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
Ilvesaro, Joanna, Attachment, polarity and communication characteristics of bone
cells
Department of Anatomy and Cell Biology, Biocenter Oulu, University of Oulu, P.O.B. 5000,
FIN-90014 University of Oulu, 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. Cadherins are transmembrane glycoproteins usually mediating homophilic calciumdependent cell-cell adhesion. In the present work we have studied the effects of the cadherin CAR
sequence HAV-containing hexapeptide AHAVSE on osteoclasts. The primary attachment of
osteoclasts to bone surface is not affected by the peptide, suggesting that it is not mediated by
cadherins. Treatment of osteoclast cultures with AHAVSE decreased the number of resorption pits
and the total resorbed area. Furthermore, we show rapid inactivation of osteoclasts with AHAVSE,
which is seen as a decrease in the percentage of osteoclasts with actin rings. Pan-cadherin antibodies
localized cadherin-like molecule in the sealing zone area of osteoclasts. 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.
We studied the polarity of mesenchymal osteoblasts using osteosarcoma cell line UMR-108 and
endosteal osteoblasts in situ in bone tissue cultures. Immunofluorescence confocal microscopy
revealed that the vesicular stomatitis virus glycoprotein (VSV G) was targeted to the culture mediumfacing surface. In endosteal osteoblasts, VSV G protein was found in the surface facing the bone
marrow and circulation. On the contrary, Influenza virus hemagglutinin (HA) was localized to the
bone substrate-facing surface of the UMR-108 cells. Electron microscopy showed that VSV particles
were budding from the culture medium-facing surface, whereas Influenza viruses budded from the
bone substrate-facing plasma membrane. These findings suggest the bone attaching plasma
membrane of osteoblasts is apical, and the circulation or bone marrow facing plasma membrane is
basolateral in nature. Gap junctions often mediate communication between different cells and cell
types. In the present work, 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. The effects of heptanol and Gap 27, known gap- junctional inhibitors, were studied using
the well-characterized pit formation assay. The inhibitors decreased the number and activity of
osteoclasts, suggesting a defect in the fusion of mononuclear osteoclast precursors to multinucleated
mature osteoclasts. Furthermore, the total resorbed area and the number of resorption pits also
decreased in the cultures. These results suggest that gap-junctional connexin-43 plays a functional
role in osteoclasts, and that the blocking of gap junctions decreases both the number and the activity
of osteoclasts.
Keywords: cell adhesion, osteoclasts, osteoblasts, cell polarity, cell communication
To my husband Vesa
Acknowledgments
This work was carried out at the Department of Anatomy and Cell Biology and Biocenter
Oulu, University of Oulu, during the years 1995-2001. It is my great pleasure to thank the
following persons, who have taken part of this work and thus made it possible:
I owe my deepest thanks to my mentor Professor Juha Tuukkanen, DDS, Ph.D. His
encouragement, support, 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. I thank my first teacher and supervisor Professor H.
Kalervo Väänänen, M.D., Ph.D., for introducing me to the research world and especially
to the world of bone. I wish to express my appreciation to Professor Hannu Rajaniemi,
M.D., Ph.D., for kindly providing me the surroundings, thus making it possible for me to
work in the Department of Anatomy and Cell Biology. I also thank Professor Juha
Peltonen, M.D., Ph.D., for his interest towards my research.
I am grateful to Professor Karl-Erik Åkerman, M.D., Ph.D., for his critical appraisal of
this thesis. Also, I am in debt to Docent Antero Salminen, Ph.D. 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.
The expertise of my co-authors, Doctors Päivi Lakkakorpi, Kalervo Metsikkö and Pasi
Tavi is most sincerely acknowledged. My humble thanks are due to the whole staff of
Department of Anatomy and Cell Biology, and, in particularly the present and former
Bone Heads for the stimulating working atmosphere. Especially Anita Kapanen, Jussi
Halleen and Teuvo Hentunen are warmly acknowledged for fun times in and outside
laboratory. I also wish to thank Sari Annunen, Allan Haimakainen, Minna Orreveteläinen,
Eero Oja and Marja Välimäki for their skilful technical assistance.
I owe my loving thanks to my best girlfriend and colleague Anne Tuomisto for the
special bond we two share - there is nobody quite like her. 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, I could do it, if I just put
my heart into it. My husband Vesa, deserves my warmest thanks. I thank him for his love
and his never-ending optimism and encouragement during hard times. Vesa’s 24-hour ITsupport is also warmly acknowledged. I am priviliged to have him as my husband.
This research was financially supported by Biocenter Oulu, Oulu University
Scholarship Foundation and the Finnish Cultural Foundation.
Oulu, March 2001
Joanna Ilvesaro
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
3,3’ 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
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
List of original publications
This thesis is based on the following articles, which are referred to in the text by their
Roman numerals:
I
Ilvesaro JM, 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. Experimental Cell Research 242:75-83.
II
Ilvesaro J, Metsikkö K, Väänänen K, Tuukkanen J (1999) Polarity of osteoblasts and
osteoblast-like UMR-108 cells. Journal of Bone and Mineral Research 14 (8): 13381344.
III
Ilvesaro J, Väänänen K, Tuukkanen J (2000) Bone-Resorbing Osteoclasts Contain
Gap-Junctional Connexin-43. Journal of Bone and Mineral Research 15 (5): 919-926.
IV
Ilvesaro J, Tavi P, Tuukkanen J Connexin-Mimetic Peptide Gap 27 Decreases
Osteoclastic Activity. Submitted.
Contents
Abstract
Dedication
Acknowledgements
Abbrevations
List of original articles
1. Introduction
2.1 Structure and function of bone tissue ..................................................................... 15
2.1.1 Bone matrix components................................................................................. 16
2.1.2 Bone cells........................................................................................................ 17
2.2 Cellular mechanisms of bone resorption ................................................................ 20
2.2.1 Osteoclastic membrane domains ..................................................................... 23
2.2.2 Attachment of osteoclasts to bone matrix........................................................ 24
2.2.3 Cadherins ........................................................................................................ 25
2.2.4 Degradation of the mineral and organic matrix............................................... 27
2.2.5 Regulation of osteoclastic bone resorption ..................................................... 28
2.2.6 Disorders of bone modeling and remodeling .................................................. 29
2.3 Cell polarity ........................................................................................................... 30
2.4 Communication between bone cells....................................................................... 31
2.4.1 Gap junctions .................................................................................................. 31
4.1 Reagents (I-IV) ...................................................................................................... 35
4.1.1 Antibodies (I-IV)............................................................................................. 35
4.2 Cell culture (I-IV) .................................................................................................. 36
4.2.1 Isolation and culture of osteoclasts (I, III-IV)................................................. 36
4.2.2 Cell line (II)..................................................................................................... 36
4.2.3 Tissue culture (II) ............................................................................................ 36
4.3 Fluorescence and microscope techniques (I-IV) .................................................... 37
4.3.1 Light microscopy (I, III-IV) ............................................................................ 37
4.3.2 Conventional fluorescence microscopy (I, III-IV) .......................................... 37
4.3.3 Confocal laser scanning microscopy (I-IV) .................................................... 38
4.3.4 Immunoelectron microscopy (II) .................................................................... 38
4.3.5 Digital image analysis (I -IV).......................................................................... 38
4.4 Viral infections (II)................................................................................................. 39
4.4.1 Metabolic labeling (II) .................................................................................... 39
4.4.2 Cell surface biotinylation (II) .......................................................................... 39
4.5 Statistical analysis (I, III-IV).................................................................................. 39
5.1 Cadherin localization in osteoclasts (I) .................................................................. 40
5.2 Effects of HAV-containing peptide (I).................................................................... 40
5.2.1 Primary attachment of osteoclasts to bone surface.......................................... 40
5.2.2 Osteoclastic bone resorption ........................................................................... 41
5.2.3 Activity of osteoclasts ..................................................................................... 41
5.3 Polarity of osteoblasts (II)...................................................................................... 41
5.3.1 Immunofluorescence data ............................................................................... 41
5.3.2 Electron microscopy data ................................................................................ 42
5.3.3 Biochemical data............................................................................................. 42
5.4 Gap junctions in osteoclasts (III and IV)................................................................ 43
5.4.1 Connexin-43 localization in osteoclasts (III) .................................................. 43
5.4.2 Effects of gap-junctional inhibitors heptanol and Gap 27 ............................... 43
6.1 Methodological aspects.......................................................................................... 45
6.2 Cadherins and attachment of osteoclasts to bone ................................................... 46
6.3 Polarity of osteoblasts ............................................................................................ 48
6.4 Gap junctions in osteoclasts ................................................................................... 49
1 Introduction
Bone tissue has three major functions in the body: it offers mechanical support to the
body, it protects major vital organs and it maintains calcium and phosphate homeostasis.
Bone also provides the environment for hematopoiesis, which takes place in the marrow
of long bones in adults. Bone is also a dynamic tissue, which remodels and repairs itself
throughout life. In normal bone tissue the formation and resorption of bone are well
balanced so that when old bone is resorbed by osteoclasts, similar amount of new bone is
formed by osteoblasts. Excessive bone resorption results in net bone loss, whereas
decreased bone resorption, or excess bone formation, results in increased bone mass.
Osteoporosis is a metabolic bone disease leading to decreased bone mass and increased
susceptibility to fractures. At the cellular level, osteoporosis is characterized by extensive
bone resorption, which leads to disturbance in the bone microarchitecture, which in turn
increases the probability of fracturing. Osteoporosis typically affects women after
menopause, when serum estrogen levels decrease. The clinical complications of
osteoporosis, fractures occur most often in the spine, femoral neck and at the wrist. The
two cell types primarily involved in bone turnover are the bone-forming osteoblasts and
bone-resorbing osteoclasts. 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.
The experimental part of this thesis work was carried out to clarify some cell
biological questions concerning different bone cells. Special interested was focused on
the attachment of osteoclasts to bone surface, the polarization of the osteoblasts and the
communication between osteoclasts and osteoblasts. All of these are interesting
biological questions, but they might also appear to be useful targets for therapy of
metabolic skeletal disorders.
2 Review of the literature
2.1 Structure and function of bone tissue
Bone consists of mineralized organic matrix and bone cells, including bone-forming
osteoblasts, bone-lining cells, osteocytes and bone-resorbing osteoclasts. There are two
morphologically distinct types of bone, cortical (compact) bone, which constitutes 85%
of total bone in the body and trabecular (cancellous) bone that constitutes the rest. The
core of each bone consists of cortex, a compact bone layer that forms the body of the
bone, which is mainly responsible for the mechanical and protective function. Trabecular
bone, a sponge-like structure of bone trabeculi, is found inside the cortex and it is
responsible for the metabolic function of bone. The remaining volume, the spaces
between the bone trabeculi in long bones, is composed of blood vessels and bone marrow,
which is the site of erythrocyte synthesis. An extensive blood circulatory system provides
bone nutrients, hormones and growth factors.
