-NHS-biotin at a concentration 0. Proteins were eluted from the streptavidinagarose by heating for 3 minutes at +95°C in SDS sample buffer.. The plasma membrane proteins of the infected cells were labeled with sulpho-NHS-biotin (Pierce Chemical Co.39 4. Brock University. after which the cells were further cultured for 3 hours (VSV) or 7 hours (WSN) in the presence of serum. 1989). The endosteal osteoblasts were infected after culturing the bisected bones overnight with same procedure as UMR-108 cells. After the SDS-PAGE. SDS-PAGE was carried out by the method of Laemmli (Laemmli 1970).4. Streptavidin-agarose (Sigma) was used to adsorb the biotinylated proteins by using end-over-end rotation overnight at +5°C.00 (Microcal Software Inc. Bucks.2 Cell surface biotinylation (II) After the chase. 35 4. Northampton. Rockford. The biotinylated proteins were isolated by the methods described by Hare & Lee (Hare et al. England) at 4 hours postinfection (VSV) or 6 hours postinfection (WSN) for 10 minutes at 500 µCi/ml and chased for 90 minutes. III-IV) All statistical analyses were performed by using Student’s t-test for independent samples and a computer program Microcal Origin version 4..4. CA) using MCID-M2 image analysis software (Imaging Research Inc. Briefly. Catherines.

E-cadherin and β-catenin antibodies were first tried in immunofluorescence staining of osteoclasts.5 Results 5. HAV. Neither of the antibodies gave positive staining in osteoclasts even though MDCK-cells as a positive control showed similar staining patterns as described earlier.2. Some staining can also be seen in the cytoplasm. both at a concentration of 1 mg/ml. Attached TRAP-positive multinucleated cells were counted after rinsing the non-attached cells . Pancadherin staining was also seen in between the vinculin double-rings. pan-cadherin antibody reacted with osteoclasts forming a ring-like structure localized in the sealing zone area. partly overlapping with them. the CAR-containing peptide AHAVSE was added to the primary attachment medium of osteoclasts. is involved in the primary attachment of osteoclasts to the bone surface. We could distinguish osteoclasts from the other cells in the cell culture by visualizing the nuclei with Hoechst stain.2 Effects of HAV-containing peptide (I) 5. Double immunofluorescence staining of pan-cadherin with F-actin revealed that pan-cadherin antibodies co-localized with the actin ring in the sealing zone area. On the contrary.1 Primary attachment of osteoclasts to bone surface To reveal if the common cadherin CAR sequence motif.1 Cadherin localization in osteoclasts (I) To localize cadherins in osteoclasts we stained the cells with different cadherin or cadherin-related antibodies. 5. or with no added peptide to act as zero-controls. When harvesting osteoclasts from rat pups the cells were allowed to attach to the bone slices with the attachment medium containing either AHAVSE or the control peptide VASAEH.

Osteoclasts were cultured for 24 hours.2. After incubation.1 Immunofluorescence data The VSV G protein localizes to the basolateral membrane of epithelial cells (RodriguezBoulan et al. after which AHAVSE or VASAEH was added to the culture medium. the cadherin CAR sequence-containing peptide AHAVSE had no effect on the primary attachment of osteoclasts to bone slices. Thus. We stained the osteoblast-like UMR-108 cells with the antibody against VSV G protein and found it to be localized to the culture medium facing plasma membrane of the cells. Similar amounts of TRAP-positive multinucleated cells were seen in AHAVSEtreated. 5.2 Osteoclastic bone resorption We studied the possible effect of AHAVSE on the ability of osteoclasts to form resorption pits. The decrease was significant at all time points from 20 minutes to 24 hours but it was not linear. The mean size and the size distribution of resorption pits were similar in all the treatments. . 5. The percentage of active osteoclasts was determined as those with actin rings. VASAEH-treated and non-treated control cultures.2. No significant differences in comparison with the zero-control were detected when the control peptide (VASAEH) was used. 5.41 away. the cells were fixed and stained for actin rings. The number of active osteoclasts decreased significantly after addition of AHAVSE.3. the numbers of active osteoclasts having actin rings were investigated. 1980.3 Activity of osteoclasts Since the AHAVSE peptide inhibited osteoclastic bone resorption. we used the VSV matrix protein antibody and found it to localize diffusely all over the cytoplasm (Nesbitt et al. 1990).3 Polarity of osteoblasts (II) 5. No morphological changes in the appearance of remaining actin rings were detected. Simons et al. Both the number of resorption pits and the total resorbed area decreased significantly after addition of the cadherin CAR sequence-containing peptide AHAVSE. 1993). To elucidate the cytoplasm of the UMR108 cells. The cells were allowed to grow for different periods of time from 20 minutes to 24 hours.

