1
Introduction
Chapter 1
10
General introduction
GENERAL INTRODUCTION
Bone and bone cells
Bones are essential for protection of the internal organs such as lungs and brains,
as an attachment site for muscles and tendons and as a reservoir for calcium,
magnesium and phosphorus[1-3]. Moreover, bones harbor hematopoietic and
mesenchymal stem cells within their marrows from which erythrocytes, leukocytes,
osteoclasts, osteoblasts and fat cells are derived [4, 5].
Bone is composed of an organic matrix and minerals. The matrix consists mainly
of fibrillar collagen type I (95%). Next to collagen, bone contains a wide variety of
non-collagenous proteins such as osteopontin and bone sialoprotein. The mineral
is composed of calcium phosphate, which is arranged in a hexagonal crystal
structure and is called hydroxyl apatite. The matrix, in particular the collagen fibrils,
makes the bone a bit flexible, whereas the mineral serves to makes the bone rigid
[1-3].
In the mammalian skeleton two types of bone formation can be identified: bones
formed by endochondral (e.g. the femur and the tibia) and those formed by
intramembranous ossification (e.g. the calvaria). Endochondral ossification is
characterized by a hyaline cartilage template of the required bone. Subsequently
this cartilage is gradually replaced by bone. This process is essential for the growth
of an organism; bones elongate and thicken during childhood. Intramembranous
bones are formed by deposition of bone matrix in a dense connective tissue. In this
type of bone not cartilage but connective tissue is used as an anlage for bone
formation by osteoblasts [1-3].
Besides the different modalities by which the bone types are formed, they also
differ in their matrix composition [6]. Some of the non-collagenous proteins (e.g.
osteopontin and vitronectin) are present in higher amounts in long bones whereas
others are more abundant in calvaria (e.g. osteoglycin, α2-HS-glycoprotein). Also
structural differences exist. For instance the cross-linking of collagen is much
higher in long bones compared to calvaria [6]. In the following paragraphs I will
summarize what is known about the process of bone turn-over, with special
emphasis on the cells involved in bone degradation.
11
Chapter 1
Bone cells
Bone tissue is characterized by a continuous remodeling due to the activity of four
different bone-associated cell types: osteoblasts, osteocytes, bone lining cells, and
osteoclasts [1-3]. Osteoblasts are cuboidal shaped cells. They are responsible for
the synthesis and deposition of the extracellular matrix of bone (e.g. collagen and
non-collagenous proteins). Initially a non-calcified layer is deposited which is
known as the osteoid layer [7]. This is followed by mineral deposition in this layer,
also due to the activity of osteoblasts [8]. During the formation of bone matrix some
of the osteoblasts become entrapped by the newly formed matrix and these cells
continue their life as osteocytes. The cuboidal appearance of the osteoblast
changes to star-shaped with long slender cytoplasmic protrusions that protrude into
the bone matrix. With these cytoplasmic processes, the osteocytes are in contact
with each other and with the cells that cover the bone surface.
The osteocyte is the most abundant cell type found in bones: >90% of the bone
cells are osteocytes. These cells are mechano-sensitive; they have the capacity to
respond to changes in loading exerted upon the bone tissue. In response to
loading or its absence osteocytes send signals to the cells lining the bone surface,
such as osteoblasts and bone lining cells, to inhibit or induce bone formation and
resorption [9]. The osteocytes are considered to be the directors of bone
remodeling. In addition to this role, recent studies indicate that osteocytes may play
a direct role in osteoclast formation by expressing osteoclast differentiation factor
RANKL [10-12].
Next to the cuboidal osteoblasts and the osteocytes, a third cell type of the
osteoblast lineage has been described. These are flattened osteoblast-like cells,
the so-called bone lining cells. They express ICAM-1 on their plasma membrane
and play a role in attraction and maturation of osteoclast precursors [13, 14].