Bone growth in embryonic development can occur in two mechanisms, either with
intramembranous ossification or endochondral ossification. The flat bones of the body,
such as bones of the skull, form by intramembranous ossification. This means that during
embryonic development mesenchymal stem cells of connective tissue differentiate
straight into bone forming osteoblasts and start to synthesize bone. On the other hand,
long bones at joints, such as the long bones of legs and arms, which bear the weight of
body, form via endochondral ossification. Mesenchymal stem cells differentiate into
chondroblasts, which first form a cartilage model of the skeleton. Later on, the cartilage
model is replaced by bone matrix with the help of osteoblasts and osteoclasts working
synchronically.
During the growth period (first 20-30 years of life), more bone is formed than is
resorbed, resulting in an increase in bone mass. Peak bone mass, meaning the maximal
amount of bone matrix an individual has attained, is reached by late adolescence
(Matkovic et al. 1994, Mundy 1995). During the next three decades (and beyond) the
adult skeleton is maintained by removing and replacing a fraction each year. This
remodeling begins with a localized resorption that is succeeded by a precisely equal
formation of bone at the same site (Parfitt 1994). Sometime after the fifth decade, the
16
formative phase of the remodeling sequence fails to keep the pace with resorptive activity
and skeletal mass, including the connectivity of trabecular bone, decreases. This reduces
skeletal strength and increases the risk of fracture over time, depending on the magnitude
by which resorption and formation are uncoupled.
2.1.1 Bone matrix components
Bone matrix consists of an organic matrix that is strengthened by deposits of inorganic
calcium salts. 70% of bone is mineral, 22% protein and 8% water (Lane 1979). Collagen,
the primary secretory product of osteoblasts, comprises about 90% of the organic bone
matrix. Type I collagen is the most abundant form of collagen found in the body and is
widely distributed in connective tissue. Type I collagen represents the majority of bone
collagen, but biochemical analysis have also indicated trace amounts of types III, V, XI
and XIII (Gehron 1989). The bone collagen fibrils are highly insoluble because of their
many covalent intra- and intermolecular cross-links, the type and pattern of which differs
from that in soft connective tissues. In the matrix, individual collagen molecules are
packed end to end with a short space (gap) between them. 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. Multiple crosslinking sites combine extracellularly to form pyridinium ring structures tying several
collagen monomer molecules together within the formed collagen fiber, thereby
rendering it completely insoluble (Eyre et al. 1988). These pyridinium cross-links are
only released on degradation of the mineralized collagen fibrils during bone resorption,
making it an excellent method to measure bone resorption rate from urine (Uebelhart et
al. 1990).
The remaining 10% of the organic matrix is composed of non-collagenous proteins
(NCPs), 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. All connective tissue cells interact with their extracellular environment in
response to chemical stimuli that direct and coordinate specific cell functions, such as
proliferation, migration and differentiation. These actions require cell attachment and
spreading by focal adhesions via integrins, that transduce signals to the cytoskeleton
(Ruoslahti et al. 1987). Bone cells synthesize four proteins that affect cell attachment:
fibronectin, thrombospondin, osteopontin and bone sialoprotein (Oldberg et al. 1986,
Robey et al. 1989, Somerman et al. 1988). Of these, bone sialoprotein is exclusively
found in bone, whereas the others can also be found in many tissues other than bone.
Proteoglycans are macromolecules that contain acidic polysaccharide side chains
(glycosaminoglycans) attached to central core protein. In bone, two types of
glycosaminoglycan are found: chondroitin sulfate and heparin sulfate. γ-Carboxylation
occurs in two bone NCPs, osteocalcin (bone gla-protein) and matrix gla-protein
(Glowacki et al. 1987, Robey et al. 1989). Osteocalcin expression is restricted to bone,
but matrix gla-protein can also be found in cartilage in addition to bone. Measurements of
17
osteocalcin in serum have proved to be valuable as markers of bone turnover in metabolic
diseases (Price et al. 1980). A number of proteins in bone appear to be associated with the
life cycle and function of osteoblasts (Ripamonti et al. 1992). These proteins may be bone
morphogenetic factors or growth factors, such as TGF-β and insulin-like growth factors,
osteoblast secretion products that can stimulate osteoblast cell growth in an autocrine or
paracrine fashion (Canalis et al. 1988, Hauschka et al. 1986, Hauschka et al. 1988, Robey
et al. 1987). Other bone cell products may be associated with the growth or
differentiation of osteoblasts in an indirect or as yet unidentified fashion. The most
abundant NCP produced by osteoblasts is osteonectin, a phosphorylated glycoprotein
accounting 2% of the total protein of developing bone in most animal species.
2+
Osteonectin has high affinity for binding Ca and hydroxyapatite and it also binds to
collagen (Termine et al. 1981) and thrombospondin (Clezardin et al. 1988).
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, about 85% phosphorous, and from 40-60%
of the body sodium and magnesium are associated with the bone crystals. These
consequently serve as the major source for the transport of these ions to and from the
extracellular fluids. Rapid responses to calcium excesses and deficiencies in the body are
crucial for a number of processes, including the regulation of muscle contraction, nerve
impulse transmission, cell membrane permeability and blood coagulation. Indeed, the
most abundant inorganic components of the bone matrix are phosphate and carbonate
salts of calcium, with traces of calcium and magnesium fluorides (Levy 1894). The
mineral is in the form of hydroxyapatite, a crystalline lattice in which the principal
components are calcium and phosphate ions (DeJong 1926). With the mineral phase, the
otherwise soft, pliable organic matrix now becomes relatively hard, rigid material which
possesses the necessary mechanical properties that permit it to withstand the forces,
stresses, and strains. 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.
2.1.2 Bone cells
2.1.2.1 Osteoblastic cells
Bone formation is the responsibility of osteoblasts, cells of mesenchymal origin lining the
new bone matrix. Osteoblasts arise from the same pluripotent stem cell with
chondroblasts, adipocytes, myoblasts and fibroblasts (Bennett et al. 1991, Grigoriadis et
al. 1988, Yamaguchi et al. 1991). Osteoblasts produce and secrete the major part of the
organic bone matrix, which in a well-regulated process becomes calcified to form
mineralized bone. Four maturational stages of osteoblast development can be
distinguished in situ – the preosteoblast, osteoblast, osteocyte and bone-lining cell (Aubin
et al. 1996). 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 are postproliferative,
18
cuboidal, strongly alkaline phosphatase-positive cells lining the bone matrix at sites of
active bone production. Osteoblasts can also be recognized by their ability to synthesize a
number of phenotype-specific or -associated macromolecules, including the bone matrix
proteins, such as osteocalcin, bone sialoprotein and osteopontin, proteoglycans, certain
hormone receptors, and cytokines and growth factors. The precise physiological role of
alkaline phosphatase is unknown, but it has been hypothesized to be involved in the
mineralization process (Wennberg et al. 2000).
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, which are considered the most mature form of osteoblastic lineage are
osteocytes. Osteocytes are smaller than osteoblasts and they have lost many of their
cytoplasmic organelles. 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, such as
osteoblasts and maybe even osteoclasts (Bonewald 1999, Doty 1981, Menton et al. 1984).
The canaliculi are small tunnels in the bone, measuring about 1-2 µm in diameter.
Although the functions of this most abundant cell type of bone, osteocyte, are poorly
understood, some evidence is gathering that osteocytes sense the mechanical stress to
bone and therefore can participate in the bone remodeling process (Burger et al. 1999,
Mikuni-Takagaki 1999). In the adult skeleton, the majority of bone surfaces that are not
being remodeled (i.e. undergoing resorption or formation), are covered by flat, thin,
elongated bone lining cells, which are thought to represent the inactive form of osteoblast
in terms of matrix production. Bone lining cells detach from the surface of bone when
osteoclasts start the resorbing of bone.
2.1.2.2 Osteoclasts
Osteoclasts, first named by Kölliker (Kölliker 1873), are multinucleated cells
differentiating from haemopoietic cells. It is generally accepted that osteoclasts are
formed by the fusion of mononuclear precursors derived from haemopoietic stem cells in
the bone marrow, rather than incomplete cell divisions (Chambers 1989, Göthling et al.
1976, Kahn et al. 1975, Suda et al. 1992, Walker 1973a, Walker 1975b, Walker 1975a).
They share a common stem cell with monocyte-macrophage lineage cells (Ash et al.
1980, Kerby et al. 1992). The differentiation of osteoclast precursors into mature
multinucleated osteoclasts requires different factors including hormonal and local stimuli
(Athanasou et al. 1988, Feldman et al. 1980, Walker 1975a, Zheng et al. 1991) and living
bone and bone cells have been shown to play a critical role in osteoclast development
(Hagenaars et al. 1989). Osteoblastic or bone marrow stromal cells are also required for
osteoclast differentiation. One of the factors produced by these cells that support
osteoclast formation is macrophage colony-stimulating factor, M-CSF (WiktorJedrzejczak et al. 1990, Yoshida et al. 1990). Receptor activator for NF-κB ligand
(RANKL, also known as TRANCE, ODF and OPGL) is the vital signal (Suda et al. 1992)
through which osteoblastic/stromal cells stimulate osteoclast formation and resorption via
19
a receptor RANK (TRANCER) located in osteoclasts (Lacey et al. 1998, Tsuda et al.
1997, Wong et al. 1997a, Wong et al. 1997b, Yasuda et al. 1998a, Yasuda et al. 1998b).
Osteoblasts also secrete protein that strongly inhibits osteoclast formation called
osteoprotegerin (OPG, also known as OCIF), which acts as a decoy receptor for the
RANKL thus inhibiting the true connection between osteoclasts and osteoblasts via
RANK and RANKL (Fig.1).
OPG Decoy receptor, soluble
TRANCE, RANKL, osteoblasts
TRANCER, RANK, osteoclasts
STIMULATION
INHIBITION
OPG
PTH
1,25(OH)2D3
M-CSF
PGE2
PGE2,
1,25(OH)2D3
Fig. 1. Regulation of osteoclast formation and resorption by TRANCE/OPG. Osteoblasts
express in their plasma membrane TRANCE. Osteoclast precursors in turn have a receptor
for TRANCE, called TRANCER, by which osteoblastic or stromal cells can stimulate
precursor differentiation into mature osteoclasts. Through this receptor osteoblastic cells can
also stimulate mature osteoclastic bone resorption. Osteoblasts also secrete a soluble protein
called OPG, which acts as a decoy receptor for TRANCE, thus blocking the stimulating effect
of TRANCE to osteoclasts or their precursors.