the ends of the processes probably communicating with neighboring cell processes showed somewhat condensed staining. the G protein immunolabeling had frequently an intense area towards circulation. at the plasma membrane in a nonpolarized manner. In the cells growing upon each other the cell surface against the bone was still deficient of virus particles. the localization of viral glycoproteins determines the site for viral budding. 5. as shown earlier (Stein et al. but as expected. The hemagglutinin antibody localized between the nucleus and bone. the budding of viruses is also polarized. where the cells were in close contact with the bone and in near confluency growing as a monolayer. as shown earlier (Odell et al. since Influenza was budding uniformly from all surfaces. where the cells did not overlap. the Influenza virus was mainly found to bud from the plasma membrane facing the bone slice. 5. On the contrary. Budding virus particles were detected at the plasma membrane of the infected cells. the superficial cells were not polarized. although in osteocyte processes extruded into the bone canaliculi. In the cases where the growing cells overlapped. Thus.3. However. In Triton-permeabilized osteocytes. Cell 35 surface biotinylation together with S-labeling revealed. the VSV G protein was localized to the bone marrow facing plasma membrane. the cells were permeabilized with Triton. the VSV G protein localization was investigated using both unpermeabilized and Triton-permeabilized cells. the VSV G protein localized apart from the Golgi complex. Influenza HA was only 22% . VSV particles were mainly budding on the medium side of UMR-108 cells. At the bone marrow or circulation side in the bone tissue cultures. In Influenza-infected UMR-108 cells the Influenza nuclear protein was used as a marker for the nucleus. Influenza HA was also found in the Golgi complex of permeabilized cells. Double staining of VSV G protein and VSV matrix protein were also performed to distinguish the cells’ outlines. which was different and opposite to the VSV G protein localization. 1993).2 Electron microscopy data We used electron microscopy to show that if the glycoprotein localization is polarized.3. although the upper cells had nonpolarized budding. The Influenza hemagglutinin was sorted to the bone substrate-facing surface of UMR-108 cells. 1993). To prove that the VSV G antibodies truly localize to the marrow facing membrane.42 In endosteal osteoblasts. Antibodies against VSV G protein still localized to bone marrow facing plasma membrane. even in these cases the cells in bone contact had virus particles budding mainly from the bone-facing surface.3 Biochemical data Sulfo-NHS-biotin binds to the cell surface proteins and does not internalize at 0°C. In unpermeabilized cells. they could also be seen in the Golgi complex.

4. whereas in inhibitor-treated cultures the number of mononuclear TRAP-positive cells was three to four times greater than that of multinucleated TRAP-positive cells. We could see no difference in the number or morphology of other cell types in the inhibitor-treated bone cell cultures. which was not available for biotin binding.1 Connexin-43 localization in osteoclasts (III) Connexin-43 antibodies were used in immunofluorescence staining of rat bone cell cultures to obtain the precise localization of Cx-43 in osteoclasts.1 Number and Activity of Osteoclasts Since osteoclasts express gap-junctional Cx-43. The numbers of both mononuclear and multinucleated TRAP-positive cells were significantly decreased both in the heptanol-treated and in the Gap 27-treated cultures. only the TRAP-positive cells seemed to be affected. The ratio of mononuclear to multinucleated TRAP-positive cells was altered in both inhibitor-treated groups. The staining could be seen in cell-cell contacts with neighboring bone marrow derived mononuclear cells at the interface of adjoining cells. that Influenza HA was hidden in the bone facing plasma membrane. This indicates that most of the VSV G protein was at the medium facing surface available for biotinylation. Even though the results are clear. double stainings with F-actin were performed. 5. we wanted to test whether the addition of gap-junctional inhibitors. . To visualize the cell outlines and the sealing zone area and thereby to distinguish active osteoclasts. affects the number or activity of osteoclasts. they indicate that polarity is not achieved in all of the UMR108 cells. 5. A significant decrease in the number of TRAP-positive cells could be seen in the heptanol-treated (3 mM) and Gap 27-treated (500 µM) cultures compared to controls.4. This indicates. The indirect immunostaining of osteoclasts revealed punctuate distribution of Cx43 immunoreactivity.43 biotinylated. VSV G protein was 55% biotinylated and thus found in the streptavidin eluate.4. The control cultures contained approximately one to two times the number of mononuclear than multinucleated TRAP-positive cells. The results of the Gap 27-treated bone cells were almost identical with heptanol-treated bone cells.2 Effects of gap-junctional inhibitors heptanol and Gap 27 5.4 Gap junctions in osteoclasts (III and IV) 5.2. but numerous immunoreactive sites were also apparent at free surfaces and within the cytoplasm of osteoclasts. either heptanol or Gap 27. On the contrary.

