MABT1517-25UG Sigma-AldrichAnti-Osteopontin Antibody, clone 2A1

Osteopontin (OPN) is a pleiotropic protein implicated in various inflammatory responses including ischemia-reperfusion (I-R) injury. Two distinct forms of the protein have been identified: an extensively studied secreted form (sOPN) and a less-well-known intracellular form (iOPN). Studies have shown that increased OPN expression parallels the time course of macrophage infiltration into injured tissue, a late event in the development of cerebral infarcts. sOPN has been suggested to promote remodeling of the extracellular matrix in the brain; the function of iOPN may be to facilitate certain signal transduction processes. Here, we studied OPN expression in adult male Sprague-Dawley rats subjected to global forebrain I-R injury. We found iOPN in the cytoplasm of both cortices and the hippocampus, but unexpectedly only the right cortex exhibited a marked increase in the iOPN level after 45 min of reperfusion. Acetaminophen, a drug recently shown to decrease apoptotic incidence, caspase-9 activation, and mitochondrial dysfunction during global I-R, significantly inhibited the increase in iOPN protein in the right cortex, suggesting a role for iOPN in the response to I-R injury in the right cortex.

Osteopontin (OPN) is a cytokine implicated in mediating responses to certain stressors, including mechanical, oxidative, and cellular stress. However, the involvement of OPN in responding to other physical and psychological stress is largely unexplored. Our previous research revealed that OPN is critical for hind limb-unloading induced lymphoid organ atrophy through modulation of corticosteroid production. In this study, we demonstrate that OPN(-/-) mice are resistant to chronic restraint stress (CRS)-induced lymphoid (largely thymus) organ atrophy; additionally, the stress-induced up-regulation of corticosterone production is significantly reduced in OPN(-/-) mice. Underlying this observation is the fact that normal adrenocorticotropic hormone levels are substantially reduced in the OPN(-/-) mice. Our data demonstrate both that injection of OPN into OPN-deficient mice enhances the CRS-induced lymphoid organ atrophy and that injection of a specific anti-OPN mAb (2C5) into wild-type mice ameliorates the CRS-induced organ atrophy; changes in corticosterone levels were also partially reversed. These studies reveal that circulating OPN plays a significant role in the regulation of the hypothalamus-pituitary-adrenal axis hormones and that it augments CRS-induced organ atrophy.

Osteopontin (OPN) is primarily a secreted phosphoglycoprotein found in a variety of tissues and body fluids. It has a wide range of reported functions, many of which are affected by the degree of post-translational modification (PTM) of the protein. These PTMs include phosphorylation, glycosylation, and cross-linking by transglutaminase. Here we describe the generation of unique monoclonal antibodies raised against recombinant OPN utilizing the OPN knockout mouse. The antibodies exhibit differential binding to OPN produced by different cell lines from the same species, as well to the multiple OPN forms in human urine. Most of the antibodies generated are able to recognize OPN produced by ras-transformed mouse fibroblasts, however only one antibody recognizes the more phosphorylated protein produced by the differentiating pre-osteoblast murine cell line MC3T3E1. Using a novel biopanning procedure combining T7 phage gene fragment display and protein G precipitation, we have epitope-mapped these antibodies. Several of the antibodies bind to regions of the OPN molecule that are phosphorylated, and one binds the region of OPN that is glycosylated. Using phosphorylated and non-phosphorylated peptides, we show that the binding of two antibodies to the C-terminal end of OPN is inhibited by phosphorylation of this region. In addition, these two antibodies are able to inhibit cell adhesion to recombinant and weakly modified OPN. The antibodies described herein may prove useful in determining the presence of modifications at specific sites and for identifying structural forms of OPN. Also, the sensitivity of these antibodies to PTMs suggests that caution must be taken when choosing anti-OPN monoclonal antibodies to detect this highly modified protein.