Osteoclast precursors attach to the bone lining cells and mature into tartrate
resistant acid phosphatase (TRAcP) positive pre-osteoclasts. Subsequently the
bone lining cells move aside to make space on the bone surface for these preosteoclasts [15]. The pre-osteoclasts migrate to the bone surface, fuse to form
multinucleated osteoclasts that start resorbing bone. When bone has been
resorbed bone lining cells enter the resorption pit and clean its surface from
collagen remnants left behind by the osteoclast [14]. Recent studies have stressed
the important role of these cells, also called reversal cells, in the remodeling cycle
12
General introduction
of bone [16]. The latter authors found that these cells play an important role in
coupling of bone formation and bone degradation [17].
The fourth cell type involved in bone remodeling is the osteoclast. Osteoclasts
are unique in their capacity to resorb the mineralized bone. They belong to the few
cell types in the body that are multinucleated. Osteoclasts originate from
mononuclear hematopoietic bone marrow cells and share this stem cell with
monocytes and macrophages [5, 18].
Formation and activity of osteoclasts
Multinucleated osteoclasts are formed by fusion of mononuclear hematopoietic
cells [19, 20]. Based on experimental evidence and on theoretical considerations,
the following sequence of events is considered to take place in vivo. The first step
in osteoclast formation is the recruitment and selection of osteoclast precursors out
of a pool of hematopoietic cells. These precursors are present in bone marrow and
blood. Chemokines like selectins and cell-cell adhesion molecules such as
integrins VLA-4 and LFA-1 and cadherins are important in this process [21-23].
After the osteoclast precursors have arrived at the bone surface, they attach to the
flattened bone lining cells. This contact stimulates the production of macrophage
colony stimulating factor (M-CSF), a cytokine secreted by the osteoblast-like bone
lining cells. M-CSF binds to its receptor c-Fms on osteoclast precursors, and this
signaling stimulates survival and proliferation of these precursors. Receptor
activator of nuclear factor kappa-B ligand (RANKL) expressed on the membrane of
these osteoblast-like cells
stimulates RANK expressed by the osteoclast
precursors [24-26]. RANK-RANKL signaling is the first essential step for activation
of an intracellular pathway mediating fusion of osteoclast precursors [27].
Osteoprotegerin (OPG), a soluble decoy receptor produced by the osteoblast-like
cells, competes with RANK for RANKL and therefore blocks osteoclastogenesis
[28, 29]. Under the influence of attached osteoclast precursors, the bone lining
cells retract from the bone surface and pre-osteoclasts migrate to the bone
surface. [15, 30]. Subsequently the differentiated osteoclast precursors fuse and
become multinucleated cells [31]. How this fusion occurs in terms of timing of the
fusion event and whether multinucleated cells can still fuse with other
multinucleated cells is not known. The phenomena related to the fusion of the cells
were studied by use of life cell imaging and described in Chapter 2 of the thesis.
13
Chapter 1
How do osteoclasts resorb bone?
The osteoclast resorbs bone by the release of acid which dissolves the mineral
crystallites, and by the release of a series of proteolytic enzymes that digest the
matrix components. First, osteoclasts attach to the bone surface; a process
mediated by the integrin αvβ3. This attachment induces actin rearrangement in
such a way that a ring-like actin filament dense area is formed [32, 33]. Due to the
lack of organelles in this area, it is called the clear zone. This zone encircles a
specialized part of the plasma membrane, the ruffled border. The ruffled border
membrane is lysosome-like due to the fusion of lysosomes and endosomes with
this membrane. These organelles release their content in the extracellular
environment, in the area where the resorption occurs [19, 34-37]. The ruffled
border membrane also harbors a proton pump v-ATPase and a chloride channel,
ClC7. Protons and chloride are released in the resorption area resulting in a very
low pH of 4-5. This acidic environment dissolves the mineral (hydroxyl apatite) of
the bone [38-42]. After demineralization, the milieu in this area is optimal for the
proteolytic dissolution of the organic matrix degradation by the cysteine proteinase
cathepsin K [43-45].
