By Giolanta Kogianni & Justin Sturge, Imperial College London *

An introduction to the biology of bone cells, their functional proteins together with a focus on the metastasis of primary tumors to bone.

1. Introduction
2. Bone cell biology
   1. Osteoclasts
   2. Osteoblasts
   3. Osteocytes
3. Bone metastasis
4. Conclusion
5. References

1. Introduction

Bone is a rigid form of connective tissue that supports locomotion, provides protection for vital organs, nurtures the bone marrow and participates in ion homeostasis. Bones consist of hydroxyapatite crystals, collagens, mainly collagen type I, and non-collagenous proteins, such as, fibronectin, osteocalcin, osteopontin, osteonectin, decorin and bone sialoprotein (BSP)^{1, 2}.

Modeling of the flat bones occurs via intramembranous ossification in which mesenchymal/stromal cells differentiate into osteoblastic cells, which synthesize woven bone that is characterized by irregular calcification and arrangement of collagen fibers; woven bone is progressively replaced by lamellar bone³. Modelling of long bones occurs via endochondral ossification during which mesenchymal cells differentiate into chondroblasts that secrete the cartilaginous matrix, bringing about longitudinal growth⁴. This process is strongly influenced by factors such as parathyroid hormone-like hormone (PTHLH) (also known as parathyroid hormone-related protein, PTHrP), Indian Hedgehog (Ihh) and the bone morphogenetic protein (BMP) family⁵ (see table). In adult bone osteoclasts act to resorb the calcified matrix, osteoblasts are responsible for forming woven bone¹ and later differentiate to become bone resident osteocytes. These three cells types are discussed below in more detail.

2. Bone cell biology

i) Osteoclasts

Osteoclasts are multinucleated cells, formed by the fusion of mononuclear progenitors (CFU-GM, colony-forming unit for the granulocyte-macrophage series) in the bone marrow, which have the capacity, in response to appropriate stimuli, to differentiate into a granulocyte, monocyte or osteoclast⁷. The osteoclast is characterized by a ruffled border that polarises the cell on the bone surface⁸. Acidification of bone is induced by an electrogenic proton ATPase, whereas the abundant Golgi complexes and a large endoplasmic reticulum compartment produce and process lysosomal and proteolytic enzymes, include members of the MMP and cathepsin families, which are secreted via the ruffled border in the localized environment and function to carry out bone degradation⁹.

Osteoclast formation and maturation is regulated by several factors, including parathyroid hormone (PTH), tumor necrosis factor-alpha (TNF-α), transforming growth factor-alpha (TGF-α), interleukin-1 (IL-1) and 1,25-dihydroxyvitamin D10. Osteoclastogenesis is also strongly influenced by the macrophage colony-stimulating factor (M-CSF) and the receptor for activation of nuclear factor kappa B ligand (RANKL), also known as osteoprotegerin ligand (OPGL), which interact with RANK or OPG expressed on osteoblast progenitor cells and marrow stromal cells¹¹.

ii) Osteoblasts

Osteoblasts are derived from bone marrow stromal cells or connective tissue mesenchymal cells¹². Central to osteoblast differentiation is the transcription factor RUNX2 (also known as core-binding factor-1, Cbfa1) expressed in osteoprogenitor cells^{13, 14} and Ihh, which regulates expression of PTH and PTHLH controlling chondrocyte hypertrophy. Other growth factors involved in osteogenesis include members of the TGF-β superfamily, such as the BMPs (including GDF15), as well as various members of the FGF and IGF families¹⁵. Expression of these factors occurs in the proliferative phase of progenitor cells, along with expression of matrix components, such as collagen type I and fibronectin¹⁴, followed by expression of bone alkaline phosphatase, matrix Gla-protein (MGP), osteopontin and osteocalcin, which result in hydroxyapatite accumulation and completion of mineralisation¹⁶. Osteoblastic activity is also influenced by Sclerostin (SOST), which binds BMP family members and suppresses their activity in bone¹⁷, low density lipoprotein-receptor related protein 5 (LRP5) and Wnt signaling, as gain of function mutations led to high bone mass while loss of function led to osteoporosis-pseudoglioma syndrome^{18, 19}. Osteocrin is a secreted bone-specific protein downregulated by vitamin D20, which acts to block naturetic peptide receptor C (NPR-C) mediated uptake of C-type naturetic peptide in bone²¹. The central nervous system has also been found to have a role in the regulation of bone formation since deficiency of leptin induces high bone mass independently of its anti-appetite effects²².

