*Interactions of normal breast tissue with bone*

The interactions of normal breast tissue with bone arise during childbearing and breastfeeding. A normal human fetus needs approximately 30 g of calcium to mineralize its skeleton during gestation (1), that leads to significant changes in calcium homeostasis during pregnancy, including adjustments in levels of parathyroid hormone (PTH), calcitonin and 1,25 dihydroxyvitamin D [1,25[OH]D] (2). These hormones exhibit their effects through three main target tissues – the intestines, kidneys and bone (3). Parathyroid hormone related peptide (PTHrP) is a hormone closely related to PTH, but which is produced by local tissues, such as breast, and is important for its differentiation (4). In addition to its role in local tissue development, PTHrP can substitute for PTH in the tissues expressing their common receptor, and thus participate in calcium homeostasis by elevating 1,25(OH)D and suppressing PTH, regulating placental calcium transport, and affecting bone resorption in the maternal skeleton (3). The regulation of calcium homeostasis by the lactating mammary gland may be of critical importance, since nursing humans secrete 300-400 mg of calcium into milk each day (5). The hormonal balance changes again during lactation, with still-reduced PTH levels, but normalized calcitonin and 1,25(OH)D, and increased PTHrP (2). During this time, increased prolactin concentrations allow for the release of breast milk, and also act to enhance bone turnover (6,7). Suckling stimulates prolactin secretion and inhibits GnRH production, both of which reduce estradiol levels, leading to bone resorption (8). Bone resorption has been shown to increase during lactation, and bone formation to decrease, resulting in a loss of 5-10% of trabecular mineral


The interactions of normal breast tissue with bone arise during childbearing and breastfeeding. A normal human fetus needs approximately 30 g of calcium to mineralize its skeleton during gestation (1), that leads to significant changes in calcium homeostasis during pregnancy, including adjustments in levels of parathyroid hormone (PTH), calcitonin and 1,25 dihydroxyvitamin D [1,25[OH]D] (2). These hormones exhibit their effects through three main target tissues – the intestines, kidneys and bone (3). Parathyroid hormone related peptide (PTHrP) is a hormone closely related to PTH, but which is produced by local tissues, such as breast, and is important for its differentiation (4). In addition to its role in local tissue development, PTHrP can substitute for PTH in the tissues expressing their common receptor, and thus participate in calcium homeostasis by elevating 1,25(OH)D and suppressing PTH, regulating placental calcium transport, and affecting bone resorption in the maternal skeleton (3). The regulation of calcium homeostasis by the lactating mammary gland may be of critical importance, since nursing humans secrete 300-400 mg of calcium into milk each day (5). The hormonal balance changes again during lactation, with still-reduced PTH levels, but normalized calcitonin and 1,25(OH)D, and increased PTHrP (2). During this time, increased prolactin concentrations allow for the release of breast milk, and also act to enhance bone turnover (6,7). Suckling stimulates prolactin secretion and inhibits GnRH production, both of which reduce estradiol levels, leading to bone resorption (8). Bone resorption has been shown to increase during lactation, and bone formation to decrease, resulting in a loss of 5-10% of trabecular mineral




Attachment proteins between breast cancer cells and the bone

Establishing of a metastatic tumor in the bone microenvironment





physiology, pathology


 Homing of breast cancer cells to bone Creation of pre-metastatic niche Migration of breast cancer cells to bone

Interactions with osteoblasts

Interactions with osteoclasts

**3.1 Breast growth and development**  *Interactions of normal breast tissue with bone* 

Therapeutic targets in the bone microenvironment

**3. Physiological regulation of breast and bone** 

Osteomimicry

 Conclusions References

content per month (9). Lactation-induced fragility fractures have been reported as a result, but are not common (10). Of interest, other important molecular mediators for the developing of lactating mammary gland are receptor activator of nuclear factorB (RANK) and its ligand RANKL, which are better known for their key role in regulating the formation of osteoclasts. Expression of RANKL in the mammary epithelium is induced by hormones increased during pregnancy, such as prolactin, progesterone, and PTHrP, and mice lacking RANKL or RANK cannot form lobuloalveolar mammary gland structures, resulting in complete inability to develop a lactating mammary gland (11). Thus, normal breast tissue can interact with bone through a system of hormonal regulators that are important during lactation, and it expresses molecular machinery that employs the same mediators to perform locally distinct functions (Figure 1).