Osteoclasts are responsible for dissolving both the mineral and organic bone matrix
(Blair et al. 1986). Osteoclasts represent terminally differentiated cells expressing a
unique polarized morphology with specialized membrane areas and several membrane
and cytoplasmic markers, such as tartrate resistant acid phosphatase (TRAP) (Anderson
et al. 1979), carbonic anhydrase II (Väänänen et al. 1983), calcitonin receptor
(Warshafsky et al. 1985) and vitronectin receptor (Davies et al. 1989). Multinucleated
osteoclasts usually contain less than 10 nuclei, but they may contain up to 100 nuclei
being between 10 and 100 µm in diameter (Göthling et al. 1976). This makes them
relatively easy to identify by light microscopy. They are highly vacuolated when in active
20
state, and also contain many mitochondria, indicating a high metabolic rate (Mundy
1990).
2.2 Cellular mechanisms of bone resorption
The process of bone remodeling is the key phenomenon in bone cell biology.
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. In the normal adult skeleton,
bone formation occurs only at sites where bone has previously been resorbed. Throughout
life old bone is continuously removed by osteoclasts and new bone is formed by
osteoblasts in a process called bone remodeling cycle. The adult skeleton is in a dynamic
state, being continuously broken down and reformed by the coordinated actions of
osteoclasts and osteoblasts on trabecular surfaces and in Haversian systems.
The remodeling cycle is a coordinated series of events, where the actions of
osteoblasts and osteoclasts are tightly coupled to retain the balance between formation
and resorption of bone. This turnover or remodeling of bone occurs in focal and discrete
packets throughout the skeleton, the rate of which varies considerably both within and
among different bones of the skeleton. The modeling of each packet takes a finite period
of time resulting that approximately 10-15% of bone surface is continuously being
remodeled. Bone remodeling occurs in focal and particular units, called basic
multicellular units (BMU) or bone remodeling units (BRU) throughout the skeleton
(Frost 1964, Parfitt 1983). The remodeling of each unit takes about 3-4 months. Bone
resorption is recognized as being a multi-step process and the work done by each BRU is
divided into four distinct phases: activation, resorption, reversal and formation (Baron
1977, Frost 1973, Frost 1986), as described below.
The term “activation” is used to describe the initiating event that converts a resting
bone surface into a remodeling surface. In the normal adult skeleton, a new BRU is
activated every 10 s (Parfitt 1983). Activation involves recruitment of osteoclast
precursors to the bone and their differentiation and fusion into functional osteoclasts. It
also involves the penetration of the bone lining cells and recognition of the bone sites to
be resorbed (Tran et al. 1982a, Tran et al. 1982b). The factors that determine why a
particular site of bone is chosen for remodeling are not clear. One of the most attractive of
the current hypotheses is that osteocytes may be involved in the activation (Chambers
1985, Frost 1973). The non-mineralized osteoid covers the mineralized bone matrix in
vivo, preventing its resorption by osteoclasts. Thus, before osteoclasts can begin the
actual resorption in vivo, the osteoid must be dissolved or mineralized, after which
osteoclasts can attach to the mineralized matrix and initiate bone resorption (Jones et al.
1986). 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. 1991).
During the resorption phase osteoclasts work in a concerted fashion, removing both
mineral and organic components of bone matrix (Blair et al. 1986). The hallmark of the
21
resorbing surface is the appearance of a scalloped erosion, also called an eroded surface
or Howship’s or resorption lacuna. In cancellous bone, osteoclasts resorb saucer-shaped
cavities some 50 µm in depth towards its center. In cortical bone, the osteoclasts drill
through the matrix in a direction that is parallel with the long axis of the bone, creating a
tunnel about 2,5 mm long and 200 µm in diameter (Eriksen 1986). The resorption phase
takes about two-three weeks (Baron et al. 1984).
Once the osteoclasts have resorbed most of the mineral and organic matrix, there is a
reversal phase, which lasts from 7-14 days and heralds the switchover from destruction to
repair. During the reversal phase macrophages may appear on the resorption surface
(Raisz 1988). The roles of these macrophages are not known, but it has been suggested
that they complete the task of bone resorption. Alternatively, they might produce factors
that help initiate osteoblastic bone formation (Raisz 1988). If the fate of osteoclasts at the
reversal site is apoptosis, the function of macrophages could be related to destruction of
osteoclast corpses. This has been shown to occur with other cells (Arends et al. 1991). It
is during the reversal phase that the coupling of bone resorption to formation takes place.
The mechanism by which osteoblasts are summoned into the resorption lacuna is
uncertain, but it is probable that a number of paracrine factors produced in or around the
remodeling site are involved. 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 factors could also be released from
the bone matrix during the resorption phase (Mundy et al. 1987). Osteoclasts are highly
motile and actively migrating cells, so that after completion of one resorption lacuna, they
can move along the bone surface to another site and restart the resorption phase
(Kanehisa et al. 1988).
Formation of new bone begins after the short reversal phase. Bone formation is a twostage process beginning with the deposition of osteoid, an organic matrix consisting
primarily of type I collagen and various other components. In normal adult bone, osteoid
is laid down in discrete lamellae about 3 µm thick. The second stage in bone formation is
mineralization of the organic matrix, which occurs after a delay of about 20 days called
the mineralization lag time. Two general theories exist for how calcification is initiated in
skeletal tissue, not necessarily excluding one another: the nucleation theory and the
matrix vesicle theory (Anderson 1984, Anderson 1989, Williams et al. 1991). 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. Originally it was believed that the initiation of bone mineralization is mainly an
extracellular, biochemical process where the presence of collagens, certain
glycosaminoglycans or lipids could trigger the initiation of calcification (Baylink et al.
1972, Irving 1958, Shuttleworth et al. 1972, Spector et al. 1972, Wuthier 1968, Wuthier
1969). 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. 1964). Later it was shown that mitochondria of bone-forming cells might
produce a local rise in the levels of mineral ions, and also a matrix, which was capable of
being mineralized (Shapiro et al. 1969). The mitochondria of columnar and hypertrophic
zones of growth plate cells were found out to contain more enzymes associated with
calcification (alkaline phosphatase, alkaline pyrophosphatase, lysozyme and lactate
dehydrogenase) than did those of the resting zone (Arsenis 1972). The matrix vesicles
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, often in contact with the
initial mineral crystals (Anderson 1984, Anderson 1995, Schwartz et al. 1987). These
vesicles have high alkaline phosphatase, alkaline pyrophosphatase and ATPase content,
but hardly any acid phosphatase, and are thus not lysosomal origin (Ali et al. 1970).
Plausible evidence has been demonstrated to support the matrix vesicle theory, 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. 1994).
However, later it has been speculated that the two mechanisms for bone mineralization
are not alternative but parallel; one is predominant in both calcified cartilage and
primitive woven bone, the other in lamellar bone (Gorski 1998, Termine 1993). Calcified
cartilage and woven bone seem to mineralize via matrix vesicles (Bonucci 1971),
membrane-bound bodies that exocytose from the plasma membrane and migrate to the
loose extracellular matrix space. The lipid-rich inner membrane of these vesicles becomes
the nidus for hydroxyapatite crystal formation and, eventually, crystallization proceeds to
the point of obliteration of the vesicle membrane producing a spherulite of clustered, tiny,
crystals. These spherulites conglomerate until a continuous mineralized mass is achieved
throughout the matrix space. The driving force for the mineral cascade, once initiated,
seems to be the mineral crystals themselves that are first associated with the matrix
vesicle membrane. The rate of mineralization in both woven bone and in lamellar bone
seems to depend on the presence of inhibitor molecules (e.g. pyrophosphate and acidic
NCPs), which in solution seem to regulate the kinetics of the mineralization process
(Termine et al. 1980). The cell buds off organelles capable of mineral accumulation and
then synthesizes proteins that can control the rate at which crystallization proceeds. 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.g. fibronectin
and osteonectin). Either because there simply is insufficient space for them or for
developmental reasons, matrix vesicles are rarely, if ever, seen in lamellar bone. Instead,
mineralization proceeds in association with the heteropolymeric matrix fibrils
themselves. 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. Other loci for the bone mineral appear to be between collagen fibrils in a brick
and mortar fashion (Weiner et al. 1986). It is unknown whether the driving force for
mineralization is the bone collagen itself or its associated NCPs. There is no doubt that in
mature calcified bone collagen and the inorganic phase are strongly bound (Irving 1973).
After the mineralization process is triggered, the mineral content of the matrix
increases rapidly over the first few days to 75% of the final mineral content (primary
mineralization), but it takes from 3 months up to a year for the matrix to reach its
maximum mineral content (secondary mineralization) (Amprino et al. 1952). The
principal component of the mature mineral phase is hydroxyapatite.
23
2.2.1 Osteoclastic membrane domains
Inactive osteoclasts, although they can still adhere to the bone surface, are not polarized
and fail to display specialized membrane domains (Holtrop et al. 1977). Actively
resorbing osteoclasts, in turn, 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. 1990, Holtrop et al. 1977, Lakkakorpi et al. 1989, Lakkakorpi et
al. 1991a, Salo et al. 1996). The membrane areas of active osteoclasts can be separated
into the following domains: sealing zone, ruffled border, basolateral membrane and
functional secretory domain (Fig. 2). In the sealing zone, also called as clear zone, the
cell membrane forms a tight attachment to the bone surface, thereby isolating the
resorption lacuna from the extracellular fluid and permitting the maintenance of a specific
microenvironment in the lacuna. The sealing zone is seen as an electro-dense area free of
organelles (Schenk et al. 1967) and it has a striated appearance in electron microscopy,
with alternating dark and light areas orientated perpendicular to the bone surface
(Malkani et al. 1973) consisting of bundles of actin filaments (King et al. 1975). Ruffled
border (RB) is a highly convoluted plasma membrane domain under which the actual
bone resorption takes place. Sealing zone surrounds the RB, thus defining the outlines of
the resorption lacuna. Acidic intracellular vesicles fuse to the cell membrane facing the
bone matrix, thereby forming RB membrane. RB does not fit to any type of previously
described plasma membrane domains, because it has both lysosomal and endosomal
features (Akamine et al. 1993, Baron et al. 1988, Palokangas et al. 1997). Bone
degradation occurs in the extracellular space between the bone matrix and the RB, which
is called the resorption lacuna. In the middle of the basolateral membrane is the fourth
membrane domain, the functional secretory domain (FSD), which appears when matrix
degradation is started (Salo et al. 1997).
1
3
2
FSD
BL
SZ
SZ
RB
BL
SZ
SZ
RB
Fig. 2. Osteoclastic membrane domains. 1) When an osteoclast is not resorbing bone, it shows
no signs of polarized membrane domains. 2) Once the osteoclast starts the resorbing, it
quickly polarizes its membrane into distinct domains. Ruffled border (RB) is a membrane
domain facing the bone surface, where the actual resorption takes place. Sealing zone (SZ)
forms a tight contact to the bone, sealing the proteolytic enzymes and acid into the forming
resorption lacuna. Basolateral membrane (BL) faces towards the bone marrow. 3) When the
osteoclast is actively resorbing bone, a fourth domain arises into the basolateral membrane,
the functional secretory domain (FSD), which acts as a route of osteoclasts to exocytose the
resorbed material.
