Titanium particles stimulate COX-2 expression in synovial fibroblasts through an oxidative stress-induced, calpain-dependent, NF-κB pathway

Abstract

In prosthetic loosening, bone resorption is induced by wear debris particles generated from the artificial joint articulation. Our prior work showed that synovial-like fibroblasts respond to titanium particles by producing receptor activator of NF-kappaB ligand (RANKL), a critical activator of osteoclastogenesis. While this effect occurs through a cyclooxygenase-2 (COX-2)-dependent pathway, the mechanism of COX-2 stimulation by titanium particles is not clear. Here we show that titanium particles induce COX-2 gene expression by activating NF-kappaB signaling. Inhibitor of NF-kappaB (IkappaBalpha) is degraded following particle treatment, permitting active NF-kappaB to translocate to the nucleus where it interacts with the COX-2 promoter and drives transcription. NF-kappaB activation is dependent on reactive oxygen species since antioxidants block the NF-kappaB signaling induced by particles. Surprisingly, IkappaBalpha degradation is independent of IKK (IkappaB kinase) and the 26S proteasome. Instead, calpain inhibitor can block the IkappaBalpha degradation induced by particles. Furthermore, the calpain-targeted COOH-terminal PEST sequence of IkappaBalpha is necessary for phosphorylation and degradation, consistent with a proteasome-independent mechanism of catabolism. Altogether, the data demonstrate a signaling pathway by which titanium particles induce oxidative stress, stimulate calpain-mediated NF-kappaB activation, and activate target gene expression, including COX-2. These findings define important targets for osteolysis but may also have importance in other diseases where fibroblasts respond to environmental particles, including pulmonary diseases.

Strontium inhibits titanium particle-induced osteoclast activation and chronic inflammation via suppression of NF-κB pathway

“…It was reported that NF-κB up-regulated TNF-α and IL-1β expression by monocytes32. Meanwhile, previous studies demonstrated that NF-κB pathway was activated by wear particles and subsequently promoted pro-inflammatory cytokines expression933. In addition, blockade NF-κB activity results in a significant reduction in pro-inflammatory cytokines.…”

Comparison of the cytotoxic and inflammatory responses of titanium particles with different methods for endotoxin removal in RAW264.7 macrophages

“…As for the ethanol-treated particles, the activation of NF-jB nuclear translocation and the production of proinflammatory cytokines are significantly greater than in the other groups when more endotoxins present on the particles. This observation is the reason that many studies have indicated that the wear particles could activate NF-jB nuclear translocation even when all particle preparations were shown to be free of endotoxin by LAL-based assay in their studies [33][34][35]. However, another paper also demonstrated that titanium-alloy particles without endotoxin did not induce NF-jB activation [36].…”

“…These results have minimized the influence of endotoxins on the particles because all particle preparations were shown to be free of endotoxin by LAL-based assay [33][34][35].However, other research has also demonstrated that the titanium-alloy particles without endotoxins did not induce NF-jB activation [36]. To examine NF-jB activation, RAW264.7 cells that were stably transfected with a luciferase reporter gene p-NF-jB-TA-Luc [37] were plated in 24-well plates at a density of 1 9 10 5 cells/well and treated with three groups of particles for 8 h. The cells were harvested, and the luciferase activity was measured using the Promega Luciferase Assay System according the manufacturer's instructions (Promega).…”

Bilirubin Inhibits Neointima Formation and Vascular Smooth Muscle Cell Proliferation and Migration

“…Cells were incubated with trypsin (0.25%), collected, diluted (1:4) with trypan blue, and examined by microscopy. Viability was assessed by the fraction of cells that excluded the dye, as previously reported (Wei et al, 2009). …”

“…These findings were subsequently extended to show that bilirubin reacts with other reactive oxygen and nitrogen species including hypochlorous acid, singlet oxygen, hydroxyl radical, nitric oxide, and peroxynitrite (see Stocker, 2004). Indeed, numerous studies have demonstrated a cytoprotective role for bilirubin in response to various oxidative insults (Yamaguchi et al, 1996; Clark et al, 2000; Dore et al, 2009; Wei et al, 2009; Jansen et al, 2010). In addition, bilirubin exerts critical anti-inflammatory actions in the vasculature.…”