Fig. 1. Physiological interactions between the functions of breast and bone. Lactation involves secretion of large amounts of calcium. Bone is a key participant in calcium homeostasis. PTH is reduced during lactation while PTHrP production by the breast tissue is increased. Suckling stimulates prolactin secretion and inhibits GnRH production, both of which reduce estradiol levels, leading to bone resorption. Prolactin and PTHrP induce breast expression of RANKL, necessary for normal lactating mammary gland function. In the bone tissue, osteoblast-produced RANKL is key regulator of osteoclastogenesis.

#### *Breast carcinoma*

Breast carcinomas may arise from the inner lining of the milk ducts or from the lobules, known, respectively, as ductal carcinomas or lobular carcinomas (12). Once a tumour exceeds 1-2 mm in diameter, it requires extensive vascularization in order to survive (13), but the speed of cancer growth often exceeds its capability to form normal vascular organization. Poor angiogenesis results in an under-vascularized microenvironment, which leads to hypoxia, acidic pH and nutrient depletion in the tumour (14). Some cancer cells may

Breast Cancer Metastases to Bone: Role of the Microenvironment 535

muscles, ligaments and tendons to allow movement in three-dimensional space. Hearing is also attributed to the mechanics of bones, with several of the body's smallest bones involved in the transmission of sound in the ear. Bone is the body's major reservoir of calcium, storing approximately 99% of it in the bone's mineral phase. Plasma calcium levels are strictly regulated in the range of 2.2-2.6 mmol/L total calcium. Such regulation is achieved by regulating calcium exchange with the environment through the kidney and intestine, and, in the absence or insufficiency of environmental sources, by regulating calcium exchange between plasma and bone through osteoblastic bone formation and osteoclastic bone destruction (32). The coordination of calcium fluxes is achived through complex hormonal regulation. Parathyroid hormone and 1,25 dihydroxy-vitamin D act to increase calcium by increasing calcium reabsorption from the kidneys and small intestine, respectively, and both act by enhancing the mobilization of calcium from bone through resorption (33). Calcitonin acts to reduce blood calcium by suppressing renal calcium reabsorption and inhibiting the mobilization from bone by preventing bone resorption (34). The combined work of these systems ensures that hypo- or hyper-calcemia is corrected, and

Bone tissue also interacts with other functionally diverse systems in the body. The endosteal surface of the medullary cavity of bones houses the haematopoietic stem cell niche, the specific location where blood stem cells best differentiate. Osteoblasts are well known to support the haematopoietic stem cell niche directly (35), and haematopoietic cells in turn regulate osteogenesis (36). Adipocyte-derived leptin regulates both appetite and bone mass accrual (37), and osteoblast-derived osteocalcin affects insulin secretion and sensitivity, as well as energy expenditure (38,39). It has most recently been shown that the skeleton regulates male fertility through osteocalcin (40), extending the breadth of bone's influence

The three cell types critical to bone's structure and function are the bone-resorbing osteoclast, the bone forming osteoblast, and the mechanosensory osteocyte. These cells work in concert to build bones, maintain mechanically sound bone tissue by replacing it on

