Content area
Full text
Introduction
A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) are secretory proteins that are involved in a variety of biological processes, such as angiogenesis, cell adhesion, proteolytic shedding and cell signaling. ADAMTS type 1 motif 9 (ADAMTS9) is involved in proteoglycan degradation (1,2). IL-1β was found to induce ADAMTS9 gene expression in OUMS-27 chondrosarcoma cells in a previous investigation (3). ADAMTS9 gene expression was synergistically induced by a combination of IL-1β and tumor necrosis factor α (TNF α), suggesting that the induction of ADAMTS9 may be associated with cartilage inflammation (4). The human ADAMTS9 promoter region contains nuclear factor of activated T cells c1 (NFATc1) consensus sites. Following treatment with IL-1β, NFATc1 was activated in human chondrocytic cells (5). A previous investigation demonstrated that, following treatment with a combination of TNF and IL-1β, the expression of activated activator protein 1 and NF-κB transcription factors was enhanced in human chondrocytic cells (6).
NF-κB is a pro-inflammatory transcription factor, the expression of which is activated by inflammatory cytokines such as TNF α and IL-1, and a number of chemokines (7–9). NF-κB activation may occur via classical or canonical pathways (10). NF-κB is composed of homodimers and heterodimers of five members of the Rel family, which exhibit different binding specificities, including p65/RelA, RelB, c-Rel, p50/p105 and p52/p100. One of the predominant types of heterodimers consists of p65 and p50 subunits. NF-κB is typically found in the cytoplasm. The nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor (IκB) kinase complex (IKK complex) is composed of two catalytic subunits (IKKα and IKKβ). The IKK complex binds with the regulatory subunit IKKγ/NF-κB essential modulator, which subsequently forms the TNF-α receptor complex, and promotes IκB phosphorylation. Phosphorylated IκB-α is rapidly ubiquitinated and degraded via a proteasome pathway. Degradation of IκB-α leads to the expression of NF-κB, which translocates into the nucleus where it binds to specific binding sites within the promoter regions of target genes (9).
In the present study, the association between NF-κB and IL-1β stimulation was examined, and the involvement of NF-κB and IL-1β in ADAMTS9 promoter activation was analyzed in OUMS-27 cells.
Materials and methods
Antibodies and reagents
Mouse monoclonal antibodies against the phosphorylated NF-κB-p65 subunit (sc-33020; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), total NF-κB-p65...