whereas in control cultures we could not see nearly any apoptotic osteoclasts. we studied the nuclei of the cells.4. To reveal whether heptanol has an effect on the activity of the remaining osteoclasts. We could see numerous apoptotic osteoclasts in the inhibitor-treated bone cells cultures. A highly significant decrease could be seen in the total resorbed area in both heptanol-treated and Gap 27 –treated cultures compared to the controls. but the cells looked absolutely healthy with intact nuclei. The activity of heptanol-treated osteoclasts was half of that of the controls. The activity of Gap 27treated osteoclasts was dramatically decreased when compared to control osteoclasts.2 Area Measurement of Excavated Pits Since heptanol and Gap 27 decreased the number and activity of osteoclasts.2. the number of resorption pits was greatly decreased. . the Gap 27 seemed to act as even more potent inhibitor of the activity. we studied the effect of these inhibitors on the ability of osteoclasts to form resorption pits.44 To find out why the number of TRAP-positive cells is decreased in the heptanoltreated bone cell cultures. we counted how many of the TRAP-positive cells had actin rings. Interestingly. In addition. 5.

at least during short period culture (Chambers et al. 1984). mineralized bone. mature osteoclasts. which are similar to UMR-106 cells that have been widely used as a model for osteoblastic cells (Koutsilieris et al. the events of the resorption cycle of isolated osteoclasts on bone slices are well characterized (Karhukorpi 1991. (Chambers et al.1 Methodological aspects We chose to use the pit formation assay as the research setting in our studies concerning osteoclasts. 1993). This method delimits our studies to monitoring mature osteoclasts. The effect of the agents used in this thesis work. originally introduced by Boyde et al. Matsumoto et al. we may safely conclude that some of the communication between osteoclasts and other bone cells occur in the pit formation assay since several cell types are present in this culture method. Regarding to the cellular events taking place at the BMU in vivo. 1984) and Chambers et al. This assay. However. enables the studying of resorption mechanism of isolated. No special structural features of active osteoclasts. In addition. when compared to those cultured on bone slices. Lakkakorpi et al. 1991a. 1993). are results from the direct effects of these agents on osteoclasts and not of the complex cellular interactions occurring in vivo. and we thus miss the complicated maturation process of osteoclast precursors. 1984). . the pit formation assay is however. This may be due to the preparation and pretreatment of bone slices resulting in inactivation of proteins and cytokines. cadherin CAR-sequence containing peptide AHAVSE or the two gap-junctional inhibitors. The setting mimics the situation in vivo: osteoclasts can resorb their natural target. biased. Different cellular mechanisms operate in osteoclasts cultured on glass.6 Discussion 6. Lakkakorpi et al. When studying the polarity of osteoblasts we chose to use an osteoblast-like cell line UMR-108 cultured on bone slices. normally liberated via resorption and which are required for the cross-talk between osteoclasts and osteoblasts. 1987. bone resorption by osteoclasts is not followed by bone formation by osteoblasts. like ruffled border or sealing zone can be seen on artificial surfaces. This is why we cultured the cells on bone slices instead of glass. thus the attachment of osteoclasts to glass may also be different from bone (Lakkakorpi et al.(Boyde et al.

