Osteopontin modulates CD44-dependent chemotaxis of peritoneal macrophages through G-protein-coupled receptors: evidence of a role for an intracellular form of osteopontin.
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.
CIHR Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.
Baoqian
B
Zhu
Keiko
K
Suzuki
Harvey A
HA
Goldberg
Susan R
SR
Rittling
David T
DT
Denhardt
Christopher A G
CA
McCulloch
Jaro
J
Sodek
D000199:Actins / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D002459:Cell Fusion; D002633:Chemotaxis / Q000502:physiology*; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism; D053495:Osteopontin; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D015415:Biomarkers; D018960:Hyaluronan Receptors; D043562:Receptors, G-Protein-Coupled; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D053495:Osteopontin