Abstract
The protease-cleaved osteopontin (OPN) was proposed to enhance the migration of memory T cells to granulomas in tuberculosis. Various forms of OPN were identified in human monocytic THP-1 cells stimulated by phorbol 12-myristate 13-acetate (PMA). Antibodies O-17, 10A16 and 34E3, which recognize N-terminus, the C-half, and thrombin-cleaved site of OPN, respectively, all detected distinct bands on Western blots following PMA stimulation. Bands corresponding to 18 and 30 kD were detected by antibodies 34E3 and 10A16, indicating that OPN cleavage occurred by endogenous proteases in the PMA-stimulated THP-1 cells. In immune-fluorescence (IF) assay, 34E3 positive signals were detected in intracellular space of non-infected and bacillus Calmette-Guérin (BCG)-infected cells; however, 10A16 positive signals were confirmed in extracellular area in PMA-stimulated cells followed by BCG infection. Small amounts of full-length (FL) and thrombin-cleaved (Tr) OPN were detected by ELISA in the supernatants of non-PMA-stimulated cells, and increased levels of all forms, including undefined (Ud) OPN, in PMA-stimulated cells. ELISA showed a decrease in OPN synthesis during BCG infection. To our knowledge, this is the first report of OPN cleavage in THP-1 macrophages after PMA stimulation, and of enhanced cleavage induced by BCG infection.
Highlights
Matricellular proteins (MCPs) constitute a family of secreted extracellular matrix (ECM) proteins that influence cell–matrix interactions [1]
Other OPN forms are detected in dengue virus (DENV) infections using a different ELISA system, which include a mixture of Full-length OPN (FL-OPN), thrombin-cleaved OPN (trOPN) and undefined OPN (Ud-OPN) [9]
Polyclonal rabbit antibody O-17 is specific to the N-terminus of OPN (Ile17–Gln31), and anti-trOPN monoclonal antibody 34E3 is specific to the epitope Ser162–Arg168, which is exposed by thrombin digestion [9,16]
Summary
Matricellular proteins (MCPs) constitute a family of secreted extracellular matrix (ECM) proteins that influence cell–matrix interactions [1]. Based on this definition, several proteins have been identified as MCPs, including connective-tissue growth factors, galectins [2] and osteopontin (OPN) [3]. Full-length OPN (FL-OPN), the intact form of OPN, is involved in the complex pathways of coagulation and fibrinolysis, where multiple sites of FL-OPN serve as targets for protease(s) cleavage. During this process, OPN fragments are produced. Other OPN forms are detected in DENV infections using a different ELISA system, which include a mixture of FL-OPN, trOPN and undefined OPN (Ud-OPN) [9]
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