and P-cadherin (Geiger et al. We noticed that the blocking of cadherin function by the peptide . 6. all of which are known to be mediated by cadherins. Synthetic peptides containing the HAV sequence have been shown to inhibit compaction of eight-cell-stage mouse embryos. 1989. Suda et al. 1996).2 Cadherins and attachment of osteoclasts to bone Since osteoclasts express the vitronectin receptor. Thus the exact nature and identity of cadherin-like molecules in the osteoclastic sealing zone remains unknown. We chose to use this antibody because it was not precisely known which cadherins. however. which further confirmed the cadherin localization to the sealing zone. Nevertheless. 1990a) and myoblast fusion (Mege et al. We used the cadherin CAR sequence containing peptide to study what the possible roles for cadherins in osteoclasts might be. were selected for antibody production. 1991a) resembles that in epithelial adherens junctions. can be found in osteoclasts and thus. have suggested that mature murine osteoclasts contain E-cadherin along their membranes at the resorbing bone surfaces. The pan-cadherin ring became localized exactly around the resorption lacuna suggesting it marks the sealing zone. 1992. Lakkakorpi et al. in osteoclastic attachment to bone. the main adherens junction proteins.46 1985. The staining pattern of pan-cadherin antibodies also rules out the possibility that the sealing zone area cannot be reached by immunoglobulins. 1995). 1995). at least. since the most homologous cytoplasmic region of the cadherin molecules. if any. Partridge et al. the final tight attachment of osteoclasts to the bone surface is mainly not mediated through it (Lakkakorpi et al. VNR has been shown to be located only in the ruffled border and basolateral membranes. We then confirmed the results using endosteal osteoblast culture to study the osteoblastic cells in situ. this antibody proved to be excellent probe for the identification of cadherins from osteoclasts. and ruffled border area. 1990). we detected no E-cadherin in mature rat osteoclasts. Horton 1990). the staining co-localized with actin-ring and in between vinculin double rings. thus suggesting that the observation concerning the lack of vitronectin receptor in the same area is not due to steric hindrance (Lakkakorpi et al. In our studies. This led us study the role of cadherins. 1991b. rat neurite outgrowth on astrocytes (Blaschuk et al. and it is missing from the area of the tight sealing zone (Lakkakorpi et al. 1991b). 1991b). The pan-cadherin antibodies should react with all (or. that the tight osteoclastic attachment to bone surface in the sealing zone area would be integrinmediated (Davies et al. It has been shown that these antibodies do react with a very broad spectrum of cadherins. Helfrich et al. most) cadherins. These data strongly emphasize the importance of the HAV sequence in the cellular functions that are dependent on cadherins. E. 1989. 1992). it has been speculated. We found that osteoclasts show a clear ring-like structure in their sealing zone area with the pan-cadherin antibody. The organization of the actin cytoskeleton in the sealing zone area of osteoclasts (Lakkakorpi et al. Mbalaviele et al. In addition. Although VNR is important in the function of osteoclasts. such as N-. with a lesser amount in the cytoplasm of these cells (Mbalaviele et al.

. This could be expected. Thus.47 does not affect the primary attachment of osteoclasts to bone matrix. Mbalaviele et al. 1999. where it was suggested that cadherins can interact with collagen type I (Mbalaviele et al. 1996) and could act as cell-matrix adhesion molecules. Tang et al. The same report suggests that E-cadherin is involved in the generation of multinucleated osteoclasts from mononuclear precursors. The emerging of actin rings to the sealing zone of osteoclast is considered as a mark of osteoclastic activity (Lakkakorpi et al. As we wanted to learn if the activity of osteoclasts is truly decreased with AHAVSE or if it is only the mode of resorption that has changed. The observed disorganization of actin rings leads to inactivation of osteoclasts. 1993). 1995). we measured the activity of osteoclasts by their morphology. 1998). We do not know what the counterpart of osteoclastic cadherin might be. 1991a). or it could speed up the degradation of actin rings and subsequent detachment of the tight sealing. Although the resorbed area is greatly decreased with the AHAVSE. 1995). which could be seen as a decrease in the number and total area of resorption pits when compared with the zero and peptide controls. Others have shown that osteoclasts express different cadherins including E-cadherin (Mbalaviele et al. but it has been shown that cadherins might also be involved in interactions between different cell types or even different cell adhesion molecules (Cepek et al. 1994. the inhibition of formation of new attachments seems to be the main reason for the decreased percentage of active osteoclasts. the true amount of bone resorbed remains open since we cannot know if the AHAVSE-treated osteoclasts have really resorbed less bone . since actin rings are an essential component of this tight attachment-mediating structure. These studies together with a recent report. since the primary attachment is shown to be mediated by integrins (Duong et al. Lakkakorpi et al. They suggest that the decrease in the resorbed area is due to inhibition of osteoclast precursor fusion. Since the sizes of the resorption pits in AHAVSE incubation were not changed when compared with those of the controls. We believe that the disorganization of actin rings after the addition of AHAVSE is likely to be too rapid to be a result of inhibition of fusion of mononuclear osteoclast precursors.or if the osteoclasts have just resorbed deeper resorption pits. inspired us to investigate the possibility that cadherins might act as cell-matrix mediator in osteoclasts. 1995) and cadherin-6 (Mbalaviele et al. When using the peptide AHAVSE. Cadherins have generally been associated with homotypic cell-cell interactions. osteoclasts resorbed markedly less bone. Our results thus suggest that AHAVSE has a specific effect on the sealing zone. we can get a percentage of osteoclasts that are actively resorbing bone. By counting the actin rings and the number of TRAP-positive cells.as it seems when looking at the decrease in the area of resorption pits . as also demonstrated earlier (Mbalaviele et al. We could see a great decrease in the activity of osteoclasts as soon as 20 minutes after AHAVSE peptide addition. 1993). we cannot quantify the depths of the resorption pits with the method used here. The HAVcontaining peptide could either prevent the formation of new actin rings and tight attachment to the bone surface. have also shown a decrease in the total resorbed area when using HAV-containing peptide (Mbalaviele et al.