*Osteoclasts:* The destruction of bone, both physiological in the case of morphogenesis and replacing old or damaged bone, and pathological in the case of osteolytic diseases such as osteoporosis, breast cancer metastasis to bone and rheumatoid arthritis, occurs through the activity of the osteoclast. Osteoclasts are cells of hematopoietic origin. The key molecular mediators of osteoclast formation from monocytic precursors are macrophage colonystimulating factor (M-SCF) acting through its receptor c-fms, and RANKL which binds to its receptor RANK (41-43). Osteoprotegerin (OPG) is the high affinity decoy receptor for RANKL and is able to prevent osteoclast differentiation by inhibiting RANK-RANKL interactions (44). RANKL binding to RANK in the presence of M-CSF induces the recruitment of adaptor molecules including TRAF6 by RANK (45), resulting in the activation of transcription factor NFB. One of the early targets of NFB is another transcription factor essential for osteoclastogenesis, nuclear factor of activated T-cells c1 (NFATc1), which later undergoes auto-amplification with the assistance of an activator protein-1 complex containing c-Fos (46-48). NFATc1 nuclear localization is regulated by

average every 10 years, and repair bones in the incidence of trauma.

ingested calcium is stored or eliminated as waste.

into reproduction as well.

*Bone cells* 

develop the ability to detach from the primary tumour and invade other areas to form secondary tumours, in a process called metastasis. Breast cancer cells favour regional lymph nodes as well as the liver, lungs, brain and bone as sites of metastasis (15). The metastatic process occurs in a complex series of interrelated steps. An epithelial-to-mesenchymaltransition (EMT) may occur whereby epithelial breast cancer cells take on a mesenchymal phenotype of reduced attachment to neighbouring cells and increased migratory capabilities (16). This may assist in their intravasation process, where the cell breaks through the epithelium into a blood vessel (17). From here, the cell migrates to a distant site, which is driven by chemotaxis and the communication between the cancer cell and a secondary site where it aims to establish (18-20). Instead of combating cancer cells, tumor-associated macrophages and T-cells may assist in the survival and dissemination of cancer cells by mitigating the immune response and promoting cancer progression (21,22). When the cell has reached its destination, it will then undergo extravasation to exit the blood vessel and establish in a new tissue (23). Bone is a preferred site for breast cancer metastases, therefore specific interactions are likely to establish between breast cancer cells and bone cells.

#### **3.2 Bone microenvironment**

Bone is a dynamic tissue that provides support and protection for organs and maintains body mineral homeostasis. All 213 bones are constantly remodelled by the coordinated action of specialized bone cells—osteoclasts that destroy bone and osteoblasts that build bone. Bone remodelling contributes to the many functions that bones provide and occurs at different rates in different areas. Higher rates of bone turnover are observed in trabecular bone compared to cortical bone (24), and at bone sites adjacent to actively hematopoietic bone marrow in the axial skeleton, where bone metastases also commonly occur (25). High bone turnover has been found to correlate to poor prognosis in patients with bone metastases (26), and prostate cancer cells have been shown to preferentially metastasize to sites of active bone turnover (27), making bone homeostasis an essential part of understanding cancer progression.

#### *Structure*

The adult skeleton is composed of 80% solid and dense cortical bone, surrounding the remaining 20% trabecular bone, a network of plates and rods through the bone marrow (28). Bone is composed of an organic phase of extracellular matrix containing collagen-1 triplehelical chains and non-collagenous proteins, and mineral phase of hydroxyapatite crystals [Ca10(PO4)6(OH)2]. Osteogenesis occurs by two distinct mechanisms – endochondral ossification, and intramembranous bone formation. Endochondral ossification occurs in most bones of mesodermal origin that form the axial skeleton, including long bones, skull, ribs and vertebrae, and involves the formation of initial mineralized cartilage template, which is first degraded by osteoclasts and then replaced with bone matrix by osteoblasts (29,30). Intramembranous ossification occurs in the flat bones and the mandible, maxilla and clavicle, where an ossification centre is created when mesenchymal stem cells condense, and directly differentiate into bone-forming osteoblasts (31).