The protons released in the resorption area by the proton pump are generated by
a reaction catalyzed by carbonic anhydrase II [46] [47-49], an enzyme present in
the cytoplasm of the osteoclast [39, 40, 42, 50]. This isoform of carbonic anhydrase
is rather specific for osteoclasts and it catalyzes the reaction of carbon dioxide into
bicarbonate and protons. The release of protons into the ruffled border area by vATPase results in a rise of the intracellular pH of the osteoclast. To compensate for
this rise in pH the osteoclast is equipped with one or more anion exchangers in the
basolateral membrane [51]. The exact role and origin of anion exchangers involved
in this process is still not known. In an attempt to shed some light on this, we
investigated in Chapter 3 whether a relatively widely expressed exchanger, anion
exchanger 2 (Ae2), plays a role in osteoclastic bone degradation [52, 53].
One of the hydrolytic enzymes present in lysosomal vacuoles is tartrate resistant
acid phosphatase (TRAcP). TRAcP is highly expressed by osteoclasts and
localizes in intracellular vesicles and vacuoles next to the ruffled border [54]. This
enzyme is also secreted and found in the resorption area outside the cell. TRAcP
is thought to play a role in dephosphorylation of non-collagenous proteins, e.g.
osteopontin, and in removal of the mannose-6-phosphate recognition marker from
lysosomal proteins [55-59]. This activity in combination with the activity of
14
General introduction
proteolytic enzymes such as cathepsin K (see above) results in a complete
digestion of the matrix constituents [43-45]. Subpopulations of osteoclasts also use
MMPs for the latter process. This is described in more detail in the section entitled
“osteoclast heterogeneity”. The inorganic and organic degradation products are
subsequently endocytosed and transcytosed in vesicles to the basolateral
membrane [54, 60] (see Figure 1). At the basolateral membrane a functional
secretory domain (FSD) is formed after matrix degradation has started [61]. Via this
secretory membrane osteoclasts remove remnants of the degraded bone matrix
and release these into the extracellular space [54, 60-62].
Figure 1. Schematic representation of an osteoclast with special emphasis on the acid
secretion. The osteoclast binds to bone matrix via the integrin αvβ3. This binding induces
actin rearrangement by which an actin ring is formed. This ring isolates the resorption area
+
from the rest of the bone surface. Protons (H ) and chloride (Cl ) are released in the
resorption area by a proton pump (v-ATPase) and chloride channel (ClC7), hereby
dissolving the bone mineral. The protons are generated by a reaction catalyzed by carbonic
anhydrase. To keep the internal pH at physiological level bicarbonate (HCO3 ) is removed
from the cell by an anion exchanger. The bicarbonate is exchanged for Cl which will be
released in the ruffled border area via the chloride channel ClC7. Degradation products are
removed in secretory vesicles through the cell and finally exocytosed through the functional
secretory domain (FSD).
15
Chapter 1
Role and function of the lysosomal protein LAMP-2 in osteoclasts
Lysosomes are not only essential for the formation of the ruffled border but high
numbers are also present in the cytoplasm of these cells [34, 36, 63, 64]. One of
the main functions of these organelles is the degradation of intracellular and
extracellular material [65]. They contain a wide variety of catabolic enzymes that
are
active at a low pH. To protect the cell from degradation, lysosomes are
surrounded by a membrane with unique properties. This membrane does not only
have the capacity to keep the catabolic enzymes enclosed within the organelle, but
it also regulates the passage of material across the membrane and establishes the
pH difference between lysosomal lumen and cytoplasm [65, 66]. Specific
lysosomal membrane proteins, play an important role in these functions. Lysosome
associated membrane protein-2 (LAMP-2) is the most abundant protein present in
the lysosomal membrane [67].