48 6. actively secreting mature osteoblasts are cuboidal or tall cells. which separate the apical and basolateral membranes from each other. In addition. et al. as epithelial cells do. In the biotinylation studies we also confirmed this conclusion showing that whereas most of the VSV G protein was at the medium-facing surface. hence strengthening our earlier results from UMR108 cells. the budding of Influenza viral particles took place in between the cell and the bone slice. 1988). Also. 1996). This led us to study whether mesenchymal osteoblasts possess polarized plasma membrane domains. 1988. These plasma membrane domains have different lipid and protein compositions (Simons et al. which could clearly be distinguished. In epithelial cells the polarity is maintained with tight junctions. van Meer et al. apart from the Golgi complex. The VSV G protein seemed to localize in a non-polarized manner at the plasma membrane of osteocytes. We could see that the VSV G protein antibodies localized to the bone marrow-facing plasma membrane of endosteal osteoblasts. we infected osteoblasts in situ with VSV. and none of the fibroblastic cell lines studied. As osteoblasts mature. Influenza glycoproteins localized in UMR-108 cells to the plasma membrane facing the bone slice. the antibodies against the basolateral marker VSV G protein localized clearly to the medium-facing plasma membrane of the cells. different plasma membrane domains could be separated in osteoblasts by viruses. however. the secretion is polarized towards the bone surface (Cooper et al. The plasma membrane of epithelial cells is divided into two domains: an apical domain. These results were thus in agreement with each other indicating that the bone surface-facing membrane domain of osteoblasts is apical in nature. 1998. where it could be biotinylated. Interestingly. which was not available for biotin binding. On the contrary.3 Polarity of osteoblasts Histologically. Unlike in fibroblasts. Our studies reveal that osteoblasts are truly polarized cells. the Influenza HA was hidden in the bone facing plasma membrane. To further prove that the polarization of UMR-108 cells resembles that of endosteal osteoblasts and is a meaningful tool. After delivery to the cell surface these two protein species spread all over the surface in a non-polarized manner (Yoshimori et al. . When they start to secrete bone. the secretion becomes non-polarized and some of the osteoblasts become trapped into the matrix as osteocytes. which faces the external milieu and a basolateral domain in contact with the internal milieu and the blood supply. Viral markers have been used in studying the different plasma membrane domains. Fibroblastic cells do not polarize their plasma membrane as the epithelial cells do. Jr. the osteocyte processes branching into the bone canaliculi showed somewhat condensed staining in the ends of the processes which probably communicate with neighboring cell processes by gap junctions. In osteoblastic UMR-108 cells. Cellular polarization is best established in epithelium. Osteocytes seem not to secrete matrix components anymore or at least the secretion is probably not polarized. Marks. have distinct domains for VSV G protein or Influenza HA on their plasma membranes. We wanted to see whether the localization of VSV G protein is changed in osteoblasts during this maturation. Vesicular Stomatitis viruses were found to bud from the upper membrane domain of these cells towards the medium corresponding the bone marrow in vivo. 1996).

