Synthesis of a Cleaved Form of Osteopontin by THP-1 Cells and Its Alteration by Phorbol 12-Myristate 13-Acetate and BCG Infection

• Published in: International journal of molecular sciences 2018-01, Vol.19 (2), p.418
• Main Authors: Bai, Gaowa, Motoda, Hirotoshi, Ozuru, Ryo, Chagan-Yasutan, Haorile, Hattori, Toshio, Matsuba, Takashi
• Format: Article
• Language: English
• Subjects: Acetic acid; Bacillus Calmette-Guerin vaccine; BCG; Biocompatibility; Cleavage; Enzyme-linked immunosorbent assay; Fluorescence; Humans; Immunological memory; Infections; Lymphocytes; Lymphocytes T; Macrophages; Macrophages - drug effects; Macrophages - metabolism; Memory cells; Monocytes; Mycobacterium bovis - physiology; N-Terminus; Osteopontin; Osteopontin - metabolism; Phorbol 12-myristate 13-acetate; protease; Protein Processing, Post-Translational; Stimulation; Tetradecanoylphorbol Acetate - pharmacology; THP-1; THP-1 Cells; Thrombin; Thrombin - metabolism; Tuberculosis; Western blotting
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms19020418

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.