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

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.

SUBMITTER: Bai G

PROVIDER: S-EPMC5855640 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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

Bai Gaowa G Motoda Hirotoshi H Ozuru Ryo R Chagan-Yasutan Haorile H Hattori Toshio T Matsuba Takashi T

International journal of molecular sciences 20180131 2

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 antibod ...[more]

PMID: 29385060

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Project description:BackgroundThe activation of NLRP3 inflammasome in macrophages has been proven to play a crucial role in the development of cardiovascular diseases. THP-1 monocytes can be differentiated to macrophages by incubation with phorbol-12-myristate 13-acetate (PMA), providing a suitable model for in vitro studies. However, PMA has been shown to have effects on the levels of IL-1β, the main mediator of NLRP3 inflammasome, while the effects on the other mediators of the inflammasome have not been reported before.MethodsTHP-1 monocytes were incubated without (THP-1), with 5ng/ml PMA for 48h (PMA48h) or with 5ng/ml PMA for 48h plus 24h in fresh medium (PMArest). Morphological changes and the expression of macrophage surface markers (CD14, CD11b, CD36 and CD204) were evaluated by flow cytometry. Changes in intracellular levels of inflammasome components (NLRP3, ASC, pro-caspase-1, pro-IL1β) were analyzed by western blot and release of mature IL-1β in cell supernatant was analyzed by ELISA. ASC speck formation was determined by immunofluorescence.ResultsAfter 48h incubation with PMA or subsequent rest in fresh medium, cells became adherent, and the differential expression of CD36, CD11b, CD14 and CD204 compared to THP-1 cells confirmed that PMArest resemble macrophages from a molecular point of view. Changes in the levels were detected in PMA48h group for all the NLRP3-related proteins, with increase of NLRP3 and pro-IL-1β and secretion of mature IL-1β. In PMArest, no pro-IL-1β and lower amounts of mature IL-1β were detected. No ASC speck was found in PMA treated groups, but the addition of a second stimulus to PMArest resulted in ASC speck formation, together with IL-1β production, confirming the responsiveness of the model.ConclusionDifferentiation of THP-1 with 5ng/ml PMA followed by 24h resting period provides a model that morphologically and molecularly resembles macrophages. However, even at low concentrations, PMA induces production of IL-1β. The 24h rest period provides for down-regulation of pro-IL-1β in PMArest group, without affecting its ability to respond to a second stimulus through activation of inflammasome.

Project description:Nasu-Hakola disease (NHD), also designated polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), is a rare autosomal recessive disorder characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by a loss-of-function mutation of DAP12 or TREM2. TREM2 and DAP12 constitute a receptor/adaptor complex expressed on osteoclasts, dendritic cells, macrophages, monocytes, and microglia. At present, the precise molecular mechanisms underlying development of leukoencephalopathy and bone cysts in NHD remain largely unknown. We established THP-1 human monocyte clones that stably express small interfering RNA (siRNA) targeting DAP12 for serving as a cellular model of NHD. Genome-wide transcriptome analysis identified a set of 22 genes consistently downregulated in DAP12 knockdown cells. They constituted the molecular network closely related to the network defined by cell-to-cell signaling and interaction, hematological system development and function, and inflammatory response, where NF-kappaB acts as a central regulator. These results suggest that a molecular defect of DAP12 in human monocytes deregulates the gene network pivotal for maintenance of myeloid cell function in NHD. We found that both DAP12 knockdown and control clones were capable of equally responding to phorbol 12-myristate 13-acetate (PMA), a known inducer of morphological differentiation of THP-1 cells, by exhibiting almost similar gene expression profiles between both, following a 24-hour exposure to 50 nM PMA. The siRNA vector construct targeting the DAP12 sequence (SI) and the control vector construct targeting the scrambled sequence (SCR) were generated by using GeneClip U1 Hairpin cloning system (Promega). The vectors were transfected in THP-1 cells by using Lipofectamine LTX reagent. The stable cell lines were selected by incubating them for approximately two months in the feeding medium with inclusion of 200 microgram/ml Hygromycin B. Then, two SI clones named SI5 and SI17, in addition to two SCR clones named SCR1 and SCR4, were selected by limiting dilution of the cells in a manner of a single cell per well plated in a 96-well cell culture plate. Microarray data for clones SCR4 and SI17 submitted in this Series.

Dataset Information

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

Publications

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

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