#### *Functions*

The mechanical functions of bone are probably their best recognized. Bones protect internal organs from damage and support the structure of the body. Bones provide anchorage for muscles, ligaments and tendons to allow movement in three-dimensional space. Hearing is also attributed to the mechanics of bones, with several of the body's smallest bones involved in the transmission of sound in the ear. Bone is the body's major reservoir of calcium, storing approximately 99% of it in the bone's mineral phase. Plasma calcium levels are strictly regulated in the range of 2.2-2.6 mmol/L total calcium. Such regulation is achieved by regulating calcium exchange with the environment through the kidney and intestine, and, in the absence or insufficiency of environmental sources, by regulating calcium exchange between plasma and bone through osteoblastic bone formation and osteoclastic bone destruction (32). The coordination of calcium fluxes is achived through complex hormonal regulation. Parathyroid hormone and 1,25 dihydroxy-vitamin D act to increase calcium by increasing calcium reabsorption from the kidneys and small intestine, respectively, and both act by enhancing the mobilization of calcium from bone through resorption (33). Calcitonin acts to reduce blood calcium by suppressing renal calcium reabsorption and inhibiting the mobilization from bone by preventing bone resorption (34). The combined work of these systems ensures that hypo- or hyper-calcemia is corrected, and ingested calcium is stored or eliminated as waste.

Bone tissue also interacts with other functionally diverse systems in the body. The endosteal surface of the medullary cavity of bones houses the haematopoietic stem cell niche, the specific location where blood stem cells best differentiate. Osteoblasts are well known to support the haematopoietic stem cell niche directly (35), and haematopoietic cells in turn regulate osteogenesis (36). Adipocyte-derived leptin regulates both appetite and bone mass accrual (37), and osteoblast-derived osteocalcin affects insulin secretion and sensitivity, as well as energy expenditure (38,39). It has most recently been shown that the skeleton regulates male fertility through osteocalcin (40), extending the breadth of bone's influence into reproduction as well.

#### *Bone cells*

534 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

develop the ability to detach from the primary tumour and invade other areas to form secondary tumours, in a process called metastasis. Breast cancer cells favour regional lymph nodes as well as the liver, lungs, brain and bone as sites of metastasis (15). The metastatic process occurs in a complex series of interrelated steps. An epithelial-to-mesenchymaltransition (EMT) may occur whereby epithelial breast cancer cells take on a mesenchymal phenotype of reduced attachment to neighbouring cells and increased migratory capabilities (16). This may assist in their intravasation process, where the cell breaks through the epithelium into a blood vessel (17). From here, the cell migrates to a distant site, which is driven by chemotaxis and the communication between the cancer cell and a secondary site where it aims to establish (18-20). Instead of combating cancer cells, tumor-associated macrophages and T-cells may assist in the survival and dissemination of cancer cells by mitigating the immune response and promoting cancer progression (21,22). When the cell has reached its destination, it will then undergo extravasation to exit the blood vessel and establish in a new tissue (23). Bone is a preferred site for breast cancer metastases, therefore

specific interactions are likely to establish between breast cancer cells and bone cells.

Bone is a dynamic tissue that provides support and protection for organs and maintains body mineral homeostasis. All 213 bones are constantly remodelled by the coordinated action of specialized bone cells—osteoclasts that destroy bone and osteoblasts that build bone. Bone remodelling contributes to the many functions that bones provide and occurs at different rates in different areas. Higher rates of bone turnover are observed in trabecular bone compared to cortical bone (24), and at bone sites adjacent to actively hematopoietic bone marrow in the axial skeleton, where bone metastases also commonly occur (25). High bone turnover has been found to correlate to poor prognosis in patients with bone metastases (26), and prostate cancer cells have been shown to preferentially metastasize to sites of active bone turnover (27), making bone homeostasis an essential part of

