Casein kinase 2 phosphorylation of recombinant rat osteopontin enhances adhesion of osteoclasts but not osteoblasts

Abstract

Osteopontin (OP) is a highly phosphorylated bone matrix protein and contains the RGD cell-binding motif, which mediates cell adhesion through integrin receptors that include alpha(v)beta3. Casein kinase 2 (CK2) is a factor-independent serine/threonine kinase, which may be the predominant physiologically relevant kinase for OP phosphorylation. This study was designed to examine the effects of unphosphorylated recombinant rat OP, and CK2-phosphorylated OP (P-OP), on the adhesion and function of mouse osteoclasts (OC) and osteoblast-like cells (UMR 201-10B and UMR 106-06) in vitro. OP significantly increased OC adhesion compared to plastic alone, and cell attachment was further increased at least twofold on OP phosphorylated with CK2. Attachment was dependent on the integrity of the RGD domain and was completely abolished in the presence of 1 mM RGD peptide. Neither CK2 phosphorylation of mutant OP, in which the RGD was converted to RGE or RAD, nor protein kinase C (PKC) phosphorylation of wild-type OP enhanced OC attachment. An antibody to the beta3 integrin subunit, but not anti-mouse CD44 antibody, specifically blocked the proportion of attachment due to phosphorylation of OP. Actin ring formation in OC was increased by plating cells onto OP, with no further increase by phosphorylation. Both OP and CK2-phosphorylated OP enhanced attachment of the two osteoblastic cell lines, compared to plastic, but in contrast to OCs, there was no significant difference with phosphorylation. Osteoblast attachment was totally blocked by 1 mM RGD peptide, but was not influenced by the beta3 integrin antibody. Plating of UMR 201-10B cells onto OP further increased retinoic acid-induced alkaline phosphatase expression. The results suggest that specific phosphorylation of OP is important for interaction with OCs, compared with osteoblastic cells, and that alternative integrins may be important in the interaction between osteoblastic cells and OP compared with OCs.

Post-translational Modifications of SIBLING Proteins and Their Roles in Osteogenesis and Dentinogenesis

“…These results indicate that phosphorylation of OPN is essential for its inhibitory role on mineralization. Two studies have suggested that phosphorylation is necessary for OPNstimulated osteoclast attachment (Ek-Rylander et al, 1994;Katayama et al, 1998). More recently, experiments performed with primary osteoclast cultures on bone slices have demonstrated that phosphates on OPN may be involved in regulating the resorptive activity of osteoclasts (Razzouk et al, 2002).…”

Complete Topographical Distribution of Both the in Vivo and in Vitro Phosphorylation Sites of Bone Sialoprotein and Their Biological Implications

“…Indeed, the covalently bound phosphates of BSP and OPN have been shown to also affect osteoclast cell attachment properties (15,30). More recent conflicting studies have utilized different forms of OPN and BSP, and the results suggest that, although phosphorylation is not important for cell attachment, it is necessary for in vitro osteoclast resorption pit formation in dead bone slices (31).…”

Osteoclast Responses to Lipopolysaccharide, Parathyroid Hormone and Bisphosphonates in Neonatal Murine Calvaria Analyzed by Laser Scanning Confocal Microscopy

“…Cell attachment molecules such as OPN and its ␣v␤3 integrin receptor are involved in osteoclast differentiation, migration to sites of resorption, fusion of postmitotic osteoclast precursors, cellular polarization, and tight sealing zone formation required for bone resorption (Reinholt et al 1990;Yamate et al 1997;Nakamura et al 1999;Duong et al 2000;McHugh et al 2000;Chellaiah and Hruska 2003). The cessation of resorption by detachment of osteoclasts from the bone matrix also involves adhesion molecules (Ek-Rylander et al 1994;Katayama et al 1998), which are important for osteoclast survival (Horton et al 2002). In addition to mediating cell adhesion, cell attachment molecules signal through receptors and affect cytoskeletal remodeling and gene transcription.…”

“…Bars ϭ 100 m. Depth in the XZ scans is 100 m. involved in the formation of a sealing zone around resorbing osteoclasts is strictly regulated by the substrata on which the cell locates and emphasizes the importance of studying these cells in their natural environment. Although our results do not directly demonstrate the importance of OPN in cytoskeletal organization, they support previous studies that have demonstrated this relationship (Katayama et al 1998;Suzuki et al 2002a). …”