Thrombin Cleavage of Osteopontin Modulates Its Activities in Human CellsIn Vitroand Mouse Experimental Autoimmune EncephalomyelitisIn Vivo

Abstract

Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α 4 β 1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases.

Osteopontin binds ICOSL promoting tumor metastasis

“…Since the thrombin cleavage site in OPN is in the middle of these putative sequences, the ability to bind ICOSL displayed by recombinant forms of the N-term (aa 17–167) and C-term (aa 168–314) portions of OPN was tested; these correspond to the two fragments generated by the physiologic thrombin cleavage of the whole OPN (Fig. 6a ) 12 . ELISA assay showed that both the OPN portions bind to ICOSL, but to a lesser extent than the full-length protein (Fig.…”

“…Osteopontin (OPN) is a phosphoprotein, secreted by several cell types, such as macrophages, DC and Th cells; it can function both as a matricellular protein and a cytokine mediating several biological functions. These include migration, adhesion, activation of inflammatory cells, and modulation of T cell activation supporting differentiation of proinflammatory type 1 (Th1) and type 17 (Th17) Th cells 12 . It is highly expressed in several types of tumors, being secreted by both tumor and microenvironment cells, and is implicated in promoting tumor invasion and metastatic dissemination 13 .…”

“…This confers to OPN the ability to adopt different functional structures by folding upon binding and to interact with multiple binding partners 17 . Thrombin cleaves OPN in the middle of the molecule, near to an RGD motif, and generates two fragments with slightly different functional activities 12 .…”

Osteopontin attenuates early brain injury through regulating autophagy‐apoptosis interaction after subarachnoid hemorrhage in rats

“…21 Gong et al reported that intranasal administration of rOPN dose-dependently increased OPN expression in brain tissue after hyperglycemic intracerebralhemorrhage. 16 In the current study, considering the reported importance of thrombin and metalloproteinases-cleaved OPN after SAH, 31,32 we used Western blot analysis to examine the amount of full-length OPN and cleaved OPN variants in the left hemisphere (the perforation side) after SAH and rOPN intranasal administration. Our results showed a significant increase in the amount of all variants of OPN, which includes both full-length and cleavage products at 24 hours after SAH.…”

Osteopontin Bridging Innate and Adaptive Immunity in Autoimmune Diseases

“…In several cell types, including vascular endothelial cells and tumor cells, OPN-N induces migration whereas OPN-C induces adhesion. By contrast, both fragments similarly induce IFN- γ secretion in T cells and tubulogenesis in vascular endothelial cells and inhibit activation-induced cell death in lymphocytes [42]. …”