The adult skeleton is composed of 80% solid and dense cortical bone, surrounding the remaining 20% trabecular bone, a network of plates and rods through the bone marrow (28). Bone is composed of an organic phase of extracellular matrix containing collagen-1 triplehelical chains and non-collagenous proteins, and mineral phase of hydroxyapatite crystals [Ca10(PO4)6(OH)2]. Osteogenesis occurs by two distinct mechanisms – endochondral ossification, and intramembranous bone formation. Endochondral ossification occurs in most bones of mesodermal origin that form the axial skeleton, including long bones, skull, ribs and vertebrae, and involves the formation of initial mineralized cartilage template, which is first degraded by osteoclasts and then replaced with bone matrix by osteoblasts (29,30). Intramembranous ossification occurs in the flat bones and the mandible, maxilla and clavicle, where an ossification centre is created when mesenchymal stem cells condense, and

The mechanical functions of bone are probably their best recognized. Bones protect internal organs from damage and support the structure of the body. Bones provide anchorage for

**3.2 Bone microenvironment** 

understanding cancer progression.

directly differentiate into bone-forming osteoblasts (31).

*Structure* 

*Functions* 

The three cell types critical to bone's structure and function are the bone-resorbing osteoclast, the bone forming osteoblast, and the mechanosensory osteocyte. These cells work in concert to build bones, maintain mechanically sound bone tissue by replacing it on average every 10 years, and repair bones in the incidence of trauma.

*Osteoclasts:* The destruction of bone, both physiological in the case of morphogenesis and replacing old or damaged bone, and pathological in the case of osteolytic diseases such as osteoporosis, breast cancer metastasis to bone and rheumatoid arthritis, occurs through the activity of the osteoclast. Osteoclasts are cells of hematopoietic origin. The key molecular mediators of osteoclast formation from monocytic precursors are macrophage colonystimulating factor (M-SCF) acting through its receptor c-fms, and RANKL which binds to its receptor RANK (41-43). Osteoprotegerin (OPG) is the high affinity decoy receptor for RANKL and is able to prevent osteoclast differentiation by inhibiting RANK-RANKL interactions (44). RANKL binding to RANK in the presence of M-CSF induces the recruitment of adaptor molecules including TRAF6 by RANK (45), resulting in the activation of transcription factor NFB. One of the early targets of NFB is another transcription factor essential for osteoclastogenesis, nuclear factor of activated T-cells c1 (NFATc1), which later undergoes auto-amplification with the assistance of an activator protein-1 complex containing c-Fos (46-48). NFATc1 nuclear localization is regulated by

Breast Cancer Metastases to Bone: Role of the Microenvironment 537

(PHEX), matrix extracellular phosphoglycoprotein (MEPE), dentin matrix protein 1 (DMP1), and fibroblast growth factor-23 (FGF23) (72,73). Osteocyte networks in the bone tissue are implicated in regulating the maintenance and mineralization of bone tissue (70,74), through expression of sclerostin, a negative regulator of bone formation (75), as well as in sensing mechanical load in part through sheer stress generated by interstitial fluid moving through the lacuno-canalicular network (76). It has also been suggested that osteocytes participate in mineral homeostasis by resorbing the lacunar walls in which they are embedded (77-79).