24
2.2.2 Attachment of osteoclasts to bone matrix
Bone resorption occurs in an extracellular compartment below the osteoclasts. This
structure has been suggested to be a late endosome-type compartment (Palokangas et al.
1997), from which different hydrolytic enzymes, together with acid, are secreted (Baron
1989). In order to retain the specific microenvironment created by the cell organelles in
the ruffled border membrane, osteoclasts need to have a tight attachment to bone matrix in
the sealing zone. The mechanism by which osteoclasts attach to bone is not fully
understood (Dresner-Pollak et al. 1994, Horton et al. 1989, Väänänen et al. 1995). A
number of cell surface glycoproteins have been identified as intercellular adhesion
molecules, and these have been classified into at least three major molecular families, the
immunoglobulin (Ig) superfamily, the integrin superfamily, and the cadherin family.
Integrins have been identified as a family of cell surface receptors that recognize
extracellular matrices (Mundy et al. 1987). 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. 1986). Osteoclasts express two
members of integrin superfamily, the vitronectin receptor αvβ3 (Davies et al. 1989, Horton
1990) and α2β1 (Horton et al. 1989). The first indications of the importance of vitronectin
receptor came from the experiments in which monoclonal antibodies against osteoclasts
(Chambers et al. 1986), later identified as anti-vitronectin receptor antibodies (Davies et
al. 1989), inhibited osteoclastic bone resorption in vitro. It has been shown that peptides
containing the RGD sequence potentially inhibit bone resorption (Horton et al. 1991,
Lakkakorpi et al. 1991b, Sato et al. 1990). It has also been shown that osteoclasts adhere
in vitro to a wide range of RGD peptide containing proteins, including bone sialoproteins,
via a β3 integrin (Helfrich et al. 1992).
Controversial data has been reported on the precise localization of vitronectin receptor
in osteoclasts. It has been suggested that vitronectin receptor mediates the tight
attachment of osteoclasts to bone matrix (Nakamura et al. 1996, Reinholt et al. 1990, Sato
et al. 1990) and that osteopontin, a bone matrix component containing the RGD sequence
(Oldberg et al. 1986) is the ligand of the osteoclast vitronectin receptor. Nevertheless,
VNR has also been shown to be located only in the ruffled border, basolateral membranes
and intracellular vesicles of osteoclasts, but missing from the area of the tight sealing
zone (Lakkakorpi et al. 1991b, Suda et al. 1995). Although VNR is important in the
function of osteoclasts, the final tight attachment of osteoclasts to the bone surface is
mainly not mediated through it, according to their known structural features (Horton
1997, Lakkakorpi et al. 1991b). Interestingly, in echistatin-treated mice, where bone
resorption is inhibited, 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. 1998, Yamamoto et al. 1998). Hence, inhibition of integrin function did
not cause detachment of osteoclasts from the bone surface or changes in the clear zone or
ruffled borders.
25
Osteoclasts can attach to and migrate on bone surface in vitro without performing bone
resorption. The attachment of these non-resorbing osteoclasts is referred as primary
attachment, and it is thought to be mediated by integrins, which allow the cells to attach
to the matrix by many simultaneous but weak bonds. This enables the cells to move along
the bone surface (Väänänen et al. 1995). Expression of vitronectin receptor has also been
detected in mononucleated tartrate resistant acid phosphatase (TRAP) positive cells
during in vitro osteoclastogenesis in culture, and RGD-containing disintegrins, such as
echistatin, inhibited the formation of multinucleated TRAP-positive cells. 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. 1991).
2.2.3 Cadherins
Cadherins are a family of structurally and functionally related molecules that usually
mediate homophilic, calcium-dependent cell-cell interactions, and are predominantly
localized at adherent junctions of epithelial cells (Boller et al. 1985, Geiger et al. 1992,
Takeichi et al. 1988, Takeichi 1991). The demonstrated role of cadherins in mediating cell
interactions, coupled with the complex spatio-temporal patterns of cadherin expression
during embryonic development, suggest that cadherins play a critical role in
embryogenesis and morphogenesis (Grunwald 1993, Takeichi 1991). The classical
cadherins are represented by a group of highly conserved integral membrane proteins
initially identified biochemically, immunochemically and functionally about 20 years
ago. These classical N-, E- and P-cadherins, along with the more recently found R, B and
EP-cadherins, posses similar structural and functional domains, which include sites for
adhesive recognition, calcium binding, membrane integration, cytoskeletal interactions,
and post-translational processing such as glycosylation, phosphorylation and proteolysis
(Geiger et al. 1992, Grunwald 1993, Kemler 1992) (Fig. 3). Cadherins are protected by
2+
2+
Ca against proteolysis but they are readily degraded if Ca is absent (Grunwald et al.
1981, Takeichi 1977).
26
Calciumbinding
site
NH2 - EC1
Adhesive
recognition
site
Phosphorylation
sites
Glycosylation
sites
EC2
EC3
EC4
TM
Cytoskeletal
binding site
CYTO - COOH
α
γ
β
Catenins
Fig. 3. Structural organization of cadherins. Indicated are the five extracellular (EC1-EC5),
transmembrane (TM), and cytoplasmic (CYTO) domains. The extracellular domains include
multiple glycosylation sites, a proteolytic cleavage site near the cell membrane and
functionally critical calcium-binding and adhesion recognition sites. The cytoplasmic domain
includes sites for interaction with catenin cytoskeletal proteins. The cadherins and catenins
both contain phosphorylation sites that regulate their function in intercellular junctions.
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. The most highly conserved region of
cadherins is the cytoplasmic domain, and the second most conserved region is around the
amino-terminal portion. 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. 1990a). 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. 1990b). 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. 1992).
Cadherins have generally been associated with homotypic cell interactions among
solid tissues. Interestingly, it has been suggested that cadherins may also be involved in
heterotypic or even heterophilic interactions (Cepek et al. 1994, Tang et al. 1993).
Moreover, a recent study has shown that cadherins can interact with collagen type I
(Mbalaviele et al. 1996) and hence could act as cell-matrix adhesion molecules.
Cadherins associate with the cytoskeleton of cells via proteins called catenins. This
protein family has at least three major classes, α, β and γ, which interact with the
cadherins (Knudsen et al. 1992, Ozawa et al. 1990). Even though cadherins have essential
roles in embryogenesis, many mature tissues also continue to express cadherins.
Cadherins may thus be partly responsible for maintaining tissue integrity. Indeed,
27
reduction of E-cadherin expression is positively correlated with progression of various
cancers and increased cellular invasiveness (Umbas et al. 1992). It seems that E-cadherin
could have tumor suppressor function.
E-cadherin is expressed in osteoblasts (Babich et al. 1994) and a new cadherin called
OB-cadherin has also been cloned from osteoblasts (Okazaki et al. 1994). 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 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. 1995). It is,
therefore, possible that since the HAV sequence inhibits osteoclast formation, the fusion
of mononuclear osteoclast precursors mediated by E-cadherin may also involve
cytoplasmic signaling events, as recently proposed (Cowin 1994).
2.2.4 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. Osteoclasts resorb bone by
generating a pH gradient between the cell and bone surface (Anderson et al. 1986, Baron
et al. 1985, Blair et al. 1993). An acidic pH favors the dissolving of the bone mineral
(hydroxyapatite) and provides optimal conditions for the action of the proteinases
secreted by osteoclasts. Inorganic bone matrix is dissolved by the acidic environment in
the resorption lacuna revealing the organic collagen network. Carbonic anhydrase II (CA
II) is a cytoplasmic enzyme hydrolyzing carbon dioxide into bicarbonate and protons
(Gay et al. 1974, Gay et al. 1983, Väänänen et al. 1983). CA II is suggested to be the
main source of protons for the acidification of the resorption lacuna (Blair et al. 1989,
Laitala et al. 1994, Minkin et al. 1972, Väänänen et al. 1990). A vacuolar-type proton
pump, V-ATPase, is present in high amounts in the membranes of a population of
intracellular vesicles. V-ATPase transports the protons generated by CA II into these
vesicles, which are then transported and fused to the RB membrane (Bekker et al. 1990,
Blair et al. 1989, Laitala et al. 1994, Sundquist et al. 1990, Väänänen et al. 1990)
releasing their proton content to the lacuna. Acidification of the extracellular resorption
lacuna is completed by passive, potential-driven chloride transport (Blair et al. 1991). RB
thus contains large amounts of V-ATPase, which probably continues to function by
transporting protons directly from the cytoplasm to the lacuna. Hydroxyapatite is first
solubilized in the acidified lacuna, after which various proteolytic enzymes, such as
lysosomal enzymes (Baron et al. 1988, Delaisse et al. 1984, Delaisse et al. 1987, Rifkin et
al. 1991) and bone-derived collagenases (Chambers et al. 1985, Delaisse et al. 1988)
secreted by osteoclasts through RB, digest the exposed organic matrix. When the
osteoclast stops resorption and moves away from the resorption lacuna, phagocytes clean
up the remains and make room for osteoblasts to begin bone formation in the newly
formed resorption cavity.
Only recently it has been shown that osteoclasts remove the degradation products of
bone matrix from the resorption lacuna by transcytosis (Nesbitt et al. 1997, Salo et al.
28
1997). The degradation products, both organic and inorganic, are endocytosed to the
resorbing osteoclast, transported through the cell in large vesicles, and finally released
into the extracellular space through the FSD. 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. This
further supports the osteoclastic penetration deeper into the bone. The transcytosis route
provides a possibility for osteoclasts to further process the endocytosed degradation
products intracellularly during their passage through the cell. The bone-specific enzyme
TRAP is located in cytoplasmic vesicles, which fuse to the transcytotic vesicles and
participates in destroying the endocytosed material in the transcytotic route (Halleen et al.
1999).
2.2.5 Regulation of osteoclastic bone resorption
A balance between bone formation and bone resorption is a necessity for the normal
function of bone. Although many acquired or environmental factors are known to affect
the adult bone mineral density (BMD) (Välimäki et al. 1994), genetic factors play a major
role as determinants of variation in BMD (Pocock et al. 1987). It has been estimated that
up to 80% of BMD is genetically controlled, and it is the rate of bone formation rather
than the rate of bone resorption that is influenced by genes (Kelly et al. 1991).