1995b. 6. Very little is known about the polarity of other than epithelial cells. which connect adjacent cells (Kumar et al. 1993. since they do not have tight junctions that usually mediate the polarity in epithelial cells. such as ions. We clearly demonstrate that osteoclasts contain Cx43. observation of the viral particle budding and the biotinylation results – we can safely conclude that osteoblastic cells clearly posses distinct polarized membrane domains. No VSV-particles can be seen to bud anywhere but the basolateral membrane domain. It has been speculated that osteoblasts. The same randomness is also detectable when looking at the biotinylation results: the biotinylation of VSV G protein is incomplete. indicating that not all of the osteoblasts bud viral particles in a polarized manner. and small metabolites. Su et al. in a non-polarized manner. Mesenchymal osteoclasts have been shown to polarize its membrane into domains (Salo et al. The Cx43 staining localizes mostly in the plasma membrane of the cells with some cytoplasmic staining and shows a staining pattern typical of a gap-junctional protein. Some reports of connexins in osteoclasts have also been published earlier (Jones et al. when comparing the degree of polarity of osteoblastic cells to that of epithelial cells we can see a certain difference. the polarization is best achieved whereas some of it is lost when the cells start growing on top of each other. In epithelial cells the polarization is absolute. 2000).4 Gap junctions in osteoclasts To establish and maintain the balance between bone formation and bone resorption. Jones et al. Cx43 staining could be seen when osteoclasts were in cell-cell contacts with bone marrow derived mononuclear cells. It seems that when osteoblasts grow in a monolayer. Although a clear tendency of polarization is easily observed in osteoblastic cells. 1994. some viral particles bud randomly from opposite membrane surfaces. The mechanism by which these non-epithelial cells organize their membrane domains and maintain the polarity is not known. osteocytes and osteoclasts may adjust the number of communicating gap junctions throughout the bone cell network. being thus apical in nature. Still. bone cells must have a way to communicate with each other. This activity is made possible by clusters of intercellular channels called gap junctions.the immunofluorescence data. whereas Influenza particles can only be seen in the apical membrane domain. Most cells communicate with their immediate neighbors through exchange of cytosolic molecules. but the functional significance has not been demonstrated earlier. Jones et al. 1996. 1997). but the special cap structure on the top of an active osteoclast has receptors for Influenza virus. Others have shown that of the bone cells osteoblasts and osteocytes express gap junctional connexins (Donahue et al. 1997). Salo et al. 1993. Most of the osteoclast membrane has receptors for VSV indicating basolateral membrane domain.49 Taking these results together . This way the cells could regulate the transmission of molecular and electrical information to support the balance of bone remodeling (Su et al. Yellowley et al. Numerous punctuate connexin stainings could be seen in the basolateral plasma membrane of . second messengers. The immunofluorescence data leaves no doubt of the Cx43 expression in osteoclasts. 1996). 1997).