Osteoblasts, osteoclasts and osteocytes must work in concert to maintain bone homeostasis (Figure 2). In normal bone physiology, the osteoclast will resorb worn or damaged bone, and then the osteoblast will form new bone in its place. The best studied example of the crosstalk between bone cells involves the RANK-RANKL-OPG triangle, where osteoblasts and osteocytes produce RANKL to promote osteoclast differentiation and survival, and OPG to prevent it, while osteoclasts express RANK, allowing them to respond to these regulatory cues. Many hormonal regulators of bone remodelling, such as PTH and estrogen, were demonstrated to act through changing the ratio of RANKL and OPG expression by osteoblasts (80). Interestingly, production of RANKL and OPG by osteoblasts is also regulated by their developmental stage, with immature osteoblasts producing more RANKL and mature osteoblasts produce more OPG, (58). Osteocytes also, at least in part, affect osteoclastogenesis through production of RANKL, which is induced in mechanicallystimulated osteocytes (81). Osteoclasts are in turn able to influence osteoblast activity. The concept of osteoclast-mediated osteoblastogenesis arose from the finding that 97% of new bone formation occurs in resorption pits (82). Several studies where osteoclasts have been genetically altered to have impaired function demonstrated diminished bone formation (83), and studies have begun to find mediators of this reversal coupling. Cardiotrophin-1 is among the first identified, and is expressed by osteoclasts and increases osteoblast activity (84). Sphingosine-1-phosphate has been shown to act earlier and induce osteoblast precursor recruitment and subsequent mature cell survival (85). Ephrin-B2/EphB4 bidirectional signaling between osteoclasts and osteoblasts, has also been identified as a key mediator of contact-dependent communication. Forward signalling by ephrin-B2 on osteoclasts to EphB4 on osteoblasts activates bone formation, whereas reverse signalling from EphB4 on osteoblasts binding to ephrin-B2 on osteoclasts inhibits osteoclastogenesis (86). Since the ability for bone cells to communicate is essential for the maintenance of bone homeostasis, it can be anticipated that disruptions in these the complex networks would lead to profound consequences. Indeed, the RANKL/OPG ratio represents one of the key mediators of

Recent evidence has led to the idea that the bone marrow supports a pre-metastatic niche - a site that receives signals from the primary tumour mass before dissemination, and changes the landscape of the target tissue to be conducive to tumour growth. It has been shown in mice treated with medium conditioned by tumour cells of different origin, the potential to home to different organs of subsequently injected cancer cells can be altered (88). In

*Communication between bone cells during normal bone remodelling* 

pathological bone destruction (87).

**4. Homing of breast cancer cells to bone** 

**4.1 Creation of the pre-metastatic niche** 

calcium signalling, which also activates calmodulin-dependent kinase, critical for further osteoclast differentiation (49). These events lead to the expression of osteoclast-specific genes including tartrate-resistant acid phosphatase (TRAP), cathepsin K, and b3 integrin (50), which are important for the degradation of bone tissue. Osteoclasts resorb bone by creating a unique microenvironment localized between this cell and bone tissue. Osteoclasts first recognize and bind to the bone matrix with integrin receptors 1 that bind collagen, fibronectin and laminin, and v3 that binds osteopontin and bone sialoprotein (51). This border forms a sealing zone over the area of bone to be resorbed, and the polarization of osteoclasts results in the formation of a ruffled border between the osteoclast and matrix (52). Targeted secretion of H+ ions through the ruffled border H+ ATPase, accompanied by movement of Cl- through chloride channels, acidifies the sealed space to a pH of approximately 4.5 (53,54), resulting in dissolution of the mineral phase of bone, and proteolytic enzymes cathepsin K and matrix metalloproteinase-9 (MMP-9) are released and activated to digest the organic matrix (55).

*Osteoblasts*: Osteoblasts are differentiated from the mesenchymal stem cells (MSC) that can also give rise to progenitors of myoblasts, adipocytes and chondrocytes (56). Commitment of MSC to become osteoprogenitors results in the upregulation of receptors for hormones, cytokines and growth factors, including PTH, prostaglandin, interleukin-11, insulin-like growth factor-1 and transforming growth factor- (57). Next, osteoprogenitor cells differentiate into preosteoblasts, cells that exhibit limited proliferation and start to express extracellular matrix proteins, such as collagen type I, bone sialoprotein and osteopontin. Preosteoblasts are also active in the production of pro-resorptive cytokine RANKL (58). Finally, mature osteoblasts do not proliferative, but actively produce and secrete collagen type I, bone sialoprotein and osteopontin as well as osteocalcin. In addition, mature osteoblasts switch to produce the RANKL inhibitor, OPG (58). Osteoblastogenesis commitment is driven by the downstream activities of Wingless-ints (Wnt) singling, the closely associated Hedgehog signalling pathway (Sonic Hedgehog, Indian Hedgehog) and bone morphogenetic proteins (BMPs), which determine where mesenchymal stem cells condense during embryonic patterning and cross-talk to induce osteoblast differentiation (59,60). Another developmentally important pathway, Notch signalling, has been shown to negatively regulate osteoblast differentiation (61-63). Important signalling events during osteoblast differentiation include the activation of the runt-related transcription factor 2 (Runx2) transcription factor, which regulates the expression of the zinc finger-containing transcription factor Osterix (64). Osterix interacts with nuclear factor for activated T cells 2 (NFATc2), and in collaboration, controls the transcription of osteoblastic target genes osteocalcin, osteopontin, osteonectin and collagen-1 (65,66). Osteoblasts anchor to newly formed bone matrix by cadherin-11 and N-cadherin, and secrete type 1 collagen and noncollagenous matrix proteins (57). The osteoblasts then regulate the subsequent mineralization of extracellular matrix (67-69).