Systemic stimulators of bone resorption include parathyroid hormone (PTH) (Barnicot
1948, Holtrop et al. 1974, Miller 1978, Raisz 1963), interleukin-1 (Chambers 1988,
Gowen et al. 1983a, Gowen et al. 1983b), tumor necrosis factor (Abe et al. 1986,
Thomson et al. 1987), transforming growth factor α (Takahashi et al. 1986) and 1,25dihydroxyvitamin D3 (1,25(OH)2D3) (Raisz et al. 1972, Tanaka et al. 1971). On the other
hand, calcitonin (CT) (Holtrop et al. 1974), gamma interferon (Gowen et al. 1986) and
transforming growth factor β (Bonewald et al. 1990, Chenu et al. 1988) are extremely
potent in inhibiting osteoclast differentiation and activity. 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 1,25(OH)2D3 or other vitamin D metabolites seem to play a role in the correction of calcium
malabsorption, which is a common feature in osteoporosis (Lamberg-Allardt 1991,
Taterossian 1973, Weisbrode et al. 1978). It has also been shown that peak bone mass is
associated with vitamin D receptor polymorphism (Viitanen et al. 1996) and is also
related to the rate of bone loss (Ferrari et al. 1995). Osteoblasts mediate the effects of
PTH and 1,25(OH)2D3 on the osteoclasts (Narbaitz et al. 1983, Silve 1982). CT, a peptide
hormone secreted by the thyroid, regulates blood calcium and phosphate levels (Kumar
1963) by causing short-lived falls in the plasma calcium. It does that by its effects to
inhibit osteoclastic bone resorption (Friedman et al. 1986) and to promote renal calcium
excretion (Ralston et al. 1985).
In the past decades, it has become apparent that the skeleton is a major target tissue for
reproductive steroid hormones, estrogen, androgen, testosterone, and to a lesser extent,
progestin. The sex steroids, especially estrogen and testosterone, play a major role in the
29
sexual dimorphism of the skeleton in mineral homeostasis during reproduction and bone
balance in adults. Estradiol and progesterone are the principal circulating sex steroids in
females but also function in males (Landers et al. 1992). Sex steroids are essential for
maintenance of normal bone volume and estrogen deficiency is an important risk factor
for osteoporosis. Ovariectomy leads to a deficit in ash weight and bone mineral density in
adult rats and monkeys (Jayo et al. 1990, Kalu et al. 1991, Lindsay et al. 1978) and it is
generally believed that treatment with estrogen of ovariectomized rats prevents the
increase of bone resorption (Anderson et al. 1970, Turner et al. 1988, Wronski et al.
1988).
2.2.6 Disorders of bone modeling and remodeling
Normal bone remodeling is initiated by local events that lead to an increase in
osteoclastic activity. This is followed eventually by an increased attraction and
proliferation of osteoblast precursors followed by the differentiation of these cells into
mature osteoblasts, which lay down new bone and repair the resorption defects caused by
the resorbing osteoclasts. Formation follows resorption at the resorption site, not at some
other location, and the amount of bone formed is almost always nearly equal to the
amount removed. Coupling is also manifest at a higher level of organization: global rate
changes in resorption are invariably followed by similar global changes in formation. The
remodeling process replaces aged bone tissue with new bone tissue. With the passage of
time, 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.
Unless microdamage is repaired, it will accumulate and lead to catastrophic structural
failure, namely, fracture. Several bone-related disorders have been mentioned in the
literature, two of which affecting the bone mass and the mechanical competence, are
shortly described here.
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, Jr. 1987, Walker 1973b). 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, with sclerosis of the base of the
skull (Elster et al. 1992a, Elster et al. 1992b). A reduced marrow space results in a
decrease in hematopoiesis even to the point of complete bone marrow failure. Nerve
compression and vascular compromise can also occur due to decrease in the caliber of
cranial nerve and vascular canals. In the mildest form the disease can produce only few,
or no symptoms, whereas the malignant form is fatal in early childhood. Although having
higher bone mass, the osteopetrotic bones are easier to fracture (Tuukkanen et al. 2000).
On the other hand, osteoporosis is a bone disease with reduction in bone mass and a
change in the microarchitecture, which increases susceptibility to fractures. In
osteoporosis, osteoclastic activity is highly increased leading to decreased bone mass.
The causal role of estrogen deficiency in this condition is well established (Odell et al.
1993, Takano-Yamamoto et al. 1990, Tuukkanen et al. 1991). Immediately after
30
menopause, the rate of osteoclastic bone loss increases as much as 10-fold, the annual
rate being about 2-3% in Caucasian women (Lindsay et al. 1980, Smith et al. 1975).
Numerous investigations have been performed to study the therapeutic approaches
treating osteoporosis (Rodan et al. 2000).
2.3 Cell polarity
Cellular polarization is best established in epithelium. 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. These plasma
membrane domains have different lipid and protein compositions (Simons et al. 1988,
van Meer et al. 1988). Epithelial cells have tight junctions, which separate the apical and
basolateral membranes from each other. The general features defining cell polarity have
been described by far in cultured (MDCK, Caco-2) and primary epithelial cells (Handler
1989, Rodriguez-Boulan et al. 1992). Many of the functions of epithelial, neuronal and
certain other cells of the immunosystem are maintained by the characteristically polarized
phenotype of these cells (Cereijido et al. 1989, Craig et al. 1992, Knust 1994, Nelson
1993, Rodriguez-Boulan et al. 1992) Polarization is also essential for embryonic
development and tissue morphogenesis. Most of the plasma membrane proteins in
epithelial cells are distributed primarily to either the apical or the basolateral plasma
membrane domains, but very few are distributed to both of these surfaces. It is probable
that only one of the two pathways to the polarized cell surface requires specific sorting
signals (Matter et al. 1994). The membrane polarity of epithelial cells has been studied by
using glycoproteins of enveloped viruses as markers (Rodriguez-Boulan et al. 1980,
Simons et al. 1990). In epithelial cells, Influenza virus targets its glycoproteins to the
apical membrane domain facing the external milieu, thus identifying it. Viral particles of
VSV-infected epithelial cells bud from the basolateral membrane, which is in contact
with the internal milieu and the blood supply. In epithelium, different plasma membrane
domains are clearly separated by tight junctions enabling the maintenance of membrane
polarity.
Most of the osteoclast membrane has receptors for VSV, representing a basolateral
membrane domain. The special cap structure on the top of an active osteoclast, the FSD,
is the target of viral glycoproteins in osteoclasts infected by Influenza A and Sendai
viruses, a feature characteristic of the apical membrane of epithelial cells, suggesting that
FSD corresponds to the apical surface of epithelial cells (Salo et al. 1997).
No exclusive reports have been presented on the polarity of osteoblastic cells.
However, it has been shown that during the secretory phase, osteoblasts look highly
polarized, and the matrix secretion is in the direction of the bone surface making it seem
like a polarized event (Cooper et al. 1998, Marks, Jr. et al. 1996). In addition, RingbomAnderson et al. have presented that during matrix formation, several matrix vesicles can
be found to bud from the long processes of U-2 OS osteoblastic cells, making the cells
look highly polarized (Ringbom-Anderson et al. 1994).
31
2.4 Communication between bone cells
A series of complex and coordinated events is required for bone remodeling (Marcus
1987, Väänänen 1993). Osteoblasts, osteocytes and osteoclasts communicate with each
other resulting in the elaborate balance of bone formation and resorption. It requires wellorganized actions by osteoblasts, osteoclasts, and osteocytes. This coordinated activity
between bone cells suggests the existence of intercellular communication, but the
mechanisms of this communication are not well characterized. Intercellular
communication can occur via secretion of soluble factors, direct cell-cell contact through
cell adhesion molecules or metabolic and electric coupling through gap junctions.
2.4.1 Gap junctions
Most cells communicate with their immediate neighbors through exchange of cytosolic
molecules, such as ions, second messengers, and small metabolites. This activity is made
possible by clusters of intercellular channels called gap junctions, which connect adjacent
cells (Kumar et al. 1996). Thus, apart from a few terminally differentiated cells, such as
skeletal muscle, erythrocytes, and circulating lymphocytes, most cells in normal tissues
generally communicate via gap junctions. Gap junctions are specialized regions of cell
membrane, 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, which interact to span the plasma
membranes of two adjacent cells and directly join the cytoplasm of one cell to that of
another (Fig. 4 and 5). Each hemichannel is composed of a hexameric array of
transmembrane proteins called connexins, a highly related multigene family consisting of
at least 13 members (Beyer et al. 1990, Goodenough et al. 1996, Saez et al. 1993).
32
Membrane
Extracellular
E2
E1
M1
M2
M3
Gap 27
M4
Cytoplasm
NH2
COOH
Fig. 4. Connexin structure. The cylinders represent transmembrane domains (M1-M4). The
loops between the first and second, as well as third and fourth, transmembrane domains are
predicted to be extracellular (E1 and E2, respectively).
Connexin channels participate in the regulation of signaling between developing and
differentiated cell types. The gap-junctional channels have an apparent selectivity based
principally on molecular size, allowing the movement of molecules smaller than 1000 Da,
such as cAMP, but preventing the movement of proteins and nucleic acids. Small
informational molecules, such as certain morphogens could be directly transmitted
between cells via gap junctions, which is important in, for instance, embryogenesis for
regulating the events between cells (Warner et al. 1984).
A spectrum of drugs has been shown to inhibit gap-junctional communication with
varying degrees of efficacy and specificity. These include for instance 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) and
glycerrhetinic acid (Davidson et al. 1995). Although a variety of drugs have been used to
block gap-junctional communication, the specificity of the blockers remains to be solved.
Bone cells mediate signals to each other through the communicating gap junctions,
which are found connecting adjacent osteoblasts together, between osteoblasts and
overlying cells or osteocytes (Akisaka et al. 1990, Jeansonne et al. 1979, Jones 1974,
Stanka 1975, Weinger et al. 1974, Yellowley et al. 2000). Osteocyte nutrition probably
takes place across the gap junctions between the cytoplasmic extensions of neighboring
osteocytes. Connexin-43 (Cx43) is widely expressed in different tissues, including brain,
heart, kidney, smooth muscle, ovary, and some epithelia, and it is actually the most
abundant connexin (Goodenough et al. 1996, Musil et al. 1990). Cx45 and Cx46 have
been identified in bone, but Cx43 is the predominant gap junction protein expressed in
bone (Donahue et al. 1995b, Vander et al. 1996).
33
On cell contact, connexons from neighboring cells may form gap junctions in a matter
of minutes or even seconds, suggesting that connexons pre-exist in the plasma membrane
(Rook et al. 1990). Gap-junctional communication would thus be an excellent method for
bone cells to rapidly propagate locally generated signals throughout the skeletal tissue, as
has been shown to happen in osteoblasts (Civitelli et al. 1993). A dependable means of
communication is utterly important for correct coupling of osteoclasts and osteoblasts.
The capability to exchange nutrients and regulatory molecules from one cell - or one cell
type - 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.
Homomeric
Homotypic
Heteromeric
Homotypic
Homomeric
Heterotypic
Heteromeric
Heterotypic
Fig. 5. Possible arrangements of connexons to form gap junction channels. Connexons consist
of six subunits, connexins. A connexon may be homomeric, when it is composed of six
identical connexin subunits or heteromeric, when it has more than one species of connexins.
