Cleavage of Osteopontin by Matrix Metalloproteinase-12 Modulates Experimental Autoimmune Encephalomyelitis Disease in C57BL/6 Mice

A role for osteopontin (OPN) in promoting disease activity of multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) has recently been suggested. As the biological activity of OPN is heavily influenced by posttranslational processing, we investigated the capacity of matrix metalloproteinase (MMP)-12 to cleave OPN and determined whether this influenced disease activity. We found that OPN mRNA and protein expression in the spinal cord increased with EAE disease in C57BL/6 mice concurrently with MMP-12 expression. A Western blot of EAE and control spinal cords revealed different OPN-immunoreactive bands, with a pattern that was similar to MMP-12 cleavage of recombinant OPN in vitro. In addition, OPN fragments in the spinal cord of EAE-afflicted mice were reduced in MMP-12^−/− mice compared with wild-type controls. However, examination of OPN^−/− mice in short- and long-term experiments revealed no difference in EAE outcomes from wild-type animals. OPN/MMP-12 double null mice were generated, and it was revealed that MMP-12^−/− mice had a worsening of disease compared with wild-type mice, which returned to wild-type levels in the OPN/MMP-12 double null mice. These results suggest that EAE disease activity may be modulated by the cleavage of OPN by MMP-12.Multiple sclerosis (MS) is a disease in which peripheral T cells infiltrate the central nervous system (CNS), where they become re-activated by presentation of CNS antigens by local antigen-presenting cells including microglia and dendritic cells to result in demyelination.^1,2,3,4 In attempts to find putative antigens and mediators of disease, studies using gene array analyses revealed that osteopontin (OPN) was highly up-regulated in lesions from patients with MS compared with control subjects.⁵ This result has prompted the investigations of OPN expression and function in MS and experimental autoimmune encephalomyelitis (EAE).OPN, also known as early T cell activation gene 1 (Eta-1), is a calcium binding phosphorylated acidic glycoprotein.^6,7 OPN has pluripotent activity and is involved in various biological roles such as extracellular matrix remodeling, tumor invasion, angiogenesis, cell-mediated immunity, and the regulation of urokinase and matrix metalloproteinase (MMP) production.⁸ Chabas et al⁵ found that OPN was expressed during EAE in various cell types and that OPN^−/− mice had reduced EAE clinical disease, which was associated with a shift toward a Th2 cytokine profile. Jansson et al⁹ also found that OPN^−/− mice had reduced mean maximal score, fewer days of paralyzing disease, and no spontaneous relapses on proteolipid protein-induced EAE. In that study, OPN^−/− mice showed reduced production of pro-inflammatory cytokines, interferon-γ (IFN-γ), and tumor necrosis factor-α. In contrast to these studies, Blom et al¹⁰ found no difference between OPN^−/− and wild-type mice for EAE outcomes.The EAE results sparked an interest in the expression of OPN in MS. Increased levels of OPN protein is reported in the serum and plasma in patients with relapsing-remitting MS compared with controls,^11,12,13,14 particularly during relapses, and in their cerebrospinal fluid.^15,16 However, there is little evidence for a genetic link between OPN and MS disease susceptibility and disease course.^{11,13,17,18,19,20} Elevated OPN levels have also been documented by immunohistochemistry around MS lesions^21,22 although this was not observed by others.²³ The role that OPN plays in EAE and MS remains uncertain, but a prevailing view is that OPN plays a destructive role during EAE since it reduces apoptosis of effector T cells and because the exogenous administration of recombinant OPN exacerbates EAE clinical disease.²⁴The biological functions of OPN are heavily influenced by posttranslational modifications such as phosphorylation, glycosylation, sulfation, and proteolytic cleavage.^25,26 Currently, there is very little knowledge about the role that proteolytic cleavage of OPN has on MS and EAE. In other disease states, the cleavage of OPN by MMPs and thrombin alters the biological functions of OPN.^27,28 For example, OPN that is cleaved by MMP-3 and MMP-7 significantly increases adhesion of tumor cells compared with full length OPN.²⁹ Recombinant MMP-12 has been demonstrated to cleave OPN, a protein that strongly influences osteoclasts activities, including attachment, spreading, and resorption.³⁰The family of MMPs is implicated in MS and EAE as several MMP members are elevated in biological samples from these conditions. These include MMP-2, -3, -8, -9, -10, -11, -12, -13, -14, and -25.^{31,32,33,34,35,36,37,38} Although the majority of these MMP members appear to have detrimental roles in MS and EAE,^39,40,41 MMP-12 is unique since its absence causes an exacerbation in EAE disease scores that is associated with up-regulation of pro-inflammatory cytokines.^31,42 Since the MMP-12^−/− mice phenotype is opposite to the reduced levels of pro-inflammatory cytokines in OPN^−/− mice with EAE,⁵ we considered the possibility that MMP-12 and OPN are inversely associated. In this article, we have tested the hypothesis that MMP-12 processes OPN to reduce its pro-inflammatory potential in EAE. We describe OPN mRNA and protein expression during EAE and have used a combination of MMP-12 and OPN single and OPN/MMP-12 double null mice to profile their EAE phenotypes.