50

osteoclasts, some of them being rather large. This finding indicates that osteoclasts are connected to the other bone cell types and thus take part in the remodeling of bone. Since the Cx43 staining was so intense in osteoclasts, we tested the effect of two gapjunctional inhibitors, heptanol and Gap 27, on osteoclasts with dramatic results. Great variety of drugs have been shown to inhibit gap-junctional communication with varying degrees of efficacy and specificity, including volatile anesthetics such as halothane (Nedergaard 1994), the straight-chain alcohols heptanol and octanol (Donahue et al. 1995a, Nedergaard 1994), anandamide (Venance et al. 1995), glycerrhetinic acid (Davidson et al. 1995) and Gap 27 (Chaytor et al. 1997, Chaytor et al. 1998). Of these we chose heptanol and Gap 27. The number of osteoclasts decreased markedly in the treated cultures. The results between the two inhibitors were very similar. When we further studied the reason for this decrease, we found to our surprise that the share of apoptotic osteoclasts was multiple to that of controls. It seems that the communication between osteoclasts and other bone marrow derived cells was essential for the survival of osteoclasts. The fact, that several osteoclasts undergo apoptosis as a result of blocking of the gap junctions, indicates the importance gap-junctional communication to the boneresorbing cells. None of the other cell types present in this bone cell culture model, excluding osteoclasts, are affected but their number is unchanged and they seem normal with healthy looking intact nuclei. When we further examined the inhibitor-treated osteoclast cultures we noticed that even though the numbers of both mononuclear and multinucleated TRAP-positive cells had reduced with the treatments, the number of TRAP-positive multinucleated cells was even more radically reduced. This could indicate that to be able to survive, multinucleated osteoclasts need contacts with the neighboring cells even more than their mononuclear precursors. Furthermore, the fusion of osteoclast precursors into mature multinucleated osteoclasts could somehow be gap junction dependent. This is by no means a far-fetched idea, since involvement of gap junctions has been demonstrated during the progress of macrophage multinucleation in vitro (Ejiri et al. 1987). In addition, Jones and Boyde (Jones et al. 1994) have also suggested the involvement of gap junctions in the fusion of osteoclast precursors to mature osteoclasts. Since the number of osteoclasts decreased significantly with the inhibitors, it was only expected that the resorption activity be decreased as well. The decrease in the resorbed area was in consistent with our expectations. We measured the activity of osteoclasts also by determining the activity of individual osteoclasts by the actin ring staining. Osteoclastic activity was found to be decreased by this method as well. The reduced activity of the remaining osteoclasts might suggest that even though some TRAP-positive mononuclear and multinucleated cells are found after Gap 27-treatment in these cultures, they may already be undergoing some changes leading eventually to apoptosis and thus inactivating the cells. Musil et al. (Musil et al. 1990) have shown that cells lacking morphologically and physiologically recognizable gap junctions can still express Cx43. This illustrates that connexin synthesis cannot automatically be taken as proof that a cell possesses functioning intercellular channels if it expresses connexin. Nevertheless, our results clearly show that inhibition of gap-junctional communication with either a commonly used gap-junctional inhibitor heptanol or with a more specific gap-junctional inhibitor Gap 27, causes osteoclastic apoptosis and hence a decrease in the bone resorption. It has

51

been suggested that the presence of Cx43 in gap junctions between osteoblasts may be a prerequisite for effective metabolic coupling between osteoblastic cells in bone cell networks. The precise role of intercellular communication in bone function is not clear, but gap junctions may modulate bone development and remodeling by coordinating the response of bone cell network to extracellular signals such as physical or hormonal signals (Palumbo et al. 1990). Indeed, it has been demonstrated that gap-junctional communication is critical for the functional responsiveness of osteoblastic network (Vander et al. 1996). Another possible role of gap-junctional intercellular communication in bone may be in the propagation of biophysical signals. A signal from cells that sense a change in mechanical load may be transmitted to other cells that do not sense the signal but are effectors of remodeling (Su et al. 1997).

7 Conclusions
Tight osteoclastic attachment to bone surface in the sealing zone is sensitive to the cadherin CAR sequence-containing peptide AHAVSE. The peptide-treatment decreased activity of osteoclasts seen as a decrease in the number of resorption pits and the total resorbed area. Staining of osteoclasts with a cadherin-recognizing antibody localized cadherin-like molecule in the sealing zone area of osteoclasts. The primary attachment of osteoclasts to bone surface is not affected by the peptide, suggesting that it is not mediated by cadherins. These results suggest that cadherin-like molecules may mediate the tight attachment of osteoclasts to the bone surface in the sealing zone and that the decrease of resorption in AHAVSE-treated osteoclast cultures is due to prevention of sealing zone formation. We demonstrate for the first time that mesenchymal osteoblastic cells have polarized plasma membrane domains. Our findings suggest that osteoblasts are polarized at some point of their life cycle. The bone attaching or bone matrix-facing plasma membrane of osteoblasts has apical characteristics, while the circulation or bone marrow-facing plasma membrane is basolateral in nature. The data from the bone cell communication studies suggest a functional role for gap junctions in osteoclastic bone resorption. We believe, that during bone remodeling, cell contacts and junctions play an important role in modulating osteoblast and osteoclast activity, recruiting osteoblasts that transform into osteocytes and forming an open canalicular network among osteocytes. Osteoclasts show connexin staining in cell-cell contacts, suggesting a possible communication mechanism to mononuclear cells. Osteoclastic activity is clearly reduced by gap junctional inhibitors and the number of osteoclasts also decreases with the treatment, which indicates the vital role of gapjunctional communication for the survival of osteoclasts. The proportional number of mononuclear TRAP-positive cells increases, which may mean that gap junctions are needed for the fusion of precursors to mature multinucleated osteoclasts. On the basis of these results we conclude that gap-junctional communication is essential for proper remodeling for bone and to the action of bone-resorbing osteoclasts.

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