Connexons associate end to end to form a double membrane gap junction channel. The
channel may be homotypic, if connexons are identical or heterotypic, when the two connexons
are different.
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
bone multicellular unit works synchronically as a cell syncytium, which is controlled and
regulated by hormonal signals and by mechanical forces adjusted to bone. The hypothesis
of this work was that when the bone cells receive a signal to start the remodeling cycle,
they have to communicate with each other in order to retain the balance between the
activities of different bone cells. This requires attaching of bone cells to the matrix,
polarizing of the cells’ plasma membranes into specific domains and contacting of bone
cells to each other. The basic mechanism of this coupling has not been previously known.
The aims of the present study work can be briefly outlined as follows:
1. To study how osteoclasts attach to bone surface. We wanted to learn what are the
proteins involved in the tight attachment in osteoclasts and what proteins are needed
from the bone surface.
2. To study if osteoblasts truly are polarized cells. The secretion of bone matrix happens
in a polarized manner. We wanted to study the mechanisms underlying this feature of
osteoblasts.
3. To study the communication mechanisms by which bone cells communicate with
each other. The communication between osteoblasts and osteoclasts is essential for
proper bone remodeling - we wanted to solve how the actual connection between the
cells happens.
4 Materials and Methods
4.1 Reagents (I-IV)
All cell culture media and reagents were purchased from Gibco BRL (Paisley, England).
Hexapeptides AHAVSE and VASAEH were synthesized in Department of Medical
Biochemistry, University of Turku, Finland. Heptanol was purchased from Sigma
Chemical Co. (St. Louis, MO). Synthetic peptide Gap 27 (SRPTEKTIFII) was
synthesized in Keck Foundation Biotechnology Resource Laboratory, Yale University
School of Medicine , USA.
4.1.1 Antibodies (I-IV)
Polyclonal rabbit antibody against pan-cadherin (Sigma Chemicals, St. Louis, MO),
monoclonal antibodies against E-cadherin (Developmental Studies Hybridoma Bank,
Iowa City, IA) and β-catenin (Transduction Laboratories, Lexington, KY) were all used at
1:50 in PBS. A monoclonal mouse antibody against chicken gizzard smooth muscle
vinculin was purchased from ICN Immuno Biologicals (Lisle, IL) and used at a dilution
1:50 (I). 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. 1991). The VSV G protein and the matrix
protein were localized using specific polyclonal antibodies produced in our own
laboratory (Metsikkö et al. 1992) (II). Monoclonal antibodies raised against Connexin-43
(Transduction Laboratories, Lexington, KY) were used at a dilution 1:100 (III).
Secondary antibodies, either tetramethylrhodamine isothiocyanate (TRITC) or
fluoresceinisothiocyanate (FITC)-conjugated, rabbit anti-mouse or swine anti-rabbit or
goat anti-rabbit were also obtained from DAKO, and used at a dilution of 1:100 (I-IV).
TRITC or FITC-conjugated phalloidin was purchased from Molecular Probes Inc.
(Eugene, OR) and was used at 5 units/ml. DNA-binding fluorochrome Hoechst 33258 (1
36
mg/ml stock, diluted 1:800, Sigma Chemical Co., St. Louis, MO) was used to visualize
nuclei (I, III-IV).
4.2 Cell culture (I-IV)
4.2.1 Isolation and culture of osteoclasts (I, 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 0.84 g of sodium bicarbonate/liter, 2 mM L-glutamine, 100 IU of
penicillin/ml, 100 µg of streptomycin/ml and 7-10% heat-inactivated fetal calf serum
(FCS). The cells were allowed to attach to sonicated bovine cortical bone slices for 30
minutes. After the attachment period, non-attached cells were rinsed away, 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. 1984, Chambers et al.
1984, Lakkakorpi et al. 1989). Cells were cultured at +37°C (5% CO2, 95% air) for up to
48 hours.
In further studies some of the following changes were made to the culture medium:
hexapeptides AHAVSE or VASAEH at concentration 1 mg/ml, heptanol 3 mM or Gap 27
at 500 µM were added to the culture medium of the cells. The cultures were usually
stopped by fixing the cells and bone samples with freshly made or refrigerated 3%
paraformaldehyde (PFA), 2% sucrose in phosphate-buffered saline (PBS) for 10 minutes
at room temperature.
4.2.2 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, 100 IU of penicillin/ml, 100 µg of streptomycin/ml and 7-10% heatinactivated fetal calf serum (FCS), which will be later referred to as MEM-FCS. The cells
were grown at +37°C (5% CO2, 95% air) in small cell culture bottles in 5 ml of medium,
and for the experiments the cells were trypsinized and plated on 150 µm thick 1 cm2
bovine bone slices.
4.2.3 Tissue culture (II)
For studying endosteal osteoblasts, 3 to 4 days old rat pup femora, tibiae and humeri were
dissected out, placed in MEM (Minimum Essential medium) containing 2 mM L-
37
glutamine, 100 IU of penicillin/ml, 100 µg of streptomycin/ml and 7-10% heatinactivated fetal calf serum (FCS). After removing the epiphyseal cartilages the
diaphyses, bones were bisected with a scalpel blade. The loose bone marrow was gently
removed by injecting culture media over it with a pipette. The bones were cultured
overnight at +37°C (5% CO2, 95% air).
4.3 Fluorescence and microscope techniques (I-IV)
4.3.1 Light microscopy (I, III-IV)
Osteoclasts were identified by staining them for tartrate-resistant acid phosphatase
(TRAP), a commonly accepted marker of osteoclasts (Minkin 1982), using a Sigma
TRAP kit (No. 386-A, Sigma Chemicals, St. Louis, MO) according to manufacturer's
instruction.
4.3.2 Conventional fluorescence microscopy (I, III-IV)
Osteoclasts were cultured in DMEM-FCS growth medium, with either hexapeptides
AHAVSE or VASAEH (I), heptanol (III) or Gap 27 (IV). After growing the cells for 48
hours, the cells were fixed and permeabilized with various methods for the
immunofluorescence staining.. 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. When using βcatenin, cells were fixed with 10 min methanol at room temperature. With pan-cadherin,
vinculin, the Influenza virus hemagglutinin, Influenza virus nucleocapsid protein, and the
VSV G protein and the matrix protein and Connexin-43 antibodies, phalloidin or Hoechst
33258, the cells were fixed in 3 % paraformaldehyde in PBS containing 2 % sucrose for
10 min at room temperature. Then the samples were rinsed in PBS and permeabilized
with 0.1% Triton X-100 in PBS for 10 minutes on ice.
For immunostaining, the bone slices or bone samples were incubated with primary
antibody one to two hours at +37°C. The samples were rinsed thoroughly several times
with PBS, and further incubated with TRITC- or FITC-labeled secondary antibodies for
30-60 min at +37°C. Non-specific binding of antibodies was blocked with swine serum.
The procedure for staining F-actin has previously been published in detail (Lakkakorpi
et al. 1991a). F-actin was stained using either TRITC or FITC-conjugated phalloidin for
20 minutes at +37°C. Finally all the samples were mounted in either Mowiol (Hoechst,
Frankfurt) in PBS containing 2.5% 1,4-diazobicyclo(2,2,2)-octane or 50% PBS-glycerol.
To visualize the nuclei, 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.
The samples were viewed with a Leitz Aristoplan microscope with appropriate filters.
38
4.3.3 Confocal laser scanning microscopy (I-IV)
Samples for confocal laser scanning microscope were prepared in the same way as for
conventional epifluorescence microscopy. The confocal laser scanning microscope
consisted of a 750 mW air-cooled argon-crypton laser (Omnichrome, Chino, CA), a Leitz
Aristoplan microscope, and 1.05-software (Leica Lasertechnik Gmbh, Heidelberg,
Germany). Scanning was performed in a XY-plane using either a 40 x 0.6 water
immersion objective (Nikon, Tokyo Japan), a PLAPO 63x objective (NA 1.4) or Fluor
40x1.3 and 50x1.0 oil immersion objectives (Leitz, Germany) with suitable zoom factors
(1x or 2x) of the scanner. Laser power and offset were adjusted according to the
fluorescence label intensity. The localization of immunostaining was acquired by
scanning serial optical xy and xz sections. 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.
4.3.4 Immunoelectron microscopy (II)
The cell cultures were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4,
for 1 to 2 days at 4°C. The bone slices were decalcified with 7% EDTA in phosphate
buffer for 4 days at +4°C with daily changes of the decalcifying medium. Ultrathin
sections (100 nm) were made after removal of mineral from the bone slice. The samples
were then postfixed with 4% OsO4, dehydrated and embedded in Epon. The samples
were examined with a Philips 410 electron microscope (Philips, The Netherlands).
4.3.5 Digital image analysis (I -IV)
MCID image analyzers utilizing M1 or M2 software (Imaging Research Inc., Brock
University, Ontario, Canada) consisting of an Intel 403 E microcomputer linked to an
Image 1280 image processor (Matrox Dorral, Quebec, Canada), were used to the
quantification of areas of the resorption lacunae. Resorption pits were visualized as
brown-colored areas after staining with peroxidase-conjugated wheat germ agglutininlectin (WGA) (Sigma Chemical Co., St. Louis, MO) (Selander et al. 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).
39
4.4 Viral infections (II)
UMR-108 cells were cultured for two days to near confluency, and the cells were infected
with wild type vesicular stomatitis virus (Indiana serotype) or Influenza virus of A/WSN
strain. Adsorption was for 1 h at 37°C with 10 plaque-forming units in MEM containing
0,1% BSA, after which the cells were further cultured for 3 hours (VSV) or 7 hours
(WSN) in the presence of serum. The endosteal osteoblasts were infected after culturing
the bisected bones overnight with same procedure as UMR-108 cells.
4.4.1 Metabolic labeling (II)
35
Cells cultured on bone slices were labeled with S-methionine (Amersham Corp., Bucks,
England) at 4 hours postinfection (VSV) or 6 hours postinfection (WSN) for 10 minutes
at 500 µCi/ml and chased for 90 minutes.
4.4.2 Cell surface biotinylation (II)
After the chase, biotinylation was performed. The plasma membrane proteins of the
infected cells were labeled with sulpho-NHS-biotin (Pierce Chemical Co., Rockford, IL)
at 0°C as described by Lisanti et al. (Lisanti et al. 1988). The biotinylated proteins were
isolated by the methods described by Hare & Lee (Hare et al. 1989). Briefly, membrane
proteins labeled with sulfo-NHS-biotin at a concentration 0.5 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 SW50.1
rotor. Streptavidin-agarose (Sigma) was used to adsorb the biotinylated proteins by using
end-over-end rotation overnight at +5°C. Proteins were eluted from the streptavidinagarose by heating for 3 minutes at +95°C in SDS sample buffer. SDS-PAGE was carried
out by the method of Laemmli (Laemmli 1970). After the SDS-PAGE, 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, Sunnyvale,
CA) using MCID-M2 image analysis software (Imaging Research Inc., Brock University,
St. Catherines, Canada).
