Osteopontin Undergoes Polymerization in Vivo and Gains Chemotactic Activity for Neutrophils Mediated by Integrin α9β1

Abstract

Osteopontin (OPN) is an integrin-binding inflammatory cytokine that undergoes polymerization catalyzed by transglutaminase 2. We have previously reported that polymeric OPN (polyOPN), but not unpolymerized OPN (OPN*), attracts neutrophils in vitro by presenting an acquired binding site for integrin ␣9␤1. Among many in vitro substrates for transglutaminase 2, only a few have evidence for in vivo polymerization and concomitant function. Although polyOPN has been identified in bone and aorta, the in vivo functional significance of polyOPN is unknown. To determine whether OPN polymerization contributes to neutrophil recruitment in vivo, we injected OPN* into the peritoneal space of mice. Polymeric OPN was detected by immunoblotting in the peritoneal wash of mice injected with OPN*, and both intraperitoneal and plasma OPN* levels were higher in mice injected with a polymerization-incompetent mutant, confirming that OPN* polymerizes in vivo. OPN* injection induced neutrophil accumulation, which was significantly less following injection of a mutant OPN that was incapable of polymerization. The importance of in vivo polymerization was further confirmed with cystamine, a transglutaminase inhibitor, which blocked the polymerization and attenuated OPN*-mediated neutrophil recruitment. The thrombin-cleaved N-terminal fragment of OPN, another ligand for ␣9␤1, was not responsible for neutrophil accumulation because a thrombin cleavage-incompetent mutant recruited similar numbers of neutrophils as wild type OPN*. Neutrophil accumulation in response to both wild type and thrombin cleavage-incompetent OPN* was reduced in mice lacking the integrin ␣9 subunit in leukocytes, indicating that ␣9␤1 is required for polymerization-induced recruitment. We have illustrated a physiological role of molecular polymerization by demonstrating acquired chemotactic properties for OPN. Osteopontin (OPN),3 an integrin-binding cytokine, plays critical roles in physiological and pathological processes, including inflammation, immunomodulation, tissue remodeling, fibrosis, mineralization (1), stem cell retention (2), and tumor metastasis (3). These functions are exerted through interactions with nine integrins and CD44 (4). OPN undergoes many types of post-translational modification, including phosphorylation (5), glycosylation, transglutamination (6), and proteolytic cleavage. Among these post-translational modifications, transglutamination (7) and thrombin-cleavage (8, 9) enhance interactions with integrins. Thrombin-cleaved N-terminal fragment of OPN (nOPN) has been found to play roles in rheumatoid arthritis (10, 11), autoimmune hepatitis (12), and stem cell retention in the bone marrow niche (2). Although OPN was found as a substrate for transglutaminase 2 (TG2; EC 2.3.2.13) earlier than thrombin, little is known about the functional role of the product of transglutamination, polymeric OPN. TG2 is a cross-linking enzyme that catalyzes isopeptide bonding between Gln and Lys residues (13) with specificity for amino acid sequences containing Gln (...

OPN‐a induces muscle inflammation by increasing recruitment and activation of pro‐inflammatory macrophages

“…OPN‐b and OPN‐c are less polymerized than OPN‐a (Nishimichi et al . 2011) owing to loss of Gln residues at exons 4 and 5, which serve as cross‐linking sites. Given that transglutaminase 2 is abundantly expressed by macrophages (Zakrzewicz et al .…”

“…2011). Recombinant human OPN‐a proteins containing: (i) RGD (arginine‐glycine‐aspartic) acid residues; and (ii) OPN‐a with the RGD domain mutated to KAE [lysine‐alanine‐glutamic; ΔRGD→KAE (OPN‐a‐KAE)] were a kind gift from Dr Larry Fisher at the National Institutes of Health.…”

“…2007; Nishimichi et al . 2011). Transglutaminase 2 catalyses OPN polymerization at multiple Gln residues coded by exons 2–5 (Christensen et al .…”

Matricellular Proteins in the Trabecular Meshwork: Review and Update

“…224,225 In addition to these active domains, it harbors bindings sites for heparin, putative thrombin cleavage sites, and an N-terminal region that is both chemotactic for neutrophils and associated with carotid plaque formation. [224][225][226][227] Osteopontin's diverse effectsincluding during development, bone resorption and calcification, wound healing, mucosal protection, and immunological responses, all of which are reviewed elsewhere 223,228 -have been conceptually grouped into 2 broad functions: regulation of acute and chronic inflammation, and regulation of macrophage-derived cells such as osteoclasts. 229 These putative functions are more or less confirmed by phenotypic traits found in osteopontin-deficient mice, including reduced bone resorption, increased dystrophic calcification, altered sympathetic control of bone mass, impaired periprosthetic osteolysis, abnormal matrix reorganization following tissue injury, and reduced T-cell-mediated immunity.…”

Functional Genomic Assessment of Phosgene-Induced Acute Lung Injury in Mice

“…Integrin a9 (Itga9), a candidate gene with a suggestive SNP association in this study using phosgene, has been associated with TGF-b1 signaling in the lung (120). Partnering with integrin b1, integrin a9b1 can interact with vascular cell adhesion molecule 1 (121), vascular endothelial growth factors (122), and secreted phosphoprotein 1 (aka osteopontin) (123) and thus could have critical roles in ALI.…”