Deletion of this protein in mice showed massive accumulation of autophagic
vacuoles in liver, heart and muscles [66, 68], and its deficiency in humans results
in accumulation of autophagic vacuoles in heart, skeletal muscle and brain, which
can lead to cardiomyopathy, myopathy and mental retardation, also known as
Danon-disease [66, 68]. LAMP-2 deficient mice show less efficient killing of
pathogens leading to periodontitis [69]. The reason that pathogens survive is due
to an inefficient fusion between lysosomes and phagosomes in polymorphonuclear
leukocytes [70]. When bacterial clearance is impaired, inflammation is probably
maintained and hence bone degradation may occur due to continuously activated
osteoclasts [69, 71]. It is known that LAMP-2 is highly expressed in the ruffled
border; however a role for LAMP-2 in osteoclast formation and/or activity has not
been described. In Chapter 4 we addressed the question whether LAMP-2 is
involved these processes by making use of mice deficient for LAMP-2.
Osteoclast heterogeneity
Besides differences in the formation and the composition of the different bone
types also the cells associated with this tissue show remarkable bone-site specific
differences [72-75]. For instance, osteoclasts in long bones were shown to use
primarily cysteine proteinases, especially cathepsin K, to degrade the bone matrix
whereas calvarial osteoclasts, use also matrix metalloproteinases (MMPs) in
addition to cathepsin K, for this process (Figure 2). This does not only happen in
calvaria [73;75], but also in intramembraneously formed scapula [76].
16
General introduction
Another remarkable difference between the osteoclasts at different bone sites is a
higher expression level (mRNA and protein) of TRAcP by calvarial osteoclasts [77].
TRAcP is synthesized as a pro-enzyme with low activity and is activated by
cysteine proteinases such as cathepsin K, L, S [78]. In contrast to the findings of
Perez et al.[76], Zenger et al.[78] found that not calvarial but long-bone osteoclasts
express a higher TRAcP activity. This discrepancy might be explained by the
method used to isolate the enzyme. \moreover, Zenger et al. indicated that
calvarial and long-bone osteoclasts expressed different TRAcP isoforms [78]. The
different isoforms may fulfill different functions in the process of resorption; after all
the composition of the bone matrix of long-bones and calvaria is quite different [6].
Another difference between osteoclasts at different bone sites is their size.
Calvarial osteoclasts were found to be larger and contained more nuclei compared
to the ones present in long bones. Since the size of an osteoclast seems to
correlate positively with the resorption activity, this may suggest that calvarial
osteoclasts express a higher resorptive activity [79].
Figure 2. Differences between osteoclasts present in
skull and long bone. Skull osteoclasts use MMPs and
cysteine proteinases for resorption whereas long bone
osteoclasts primarily use cysteine proteinases.
Expression of cysteine proteinases is much higher in
long bone osteoclasts compared to calvarial osteoclasts
whereas TRAcP expression is higher in calvarial
osteoclasts. Calvarial osteoclasts are larger with more
nuclei compared to long bone osteoclasts. Adapted from
Everts et al. [80]
Although these findings
indicate bone-site related differences between
osteoclasts, hardly anything is known about the response of these different cells to
compounds like hormones, cytokines and growth factors. Recently it was shown
that marrow cells derived from different bones respond differently to PTH in
combination with vitamin D. Marrow cells were isolated from long bones and from
jaws (mandible) and the effect of PTH and vitamin D on osteoclastogenesis was
investigated. Marrow cultures from long bones proved to generate higher numbers
17
Chapter 1
of osteoclasts than those from mandibles, thus indicating differences in the
response to PTH and vitamin D [81]. Yet, thus far this is the only study in which the
question is addressed whether different precursor populations respond differently
to agents known to affect osteoclastogenesis. In the present thesis we explored
this issue further and investigated effects of the cytokine IL-1β (Chapter 5) and the
osteoclast inhibiting drugs, the bisphosphonates (Chapter 6).
