Inhibition by pentoxifylline of TNF-α-stimulated fractalkine production in vascular smooth muscle cells: Evidence for mediation by NF-κB down-regulation

1. Fractalkine is a CX ₃C 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. 2. 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. 3. The ubiquitin/proteosome inhibitors, MG132 (10 αM) and pyrrolidine dithiocarbamate (200 αM), suppressed activation of NF-κB as well as stimulation of fractalkine mRNA and protein expression by TNF-α. 4. 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. 5. 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-αBα and p65/NF-κB nuclear translocation. 6. 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.