*Osteocytes:* While each cell type is essential for the maintenance of bone homeostasis, osteocytes are the most populous and account for over 95% of all cells in the skeleton, covering 94% of all bone surface (70). Osteocytes are differentiated from osteoblasts embedded in the bone matrix. During differentiation, the osteocyte cell body size decreases, and the number of long dendrite-like cell processes increases and they extend, connecting the cell with other osteocytes (70,71). Osteocyte-specific genes are activated, including phosphate-regulating gene with homologies to endopeptidases on the X chromosome

calcium signalling, which also activates calmodulin-dependent kinase, critical for further osteoclast differentiation (49). These events lead to the expression of osteoclast-specific genes including tartrate-resistant acid phosphatase (TRAP), cathepsin K, and b3 integrin (50), which are important for the degradation of bone tissue. Osteoclasts resorb bone by creating a unique microenvironment localized between this cell and bone tissue. Osteoclasts first recognize and bind to the bone matrix with integrin receptors 1 that bind collagen, fibronectin and laminin, and v3 that binds osteopontin and bone sialoprotein (51). This border forms a sealing zone over the area of bone to be resorbed, and the polarization of osteoclasts results in the formation of a ruffled border between the osteoclast and matrix (52). Targeted secretion of H+ ions through the ruffled border H+ ATPase, accompanied by movement of Cl- through chloride channels, acidifies the sealed space to a pH of approximately 4.5 (53,54), resulting in dissolution of the mineral phase of bone, and proteolytic enzymes cathepsin K and matrix metalloproteinase-9 (MMP-9) are released and

*Osteoblasts*: Osteoblasts are differentiated from the mesenchymal stem cells (MSC) that can also give rise to progenitors of myoblasts, adipocytes and chondrocytes (56). Commitment of MSC to become osteoprogenitors results in the upregulation of receptors for hormones, cytokines and growth factors, including PTH, prostaglandin, interleukin-11, insulin-like growth factor-1 and transforming growth factor- (57). Next, osteoprogenitor cells differentiate into preosteoblasts, cells that exhibit limited proliferation and start to express extracellular matrix proteins, such as collagen type I, bone sialoprotein and osteopontin. Preosteoblasts are also active in the production of pro-resorptive cytokine RANKL (58). Finally, mature osteoblasts do not proliferative, but actively produce and secrete collagen type I, bone sialoprotein and osteopontin as well as osteocalcin. In addition, mature osteoblasts switch to produce the RANKL inhibitor, OPG (58). Osteoblastogenesis commitment is driven by the downstream activities of Wingless-ints (Wnt) singling, the closely associated Hedgehog signalling pathway (Sonic Hedgehog, Indian Hedgehog) and bone morphogenetic proteins (BMPs), which determine where mesenchymal stem cells condense during embryonic patterning and cross-talk to induce osteoblast differentiation (59,60). Another developmentally important pathway, Notch signalling, has been shown to negatively regulate osteoblast differentiation (61-63). Important signalling events during osteoblast differentiation include the activation of the runt-related transcription factor 2 (Runx2) transcription factor, which regulates the expression of the zinc finger-containing transcription factor Osterix (64). Osterix interacts with nuclear factor for activated T cells 2 (NFATc2), and in collaboration, controls the transcription of osteoblastic target genes osteocalcin, osteopontin, osteonectin and collagen-1 (65,66). Osteoblasts anchor to newly formed bone matrix by cadherin-11 and N-cadherin, and secrete type 1 collagen and noncollagenous matrix proteins (57). The osteoblasts then regulate the subsequent

