Osteopontin localizes to the nucleus of 293 cells and associates with polo-like kinase-1

Abstract

Osteopontin (OPN) is a secreted phosphoprotein involved in cellular proliferation and associated with tumor progression. Although an intracellular form of OPN has been described, its function remains unknown. In this study, a novel nuclear location for intracellular OPN and a correlation with cell division were demonstrated. OPN distinctly localized to the nucleus in a subset of transiently transfected human embryonic kidney 293 cells. Immunoblotting confirmed the nuclear location of native OPN, and results from immunofluorescence studies suggested an association between nuclear OPN and cell cycle progression. Flow cytometry revealed that nuclear and cellular OPN content rose significantly during the S and G(2)/M phases, respectively. Treatment of cells with the DNA polymerase inhibitor aphidicolin prevented cell cycling and greatly reduced cellular OPN content. The intracellular location of OPN coincided with polo-like kinase-1 (Plk-1), a member of the polo-like kinase family, which, in part through their regulation of centrosome-related events, are integral to successful cellular mitosis. OPN and Plk-1 were coimmunoprecipitated from nuclear, but not cystoslic, extracts, demonstrating an interaction that is limited to the nucleus, presumably during mitosis. Deletion of the COOH terminus of OPN militated against nuclear localization and Plk-1 interaction. Elevated expression of OPN was also associated with an increase in the number of multinucleate 293 cells, whereas transfection of the COOH-terminal-deleted OPN decreased the percentage of multinucleate cells below basal levels. These findings implicate intranuclear OPN as a participant in the process of cell duplication.

Alternative translation of osteopontin generates intracellular and secreted isoforms that mediate distinct biological activities in dendritic cells

“…This observation opened the possibility that the difference in size (Ϸ5 kDa) reflected deletion of the 16-aa signal sequence. We then analyzed proteins translated in vitro from full-length Opn mRNA and an Opn mRNA truncation mutant (⌬48Koz) that lacked the first 48 nucleotides encoding the Opn signal sequence (amino acid [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]). An artificial AUG/Kozak sequence was introduced to initiate translation ( Fig.…”

“…However, a nonsecreted species of Opn has been implicated in a growing number of cellular processes, including migration, fusion, and motility (8)(9)(10)(11). Recent studies have revealed that an intracellular form of Opn (Opn-i) in plasmacytoid dendritic cells (pDC) interacts with IRF7 to induce IFN␣ expression (12), whereas expression of Opn-i in conventional DC (cDC) promotes differentiation of IL-17-producing T helper cells (Th17 cells) (13).…”

Intracellular Osteopontin Inhibits Toll-like Receptor Signaling and Impedes Liver Carcinogenesis

“…iOPN takes part in several biologic functions. In the nucleus of 293 cells, iOPN colocalizes with polo-like kinase 1 and participates in cell duplication (19). In fibroblasts, iOPN colocalizes with the hyaluronan-CD44-ERM (ezrin/radixin/moesin) complex at perimembrane regions and plays a role in cell migration (20).…”

Embracing the complexity of matricellular proteins: the functional and clinical significance of splice variation

“…Nuclear localisation of OPN has been described (81) and an intracellar isoform of osteopontin (OPNi) has also been characterised. This intracellular form is a truncated protein that lacks the N-terminus, including the signal peptide, and is instead localised to the cytoplasm.…”