Studies on the phenotype and function of osteoclasts using osteopetrotic and rachitic animal models

Abstract: Osteoclasts are multinucleated bone-resorbing cells that have been implicated in a variety of skeletal diseases e.g. osteoporosis, rheumatoid arthritis and skeletal metastasis. Under physiological conditions, the osteoclast participates in ossification during longitudinal bone growth as well as in bone remodelling during adulthood. Bone resorption is initiated by attachment of osteoclasts to the surface of bone to be resorbed by interaction with mineral-associated adhesive proteins. This interaction also drives formation of specialized membrane domains called ruffled borders and sealing zones leading to cell polarization necessary for enclosement of the degradative process in a discrete extracellular area. In this resorption lacuna, local acidification leads to dissolution of mineral, and secretion of proteolytic enzymes e.g. cathepsin K (Ctsk) and matrix metalloproteinase (MMP)-9 completes degradation of the exposed collagen. Another secreted osteoclast enzyme, tartrate-resistant acid phosphatase (TRAP), is a protein phosphatase with suggested roles in regulating osteoclast adhesion, migration and matrix degradation. In this thesis, animal models with the skeletal diseases rickets and osteopetrosis were employed to study the relation between ultrastructural morphological characteristics of osteoclasts and secretion of biochemical resorption markers e.g. Ctsk, MMP-9 and TRAP. Using two different models of rickets e.g. induced by dietary restriction of phosphate and vitamin D in young rats and transgenic mice overexpressing the phosphate-regulating FGF-23 in osteoblasts, it was demonstrated that mineralization of bone matrix is required for osteoclast polarization but not for secretion of proteolytic enzymes and that matrix mineralization is protective for and limits collagen degradation. It is concluded that the validity of osteoclast polarization as an ultrastructural morphological indicator of osteoclast resorptive activity is restricted to bone with normal mineralization. Moreover, genes encoding mineralization-promoting proteins are activated in osteoblasts from mice with hypophosphatemic rickets, indicative of a compensatory mechanism to favour mineralization. TRAP is synthesized as an inactive monomeric precursor requiring proteolytic processing to become an active enzyme. Proteolytic processing of TRAP was altered in a subpopulation of metaphyseal osteoclasts in a mouse strain where the Ctsk gene was inactivated, implicating Ctsk as a regulator of TRAP processing in vivo and supporting the concept of functional heterogeneity of osteoclasts depending on their precise anatomical localization within the skeleton. Moreover, the intracellular distribution and secretion of monomeric and proteolytically processed TRAP was altered in Ctsk-deficient osteoclasts, providing evidence for a novel role for Ctsk in the regulation of intracellular membrane traffic in osteoclasts. The possibility that TRAP is the common denominator for this vesicular transport regulation was indicated by the observation of an accumulation of cytoplasmic vesicles in osteoclasts from mice with an inactivated TRAP gene. In addition, whereas Ctsk was secreted by both polarized and non-polarized osteoclasts, TRAP was secreted only by polarized osteoclasts under certain conditions, suggesting different regulation for secretion of these enzymes as well as an action of TRAP later than Ctsk in the matrix degradative phase of the resorption sequence.