4.5 Statistical analysis (I, III-IV)
All statistical analyses were performed by using Student’s t-test for independent samples
and a computer program Microcal Origin version 4.00 (Microcal Software Inc.,
Northampton, MA).
5 Results
5.1 Cadherin localization in osteoclasts (I)
To localize cadherins in osteoclasts we stained the cells with different cadherin or
cadherin-related antibodies. E-cadherin and β-catenin antibodies were first tried in
immunofluorescence staining of osteoclasts. Neither of the antibodies gave positive
staining in osteoclasts even though MDCK-cells as a positive control showed similar
staining patterns as described earlier. We could distinguish osteoclasts from the other
cells in the cell culture by visualizing the nuclei with Hoechst stain.
On the contrary, pan-cadherin antibody reacted with osteoclasts forming a ring-like
structure localized in the sealing zone area. Some staining can also be seen in the
cytoplasm. 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. Pancadherin staining was also seen in between the vinculin double-rings, partly overlapping
with them.
5.2 Effects of HAV-containing peptide (I)
5.2.1 Primary attachment of osteoclasts to bone surface
To reveal if the common cadherin CAR sequence motif, HAV, is involved in the primary
attachment of osteoclasts to the bone surface, the CAR-containing peptide AHAVSE was
added to the primary attachment medium of osteoclasts. 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, both at a
concentration of 1 mg/ml, or with no added peptide to act as zero-controls. Attached
TRAP-positive multinucleated cells were counted after rinsing the non-attached cells
41
away. Similar amounts of TRAP-positive multinucleated cells were seen in AHAVSEtreated, VASAEH-treated and non-treated control cultures. Thus, the cadherin CAR
sequence-containing peptide AHAVSE had no effect on the primary attachment of
osteoclasts to bone slices.
5.2.2 Osteoclastic bone resorption
We studied the possible effect of AHAVSE on the ability of osteoclasts to form resorption
pits. Both the number of resorption pits and the total resorbed area decreased significantly
after addition of the cadherin CAR sequence-containing peptide AHAVSE. No significant
differences in comparison with the zero-control were detected when the control peptide
(VASAEH) was used. The mean size and the size distribution of resorption pits were
similar in all the treatments.
5.2.3 Activity of osteoclasts
Since the AHAVSE peptide inhibited osteoclastic bone resorption, the numbers of active
osteoclasts having actin rings were investigated. Osteoclasts were cultured for 24 hours,
after which AHAVSE or VASAEH was added to the culture medium. The cells were
allowed to grow for different periods of time from 20 minutes to 24 hours. After
incubation, the cells were fixed and stained for actin rings. The percentage of active
osteoclasts was determined as those with actin rings. The number of active osteoclasts
decreased significantly after addition of AHAVSE. The decrease was significant at all
time points from 20 minutes to 24 hours but it was not linear. No morphological changes
in the appearance of remaining actin rings were detected.
5.3 Polarity of osteoblasts (II)
5.3.1 Immunofluorescence data
The VSV G protein localizes to the basolateral membrane of epithelial cells (RodriguezBoulan et al. 1980, Simons et al. 1990). 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. To elucidate the cytoplasm of the UMR108 cells, we used the VSV matrix protein antibody and found it to localize diffusely all
over the cytoplasm (Nesbitt et al. 1993).
42
In endosteal osteoblasts, the VSV G protein localization was investigated using both
unpermeabilized and Triton-permeabilized cells. In unpermeabilized cells, the VSV G
protein was localized to the bone marrow facing plasma membrane. To prove that the
VSV G antibodies truly localize to the marrow facing membrane, the cells were
permeabilized with Triton. Antibodies against VSV G protein still localized to bone
marrow facing plasma membrane, but as expected, they could also be seen in the Golgi
complex. Double staining of VSV G protein and VSV matrix protein were also performed
to distinguish the cells’ outlines. At the bone marrow or circulation side in the bone tissue
cultures, the G protein immunolabeling had frequently an intense area towards
circulation.
In Triton-permeabilized osteocytes, the VSV G protein localized apart from the Golgi
complex, at the plasma membrane in a nonpolarized manner, although in osteocyte
processes extruded into the bone canaliculi, the ends of the processes probably
communicating with neighboring cell processes showed somewhat condensed staining.
In Influenza-infected UMR-108 cells the Influenza nuclear protein was used as a
marker for the nucleus, as shown earlier (Stein et al. 1993). The Influenza hemagglutinin
was sorted to the bone substrate-facing surface of UMR-108 cells. The hemagglutinin
antibody localized between the nucleus and bone, which was different and opposite to the
VSV G protein localization. Influenza HA was also found in the Golgi complex of
permeabilized cells.
5.3.2 Electron microscopy data
We used electron microscopy to show that if the glycoprotein localization is polarized,
the budding of viruses is also polarized, as shown earlier (Odell et al. 1993). Thus, the
localization of viral glycoproteins determines the site for viral budding. Budding virus
particles were detected at the plasma membrane of the infected cells.
VSV particles were mainly budding on the medium side of UMR-108 cells, where the
cells did not overlap. In the cells growing upon each other the cell surface against the
bone was still deficient of virus particles, although the upper cells had nonpolarized
budding. On the contrary, the Influenza virus was mainly found to bud from the plasma
membrane facing the bone slice, where the cells were in close contact with the bone and
in near confluency growing as a monolayer. In the cases where the growing cells
overlapped, the superficial cells were not polarized, since Influenza was budding
uniformly from all surfaces. However, even in these cases the cells in bone contact had
virus particles budding mainly from the bone-facing surface.
5.3.3 Biochemical data
Sulfo-NHS-biotin binds to the cell surface proteins and does not internalize at 0°C. Cell
35
surface biotinylation together with S-labeling revealed, Influenza HA was only 22%
43
biotinylated. This indicates, that Influenza HA was hidden in the bone facing plasma
membrane, which was not available for biotin binding. On the contrary, VSV G protein
was 55% biotinylated and thus found in the streptavidin eluate. This indicates that most
of the VSV G protein was at the medium facing surface available for biotinylation. Even
though the results are clear, they indicate that polarity is not achieved in all of the UMR108 cells.
5.4 Gap junctions in osteoclasts (III and IV)
5.4.1 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. To visualize the cell
outlines and the sealing zone area and thereby to distinguish active osteoclasts, double
stainings with F-actin were performed. The indirect immunostaining of osteoclasts
revealed punctuate distribution of Cx43 immunoreactivity. The staining could be seen in
cell-cell contacts with neighboring bone marrow derived mononuclear cells at the
interface of adjoining cells, but numerous immunoreactive sites were also apparent at free
surfaces and within the cytoplasm of osteoclasts.
5.4.2 Effects of gap-junctional inhibitors heptanol and Gap 27
5.4.2.1 Number and Activity of Osteoclasts
Since osteoclasts express gap-junctional Cx-43, we wanted to test whether the addition of
gap-junctional inhibitors, either heptanol or Gap 27, affects the number or activity of
osteoclasts. 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.
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.
We could see no difference in the number or morphology of other cell types in the
inhibitor-treated bone cell 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 control cultures contained approximately one to two times
the number of mononuclear than multinucleated TRAP-positive cells, 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. The results of the Gap
27-treated bone cells were almost identical with heptanol-treated bone cells.
44
To find out why the number of TRAP-positive cells is decreased in the heptanoltreated bone cell cultures, we studied the nuclei of the cells. We could see numerous
apoptotic osteoclasts in the inhibitor-treated bone cells cultures, whereas in control
cultures we could not see nearly any apoptotic osteoclasts, but the cells looked absolutely
healthy with intact nuclei.
To reveal whether heptanol has an effect on the activity of the remaining osteoclasts,
we counted how many of the TRAP-positive cells had actin rings. 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.
Interestingly, the Gap 27 seemed to act as even more potent inhibitor of the activity.
5.4.2.2 Area Measurement of Excavated Pits
Since heptanol and Gap 27 decreased the number and activity of osteoclasts, we studied
the effect of these inhibitors on the ability of osteoclasts to form resorption pits. 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. In addition, the number of resorption
pits was greatly decreased.
6 Discussion
6.1 Methodological aspects
We chose to use the pit formation assay as the research setting in our studies concerning
osteoclasts. This assay, originally introduced by Boyde et al.(Boyde et al. 1984) and
Chambers et al. (Chambers et al. 1984), enables the studying of resorption mechanism of
isolated, mature osteoclasts. The setting mimics the situation in vivo: osteoclasts can
resorb their natural target, mineralized bone. In addition, the events of the resorption
cycle of isolated osteoclasts on bone slices are well characterized (Karhukorpi 1991,
Lakkakorpi et al. 1991a, Lakkakorpi et al. 1993). Regarding to the cellular events taking
place at the BMU in vivo, the pit formation assay is however, biased; bone resorption by
osteoclasts is not followed by bone formation by osteoblasts. This may be due to the
preparation and pretreatment of bone slices resulting in inactivation of proteins and
cytokines, normally liberated via resorption and which are required for the cross-talk
between osteoclasts and osteoblasts. This method delimits our studies to monitoring
mature osteoclasts, and we thus miss the complicated maturation process of osteoclast
precursors, at least during short period culture (Chambers et al. 1984). The effect of the
agents used in this thesis work, cadherin CAR-sequence containing peptide AHAVSE or
the two gap-junctional inhibitors, are results from the direct effects of these agents on
osteoclasts and not of the complex cellular interactions occurring in vivo. However, 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. Different cellular mechanisms operate in osteoclasts cultured on glass, when
compared to those cultured on bone slices. No special structural features of active
osteoclasts, like ruffled border or sealing zone can be seen on artificial surfaces, thus the
attachment of osteoclasts to glass may also be different from bone (Lakkakorpi et al.
1993). This is why we cultured the cells on bone slices instead of glass.
When studying the polarity of osteoblasts we chose to use an osteoblast-like cell line
UMR-108 cultured on bone slices, which are similar to UMR-106 cells that have been
widely used as a model for osteoblastic cells (Koutsilieris et al. 1987, Matsumoto et al.
46
1985, Partridge et al. 1996). We then confirmed the results using endosteal osteoblast
culture to study the osteoblastic cells in situ.
6.2 Cadherins and attachment of osteoclasts to bone
Since osteoclasts express the vitronectin receptor, it has been speculated, that the tight
osteoclastic attachment to bone surface in the sealing zone area would be integrinmediated (Davies et al. 1989, Helfrich et al. 1992, Horton 1990). Nevertheless, VNR has
been shown to be located only in the ruffled border and basolateral membranes, and it is
missing from the area of the tight sealing zone (Lakkakorpi et al. 1991b, Suda et al.
