Research ArticleOsteoclast migration on phosphorylated osteopontin is regulated by endogenous tartrate-resistant acid phosphatase
Introduction
During later stages of osteoclast differentiation, osteoclasts are targeted to specific sites on the bone surface where resorption is initiated. The attachment of osteoclasts and the subsequent polarization of the cell, i.e., formation of a sealing zone and a ruffled border, are dependent on the interaction between cell surface receptors and matrix surface molecules leading to changes in intracellular protein phosphorylation, cytoskeletal rearrangements, and vesicular transport [1]. The integrin αvβ3 appears to be particularly important for initiating signaling events leading to osteoclast polarization, since in its absence the osteoclasts fail to develop normal sealing zones and ruffled borders, which are essential for normal osteoclast function [2].
Osteopontin (OPN) is a ligand for the αvβ3-integrin through an RGD sequence and is particularly abundant in bone at sites where the osteoclasts attach [3]. Binding of OPN to the αvβ3-integrin in the sealing zone or podosomes of osteoclasts activates a gelsolin-dependent signal transduction pathway that involves activation of RhoA and appears necessary for reorganization of the actin cytoskeleton for osteoclast motility [4]. Thus, OPN not only activate the osteoclast for bone matrix resorption but can also induce osteoclast migration in a manner dependent on the αvβ3-integrin as well as the hyaluronan receptor CD 44 [5], [6].
OPN is a phosphorylated glycoprotein with a variable extent of phosphorylation, where the number of phosphates ranges from 28 to 32 in the OPN from milk, 12–13 in the OPN from bone, and 8 in the urine OPN [7], [8], [9], [10]. The reason for the occurrence of heterogeneous phosphorylation of OPN in different sites is not well understood but may conceivably be related to differential actions of protein kinases and/or phosphatases. The phosphorylation of OPN appears to control several of its biological activities, e.g., cell adhesion and migration [11], [12], [13], [14], mineralization [15], [16], and immune and cytokine responses [14]. For instance, αvβ3-integrin-dependent interaction of osteoclasts and macrophages with OPN are enhanced on phosphorylated compared with unphosphorylated OPN [13], [14]. Moreover, chemotactic and chemokinetic migration of macrophages as well as choriocarcinoma cells was recently found to be stimulated by OPN phosphorylation [11], [14].
During bone resorption, the osteoclasts alternate between phases of matrix degradation and non-resorptive stages of migration [17]. The mechanisms responsible for the transition between these stages presumably involve an initial phase of osteoclast detachment from the resorbed bone surface followed by re-attachment to a neighboring unresorbed area. OPN has been implicated both as an adhesive as well as a migratory protein for monocyte–macrophage lineage cells, including osteoclasts [6], [18], [19]. Phosphorylations on OPN appear to enhance both αvβ3-integrin-dependent adhesion as well as migration of cells [11], [12], [13], [14]. Moreover, migrating cells tend to bind with intermediate strength to their adhesive receptors [20], [21]. In order to control the transition between firm attachment and migration, mechanisms responsible for regulating the phosphorylations on OPN, in effect generating forms with variable avidity for binding the αvβ3-integrin, need to be implicated. In this regard, modifying phosphorylation of OPN extracellularly by the action of protein phosphatases could therefore be considered as a potentially important mechanism.
Tartrate-resistant acid phosphatase (TRAP), a metallophosphatase highly expressed in osteoclasts, is secreted into the resorption lacuna and associated with the resorbing matrix [22], [23], [24], [25]. When activated by proteolytic processing, TRAP exhibits protein phosphatase activity towards several bone matrix proteins, including OPN [24], [25]. Dephosphorylation of OPN from rat bone by TRAP rendered the protein less adhesive for osteoclasts [12] , and phosphorylation reversed this effect [13]. In this study, we addressed whether phosphorylations on OPN were involved in migration of osteoclasts and if endogenous TRAP from osteoclasts modulated OPN-dependent osteoclast migration.
Section snippets
Materials
Minimum Essential Medium Eagle (M4526), alkaline phosphatase-conjugated goat anti-rabbit IgG (A7539), leukocyte detection kit (387-A), BSA (bovine serum albumin, A2153) and pNPP (4-nitrophenyl phosphate bis[tris(hydroxymethyl)-aminomethane] salt, 73737) came from Sigma-Aldrich Co (St. Louis, MO, USA). l-Glutamine (25030), gentamycin (15710), HEPES (15630), and anti-β3 (anti-rat CD61, 554950) were from Invitrogen (Paisley, UK). BCIP/NBT (11681451001) and E-64 (N-[N-(l-3-trans
Post-translational modifications of osteopontin promote αvβ3 integrin-dependent osteoclast adhesion
Dose–response curves comparing the capacity of osteopontin purified from bovine milk (bmOPN), containing on the average 28 phosphates and 5 O-glycosidic oligosaccharides [10], with human recombinant osteopontin (hrOPN) expressed in Escherichia coli, lacking these post-translational modifications, to promote adhesion of primary rat osteoclasts isolated from long bones of newborn rat pups are shown in Fig. 1A. At coating concentrations of 1–3 μg/ml, the numbers of osteoclasts increased
Discussion
The post-translational phosphorylations of OPN have been implicated in the regulation of various biological events, for example, cellular adhesion and motility, involving OPN [37]. Both macrophages and osteoclasts are derived from a common myeloid progenitor cell, and have been shown to adhere to OPN involving integrins in a phosphorylation-dependent manner [12], [14]. However, whereas the phosphorylation-dependent interaction with OPN induced cell spreading and haptotactic migration of
Acknowledgments
This study was supported by grants from the Swedish Research Council, Stockholm County Council (ALF), and Karolinska Institutet. We thank Drs. Ian Cassady and David Hume, Institute for Molecular Bioscience, University of Queensland, St. Lucia, Australia, for providing access to the transgenic TRAP-overexpressing mice and to Dr. Esben Sörensen, University of Aarhus, Denmark, for a sample of human milk OPN.
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