Osteopontin modulates CD44‐dependent chemotaxis of peritoneal macrophages through G‐protein‐coupled receptors: Evidence of a role for an intracellular form of osteopontin

Abstract

Expression of osteopontin (OPN) by activated T-cells and macrophages is required for the development of cell-mediated inflammatory responses. Acting through integrin alpha(v)beta(3) and CD44 receptors, OPN can promote chemoattraction and pro-inflammatory cytokine expression by macrophages. In this study, we have used peritoneal macrophages from OPN-/, CD44-/-, and WT mice to study the relationship between OPN and CD44 in macrophage migration. Using confocal microscopy, we show that OPN co-distributes with CD44 inside macrophages at cell edges and in cell processes in a mutually dependent manner. The existence of an intracellular form of OPN is supported by pulse-chase studies in which a thrombin-sensitive, phosphorylated protein immunoprecipitated with OPN antibodies is retained inside macrophages. In OPN-/- and CD44-/- macrophages, the absence of CD44 and OPN, respectively, is associated with the formation of fewer cell processes, reduced cell fusion required to form functional multinucleated osteoclasts in the presence of CSF-1 and RANKL, and impaired chemotaxis. Whereas the chemotaxis of CD44-/- cells to various chemoattractants is almost completely abrogated, a differential effect is seen with the OPN-/- cells. Thus, OPN-/- cells migrate normally towards CSF-1 but not towards fMLP and MCP-1, which signal through G-protein coupled receptors (GPCRs). That the GPCR-mediated migration is dependent upon the level of cell-surface CD44 is indicated by the reduced cell-surface expression of CD44 in OPN-/- cells and a comparable impairment in the chemotaxis of CD44+/- cells. Although chemotaxis of OPN-/- cells could be rescued by an OPN substratum, or by addition of high levels of OPN in solution, no response is evident with physiological levels of OPN, indicating a requirement for the CD44-associated intracellular OPN in CD44 cell-surface expression. These studies indicate, therefore, that the level of cell surface CD44 is critical for GPCR-mediated chemotaxis by peritoneal macrophages and suggest that a novel intracellular form of OPN may modulate CD44 activities involved in these processes.

Osteopontin: Role in immune regulation and stress responses

“…In the absence of OPN expression, macrophage migration and cytokine production are impaired Sodek et al, 2006). Zhu et al (2004) showed that in macrophages OPN both regulates the distribution of CD44 and, as iOPN, co-localizes with CD44 at the inner surface of the plasma membrane. OPN-deficient macrophages have a more diffuse distribution of CD44, suggesting that CD44 cooperates with OPN in regulating macrophage migration.…”

Role of osteopontin in tumour progression

“…Secondly, the ability of cells to migrate may be directly tied to their tumorigenicity and OPN clearly participates in pathways regulating migration in diverse cell types including osteoclasts, fibroblasts, macrophages and tumour cells (Tuck et al, 2000). Interestingly, in macrophages, OPN regulates migration toward some chemokines but not others, suggesting that the protein may function in a subset of migratory pathways (Zhu et al, 2003): perhaps, this observation may help to explain some of the diverse effects of OPN in different tumour systems. Invasiveness is clearly related to migration, but not only do cells need to be motile to invade, they also need to degrade the extracellular matrix.…”

“…Early on, it was recognised that OPN had adhesive activity, confirmed by the observation that its receptors all mediate cell adhesion. An additional well-characterised function of OPN is in regulating migration: not only is it chemotactic for many cell types but also OPN-deficient cells are hypomotile (Zhu et al, 2003), suggesting that the protein plays an intrinsic role in migration. The protein regulates cytokine production by macrophages, and in several diverse systems, it has been shown to act as a survival factor ).…”

CD44 in hematological neoplasias

“…CD44-/-macrophages show decreased response to chemoattractants, implied by impaired migration [82]. Ligation of CD44v6 and v7 on monocytes trigger the release of GM-CSF and IL-6, which thus leads to increased proliferation of myeloid and lymphoid progenitor cells [72].…”