*Osteocytes:* While each cell type is essential for the maintenance of bone homeostasis, osteocytes are the most populous and account for over 95% of all cells in the skeleton, covering 94% of all bone surface (70). Osteocytes are differentiated from osteoblasts embedded in the bone matrix. During differentiation, the osteocyte cell body size decreases, and the number of long dendrite-like cell processes increases and they extend, connecting the cell with other osteocytes (70,71). Osteocyte-specific genes are activated, including phosphate-regulating gene with homologies to endopeptidases on the X chromosome

activated to digest the organic matrix (55).

mineralization of extracellular matrix (67-69).

(PHEX), matrix extracellular phosphoglycoprotein (MEPE), dentin matrix protein 1 (DMP1), and fibroblast growth factor-23 (FGF23) (72,73). Osteocyte networks in the bone tissue are implicated in regulating the maintenance and mineralization of bone tissue (70,74), through expression of sclerostin, a negative regulator of bone formation (75), as well as in sensing mechanical load in part through sheer stress generated by interstitial fluid moving through the lacuno-canalicular network (76). It has also been suggested that osteocytes participate in mineral homeostasis by resorbing the lacunar walls in which they are embedded (77-79).

#### *Communication between bone cells during normal bone remodelling*

Osteoblasts, osteoclasts and osteocytes must work in concert to maintain bone homeostasis (Figure 2). In normal bone physiology, the osteoclast will resorb worn or damaged bone, and then the osteoblast will form new bone in its place. The best studied example of the crosstalk between bone cells involves the RANK-RANKL-OPG triangle, where osteoblasts and osteocytes produce RANKL to promote osteoclast differentiation and survival, and OPG to prevent it, while osteoclasts express RANK, allowing them to respond to these regulatory cues. Many hormonal regulators of bone remodelling, such as PTH and estrogen, were demonstrated to act through changing the ratio of RANKL and OPG expression by osteoblasts (80). Interestingly, production of RANKL and OPG by osteoblasts is also regulated by their developmental stage, with immature osteoblasts producing more RANKL and mature osteoblasts produce more OPG, (58). Osteocytes also, at least in part, affect osteoclastogenesis through production of RANKL, which is induced in mechanicallystimulated osteocytes (81). Osteoclasts are in turn able to influence osteoblast activity. The concept of osteoclast-mediated osteoblastogenesis arose from the finding that 97% of new bone formation occurs in resorption pits (82). Several studies where osteoclasts have been genetically altered to have impaired function demonstrated diminished bone formation (83), and studies have begun to find mediators of this reversal coupling. Cardiotrophin-1 is among the first identified, and is expressed by osteoclasts and increases osteoblast activity (84). Sphingosine-1-phosphate has been shown to act earlier and induce osteoblast precursor recruitment and subsequent mature cell survival (85). Ephrin-B2/EphB4 bidirectional signaling between osteoclasts and osteoblasts, has also been identified as a key mediator of contact-dependent communication. Forward signalling by ephrin-B2 on osteoclasts to EphB4 on osteoblasts activates bone formation, whereas reverse signalling from EphB4 on osteoblasts binding to ephrin-B2 on osteoclasts inhibits osteoclastogenesis (86). Since the ability for bone cells to communicate is essential for the maintenance of bone homeostasis, it can be anticipated that disruptions in these the complex networks would lead to profound consequences. Indeed, the RANKL/OPG ratio represents one of the key mediators of pathological bone destruction (87).
