Fractalkine is a CX3C chemokine for mononuclear leukocytes that is expressed mainly by vascular cells, and regulated by pro-inflammatory cytokines. This study investigated signal transduction mechanisms by which tumor necrosis factor (TNF)-α stimulated fractalkine expression in cultured rat vascular smooth muscle cells (VSMCs), and the modulatory effect of a haemorrheologic agent, pentoxifylline, on its production.
TNF-α (1–50 ng ml−1) stimulated fractalkine mRNA and protein expression in concentration- and time-dependent manners. Pretreatment with calphostin C (0.4 μM, a selective inhibitor of protein kinase C (PKC), and PD98059 (40 μM), a specific inhibitor of p42/44 mitogen-activated protein kinase (MAPK) kinase, attenuated TNF-α-stimulated fractalkine mRNA and protein expression. In contrast, H-89 (2 μM), a selective inhibitor of cAMP-dependent protein kinase, wortmannin (0.5 μM), a selective inhibitor of phosphatidylinositol 3-kinase, and SB203580 (40 μM), a specific inhibitor of p38 MAPK, had no discernible effect.
The ubiquitin/proteosome inhibitors, MG132 (10 μM) and pyrrolidine dithiocarbamate (200 μM), suppressed activation of NF-κB as well as stimulation of fractalkine mRNA and protein expression by TNF-α.
TNF-α-activated phosphorylation of PKC was blocked by calphostin C, whereas TNF-α-augmented phospho-p42/44 MAPK and phospho-c-Jun levels were reduced by PD98059. Neither calphostin C nor PD98059 affected TNF-α-induced degradation of I-κBα or p65 nuclear translocation.
Pretreatment with pentoxifylline (0.1–1 mg ml−1) decreased TNF-α-stimulated fractalkine mRNA and protein expression, which was preceded by a reduction in TNF-α-activated phosphorylation of PKC, p42/44 MAPK and c-Jun as well as degradation of I-κBα and p65/NF-κB nuclear translocation.
These data indicate that activation of PKC, p42/44 MAPK kinase, and NF-κB are involved in TNF-α-stimulated fractalkine production in VSMCs. Down-regulation of the PKC, p42/44 MAPK, and p65/NF-κB signals by PTX may be therapeutically relevant and provide an explanation for the anti-fractalkine effect of this drug.
British Journal of Pharmacology (2003) 138, 950–958. doi:10.1038/sj.bjp.0705088