1995). Although VNR is important in the function of osteoclasts, the final tight
attachment of osteoclasts to the bone surface is mainly not mediated through it
(Lakkakorpi et al. 1991b). The organization of the actin cytoskeleton in the sealing zone
area of osteoclasts (Lakkakorpi et al. 1989, Lakkakorpi et al. 1991a) resembles that in
epithelial adherens junctions. This led us study the role of cadherins, the main adherens
junction proteins, in osteoclastic attachment to bone.
We found that osteoclasts show a clear ring-like structure in their sealing zone area
with the pan-cadherin antibody. The pan-cadherin antibodies should react with all (or, at
least, most) cadherins, since the most homologous cytoplasmic region of the cadherin
molecules, were selected for antibody production. It has been shown that these antibodies
do react with a very broad spectrum of cadherins, such as N-, E- and P-cadherin (Geiger
et al. 1990). We chose to use this antibody because it was not precisely known which
cadherins, if any, can be found in osteoclasts and thus, this antibody proved to be
excellent probe for the identification of cadherins from osteoclasts. The pan-cadherin ring
became localized exactly around the resorption lacuna suggesting it marks the sealing
zone. In addition, the staining co-localized with actin-ring and in between vinculin double
rings, which further confirmed the cadherin localization to the sealing zone. The staining
pattern of pan-cadherin antibodies also rules out the possibility that the sealing zone area
cannot be reached by immunoglobulins, thus suggesting that the observation concerning
the lack of vitronectin receptor in the same area is not due to steric hindrance
(Lakkakorpi et al. 1991b). Mbalaviele et al. have suggested that mature murine
osteoclasts contain E-cadherin along their membranes at the resorbing bone surfaces, and
ruffled border area, with a lesser amount in the cytoplasm of these cells (Mbalaviele et al.
1995). In our studies, however, we detected no E-cadherin in mature rat osteoclasts. Thus
the exact nature and identity of cadherin-like molecules in the osteoclastic sealing zone
remains unknown.
We used the cadherin CAR sequence containing peptide to study what the possible
roles for cadherins in osteoclasts might be. Synthetic peptides containing the HAV
sequence have been shown to inhibit compaction of eight-cell-stage mouse embryos, rat
neurite outgrowth on astrocytes (Blaschuk et al. 1990a) and myoblast fusion (Mege et al.
1992), all of which are known to be mediated by cadherins. These data strongly
emphasize the importance of the HAV sequence in the cellular functions that are
dependent on cadherins. We noticed that the blocking of cadherin function by the peptide
47
does not affect the primary attachment of osteoclasts to bone matrix, as also demonstrated
earlier (Mbalaviele et al. 1995). This could be expected, since the primary attachment is
shown to be mediated by integrins (Duong et al. 1999, Lakkakorpi et al. 1993).
When using the peptide AHAVSE, osteoclasts resorbed markedly less bone, which
could be seen as a decrease in the number and total area of resorption pits when
compared with the zero and peptide controls. Although the resorbed area is greatly
decreased with the AHAVSE, we cannot quantify the depths of the resorption pits with
the method used here. Thus, the true amount of bone resorbed remains open since we
cannot know if the AHAVSE-treated osteoclasts have really resorbed less bone - as it
seems when looking at the decrease in the area of resorption pits - or if the osteoclasts
have just resorbed deeper resorption pits. Mbalaviele et al. have also shown a decrease in
the total resorbed area when using HAV-containing peptide (Mbalaviele et al. 1995). They
suggest that the decrease in the resorbed area is due to inhibition of osteoclast precursor
fusion. The same report suggests that E-cadherin is involved in the generation of
multinucleated osteoclasts from mononuclear precursors.
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, we measured the activity of
osteoclasts by their morphology. The emerging of actin rings to the sealing zone of
osteoclast is considered as a mark of osteoclastic activity (Lakkakorpi et al. 1991a). By
counting the actin rings and the number of TRAP-positive cells, we can get a percentage
of osteoclasts that are actively resorbing bone. We could see a great decrease in the
activity of osteoclasts as soon as 20 minutes after AHAVSE peptide addition. The
observed disorganization of actin rings leads to inactivation of osteoclasts. 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. Our
results thus suggest that AHAVSE has a specific effect on the sealing zone, since actin
rings are an essential component of this tight attachment-mediating structure. The HAVcontaining peptide could either prevent the formation of new actin rings and tight
attachment to the bone surface, or it could speed up the degradation of actin rings and
subsequent detachment of the tight sealing. Since the sizes of the resorption pits in
AHAVSE incubation were not changed when compared with those of the controls, the
inhibition of formation of new attachments seems to be the main reason for the decreased
percentage of active osteoclasts.
We do not know what the counterpart of osteoclastic cadherin might be. Cadherins
have generally been associated with homotypic cell-cell interactions, 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, Tang et al. 1993). These
studies together with a recent report, where it was suggested that cadherins can interact
with collagen type I (Mbalaviele et al. 1996) and could act as cell-matrix adhesion
molecules, inspired us to investigate the possibility that cadherins might act as cell-matrix
mediator in osteoclasts. Others have shown that osteoclasts express different cadherins
including E-cadherin (Mbalaviele et al. 1995) and cadherin-6 (Mbalaviele et al. 1998).
48
6.3 Polarity of osteoblasts
Histologically, actively secreting mature osteoblasts are cuboidal or tall cells. When they
start to secrete bone, the secretion is polarized towards the bone surface (Cooper et al.
1998, Marks, Jr. et al. 1996). This led us to study whether mesenchymal osteoblasts
possess polarized plasma membrane domains, as epithelial cells do. Cellular polarization
is best established in epithelium. Viral markers have been used in studying the different
plasma membrane domains. The plasma membrane of epithelial cells is divided into two
domains: an apical domain, which faces the external milieu and a basolateral domain in
contact with the internal milieu and the blood supply. These plasma membrane domains
have different lipid and protein compositions (Simons et al. 1988, van Meer et al. 1988).
In epithelial cells the polarity is maintained with tight junctions, which separate the apical
and basolateral membranes from each other.
Our studies reveal that osteoblasts are truly polarized cells. Unlike in fibroblasts,
different plasma membrane domains could be separated in osteoblasts by viruses.
Fibroblastic cells do not polarize their plasma membrane as the epithelial cells do, and
none of the fibroblastic cell lines studied, have distinct domains for VSV G protein or
Influenza HA on their plasma membranes. After delivery to the cell surface these two
protein species spread all over the surface in a non-polarized manner (Yoshimori et al.
1996). In osteoblastic UMR-108 cells, however, the antibodies against the basolateral
marker VSV G protein localized clearly to the medium-facing plasma membrane of the
cells. In addition, Vesicular Stomatitis viruses were found to bud from the upper
membrane domain of these cells towards the medium corresponding the bone marrow in
vivo. To further prove that the polarization of UMR-108 cells resembles that of endosteal
osteoblasts and is a meaningful tool, we infected osteoblasts in situ with VSV. We could
see that the VSV G protein antibodies localized to the bone marrow-facing plasma
membrane of endosteal osteoblasts, hence strengthening our earlier results from UMR108 cells.
As osteoblasts mature, the secretion becomes non-polarized and some of the
osteoblasts become trapped into the matrix as osteocytes. Osteocytes seem not to secrete
matrix components anymore or at least the secretion is probably not polarized. We
wanted to see whether the localization of VSV G protein is changed in osteoblasts during
this maturation. The VSV G protein seemed to localize in a non-polarized manner at the
plasma membrane of osteocytes, apart from the Golgi complex, which could clearly be
distinguished. Interestingly, 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.
On the contrary, Influenza glycoproteins localized in UMR-108 cells to the plasma
membrane facing the bone slice. Also, the budding of Influenza viral particles took place
in between the cell and the bone slice. These results were thus in agreement with each
other indicating that the bone surface-facing membrane domain of osteoblasts is apical in
nature. In the biotinylation studies we also confirmed this conclusion showing that
whereas most of the VSV G protein was at the medium-facing surface, where it could be
biotinylated, the Influenza HA was hidden in the bone facing plasma membrane, which
was not available for biotin binding.
49
Taking these results together - the immunofluorescence data, observation of the viral
particle budding and the biotinylation results – we can safely conclude that osteoblastic
cells clearly posses distinct polarized membrane domains. Still, when comparing the
degree of polarity of osteoblastic cells to that of epithelial cells we can see a certain
difference. In epithelial cells the polarization is absolute. No VSV-particles can be seen to
bud anywhere but the basolateral membrane domain, whereas Influenza particles can only
be seen in the apical membrane domain. Although a clear tendency of polarization is
easily observed in osteoblastic cells, some viral particles bud randomly from opposite
membrane surfaces, in a non-polarized manner. 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. It seems that when osteoblasts grow in a monolayer, the polarization is best
achieved whereas some of it is lost when the cells start growing on top of each other. Very
little is known about the polarity of other than epithelial cells. Mesenchymal osteoclasts
have been shown to polarize its membrane into domains (Salo et al. 1996, Salo et al.
1997). Most of the osteoclast membrane has receptors for VSV indicating basolateral
membrane domain, but the special cap structure on the top of an active osteoclast has
receptors for Influenza virus, being thus apical in nature. The mechanism by which these
non-epithelial cells organize their membrane domains and maintain the polarity is not
known, since they do not have tight junctions that usually mediate the polarity in
epithelial cells.
6.4 Gap junctions in osteoclasts
To establish and maintain the balance between bone formation and bone resorption, bone
cells must have a way to communicate with each other. Most cells communicate with
their immediate neighbors through exchange of cytosolic molecules, such as ions, second
messengers, and small metabolites. This activity is made possible by clusters of
intercellular channels called gap junctions, which connect adjacent cells (Kumar et al.
1996). Others have shown that of the bone cells osteoblasts and osteocytes express gap
junctional connexins (Donahue et al. 1995b, Jones et al. 1993, Yellowley et al. 2000).
Some reports of connexins in osteoclasts have also been published earlier (Jones et al.
1993, Jones et al. 1994, Su et al. 1997), but the functional significance has not been
demonstrated earlier. It has been speculated that osteoblasts, osteocytes and osteoclasts
may adjust the number of communicating gap junctions throughout the bone cell
network. This way the cells could regulate the transmission of molecular and electrical
information to support the balance of bone remodeling (Su et al. 1997).
We clearly demonstrate that osteoclasts contain Cx43. The immunofluorescence data
leaves no doubt of the Cx43 expression in osteoclasts. 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. Cx43 staining could be seen when osteoclasts
were in cell-cell contacts with bone marrow derived mononuclear cells. Numerous
punctuate connexin stainings could be seen in the basolateral plasma membrane of
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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