iii) Osteocytes

Osteocytes are terminally differentiated, postmitotic cells, derived from bone surface osteoblasts that have become encased by their own matrix production, which eventually calcifies²³. Osteocytes are characterised by low bone alkaline phosphatase activity and collagen type I expression and high osteocalcin, osteopontin and connexin 43 expression, of which the latter is important for communication between cells²⁴. Osteocytes express integrins, such as, β1, αvβ3 and β3, which assist in adhesion to bone matrix. Osteocytes are influenced by the activity of 1,25(OH)2D3 and hormones like PTH, PTHLH, estrogen, glucocorticoids, mineralocorticoids and prostaglandins, while their function as regulators of bone remodeling and microdamage repair is strongly dependent on mechanical stimulation (for review see Kogianni et al., 200725). Osteocytes also express receptors associated with the nervous system, such as neurokinin-1 receptor26, EAAT1 (also known as glutamate and aspartate transporter, GLAST)²⁷ and the serotonin responsive 5HT2B receptor²⁸.

3. Bone metastasis

Several cancers, both solid tumors and haematopoietic malignancies, have profound effects upon the skeleton, causing an increase in osteoclast formation and activity, either systemically as in humoral hypercalcaemia of malignancy or locally in bone metastases. Of particular significance bone is a preferential site for tumor cell metastasis of the three major cancer types i.e. adenocarcinomas of the lung, breast and prostate gland. Bone metastases with an osteoblastic (bone-forming) phenotype (eg prostate) arises due to the activation of osteoblasts or inhibition of osteoclasts (or both) by the cancer cells; in contrast metastases with an osteolytic (bone-degrading) phenotype result from increased activity of osteoclasts or decreased osteoblast function (or both) induced by the cancer cells²⁹. For example, release of factors such as TGF-beta can profoundly affect the growth of tumor cells and their production of bone-resorbing or forming cytokines. On the other hand cancer cells are able to produce factors, including PTHLH, IL6, IL11, and COX2-generated prostanoids, RANKL/RANK or OPG, which strongly alter the activity/function of stromal/osteoblastic or osteoclastic cells. Metastases from prostate cancer, most of which are adenocarcinomas, nearly always form osteoblastic lesions in bone, although the concept of an accompanying osteolytic phenotype is becoming increasingly accepted, presumably to facilitate prostate cancer establishment and expansion. By contrast, bone metastases from lung or breast cancers more often are osteolytic. However, metastases from the relatively uncommon neuroendocrine tumors of the prostate also produce osteolytic lesions.

It is the acquisition of invasive properties by tumor cells that facilitates their metastatic spread and this involves their increased expression of proteins involved in cell-matrix adhesions (eg. integrin family members, uPAR and CD44), remodeling of the extracellular matrix by protease systems (eg. MMPs/TIMPs and uPA/PAI1/PAI2) and promotion of cell migration/chemotaxis (Rho, Rac, Cdc42, Rho kinase/ROCK, PAK)^30-32. Of particular note the organ specific metastasis of certain primary tumors to bone has been attributed to the ability of these cancer cells to upregulate the expression of certain chemokine receptors (eg CXCR4) and local production of chemokine receptor ligands in the bone (eg stromal cell derived factor-1, SDF1)³³. A similar chemotactic receptor/ligand signaling axis has been uncovered for RANK/RANKL in prostate cancer, breast cancer and melanoma cells and this represents another important promotor of metastasis to bone³⁴. Other multifunctional receptor/ligand signalling axes that are important in tumor progression, such as epidermal growth factor and its receptor, EGF/EGFR, and uPA/uPAR are also able to drive tumor cell chemotaxis^35-37.

4. Conclusion

The processes of bone modeling and remodeling, which is the process by which old or damaged bone is replaced by new bone are regulated by a variety of osteotropic cytokines and hormones, which influence the activity of bone cells in order to maintain coupling between bone resorption and formation. Imbalances in these interactions result in abnormal turnover cycles, characterized by insufficient formation of bone resorbing or forming cells, increased resorptive or forming activity or abnormal formation of mineral crystals, leading to diseases such as osteoporosis and Paget’s disease^38-40. It is evident that the osteolytic and osteoblastic characterstics of these diseases are also the hallmarks of secondary tumors which have established in the skeleton.

Bone metastasis

5. References

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Giolanta Kogianni & Justin Sturge, Imperial College London