IL-1β
IL-1β is a pro-inflammatory cytokine involved in the immune defense against
infection. IL-1β also stimulates osteoclast formation, differentiation and bone
resorption and inhibits bone formation [82-84]. Under the influence of IL-1β the
expression of M-CSF and RANKL is upregulated by cells at the bone surface, thus
activating osteoclastogenesis [85-87]. Binding of IL-1β to its receptor (IL-1R1) can
directly activate osteoclastogenesis by enhancing transcription factors such as
NFATc1 and osteoclast specific genes like TRAcP and cathepsin K. There is,
however, also another receptor, IL-1R2. This receptor antagonizes IL-1R1 and
causes a transcription blockade [88, 88, 89].
Thus far all studies on the effect of IL-1β on osteoclastogenesis and bone
resorption, made use of precursors in bone marrow obtained from long bones or
those from peripheral blood. Whether precursors obtained from other bone sites
respond similarly to this cytokine, is unknown. Therefore, we investigated in
Chapter 5 whether osteoclasts and their precursors from different bone sites
respond differently to IL-1β in vitro.
Bisphosphonates
Bisphosphonates are used to inhibit bone degradation that occurs e.g. during
osteoporosis. Osteoporosis is characterized by a reduced bone mass and a lower
mineral density. The two most common causes for osteoporosis are the use of
medications such as glucocorticosteroids and the lack of estrogen due to
menopause. Osteoporosis induced by medication, may thus arise at any age and
is likely to affect men and women equally. Glucocorticosteroids inhibit osteoblast
activity, which results in a suppressed bone remodeling. This means that bone
formation as well as bone degradation is suppressed, eventually resulting in bones
that are more brittle [90, 91]. Others stated that glucocorticosteroids act directly on
18
General introduction
osteoclasts by increasing their life span and therefore reducing the bone density
[92].
Yet, the most common form of osteoporosis is age-related postmenopausal
osteoporosis. This occurs in 5-20% of postmenopausal women and rapidly
increases over age. This incidence rate is eight times higher than in men. Estrogen
deficiency is thought to be the cause of this type of osteoporosis. Estrogen
deficiency causes a higher activity of bone formation and bone degradation, but the
relative activity of osteoclasts is higher than that of osteoblasts, resulting in a lower
bone mass [91, 93]. Bisphosphonates block bone resorption by inducing apoptosis
of osteoclasts [94]. Two types of bisphosphonates are used: the nitrogen- and the
non-nitrogen-containing
bisphosphonates.
The
nitrogen-containing
bisphosphonates inhibit the transport of GTPases to the membranes, thereby
disturbing the formation of actin filaments, which are essential for osteoclast
activity [95, 96]. The non-nitrogen containing bisphosphonates cause apoptosis by
the formation of a cytotoxic ATP analog [97].
Patients treated with high dose of bisphosphonates, such as cancer patients with
bone metastases, develop in 0.1-10% of the cases osteonecrosis of the jaw [98].
Why this particularly occurs in the jaw is not clear. A possible explanation for this
site-specific effect of the drug could be the heterogeneity of the osteoclasts and
their local precursors. In Chapter 6 we investigated the effect of bisphosphonates
on osteoclastogenesis by bone marrow cells obtained from jaw and long bone.
Outline of this thesis
The common denominator of the thesis is osteoclast heterogeneity. Basically, three
approaches were used to shed light on this topic. First, life cell imaging was used
to explore how osteoclasts were formed (Chapter 2). Next, making use of Ae2 and
LAMP-2 knock-out mice, we addressed the question whether osteoclast precursors
and osteoclasts from different bone sites were differently affected by the deletion of
these proteins (Chapters 3 and 4). Finally, it was hypothesized that osteoclasts
and their precursors may respond differently to the inflammatory cytokine IL-1β
(Chapter 5) and to bisphosphonates (Chapter 6). In the final chapter (Chapter 7)
the findings of the thesis will be discussed with particular emphasis on the role of
different osteoclasts at different skeletal sites in physiology and pathology.
19
Chapter 1
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