Tumor necrosis factor-α stimulates fractalkine production by mesangial cells and regulates monocyte transmigration:: Down-regulation by cAMP

Background. Fractalkine is a CX3C chemokine for mononuclear cells that has been implicated in the recruitment and accumulation of monocytes seen in glomerular diseases. We investigated the mechanisms by which tumor necrosis factor (TNF)-alpha stimulates mesangial cell (MC) fractalkine expression, and the effects of MC-derived fractalkine on monocyte transmigration. Methods. Cultured rat MCs were incubated with TNF-alpha, with or without pretreatment with pharmacologic inhibitors of protein kinases or transcriptional factors downstream to TNF-alpha. Fractalkine mRNA and protein were analyzed by Northern and Western blotting. Translocation of nuclear factor (NF)-kappaB was evaluated by immunocytochemical staining. Monocyte transmigration was determined by in vitro chemotaxis assay. Results. TNF-alpha stimulated MC fractalkine mRNA as well as cell-bound and soluble protein expression in a dose- and time-dependent manner. The soluble fractalkine was shed from the cell-bound form via metalloproteinase-dependent cleavage, and mediated in part TNF-alpha-induced monocyte transmigration in vitro. The incubation of MCs with calphostin C [a selective inhibitor of protein kinase C (PKC)] or PD98059 [a selective inhibitor of p42/44 mitogen-activated protein kinase (MAPK) kinase] attenuated TNF-alpha-stimulated fractalkine mRNA and protein expression. Coincubation of MCs with calphostin C and PD98059 resulted in a synergistic inhibition of TNF-alpha-stimulated fractalkine mRNA and protein expression. Incubation of MCs with phorbol myristate acetate (PMA) for four hours resulted in an increase in fractalkine mRNA expression that could be suppressed by calphostin C or depletion of PKC by pretreatment with PMA for 24 hours. Further, activation of PKC-depleted MCs with TNF-alpha stimulated fractalkine mRNA expression that could be blocked by calphostin C. PD 98059, but not calphostin C, inhibited TNF-alpha-activated phospho-p42/44 MAPK and phospho-c-Jun levels, whereas only calphostin C inhibited TNF-alpha-activated phosphorylation of PKCzeta/iota. The incubation of MCs with MG132, a NF-kappaB inhibitor, abolished TNF-alpha-induced degradation of inhibitory protein of NF-kappaB (I-kappaB)alpha, nuclear translocation of NF-kappaB, and fractalkine expression, without affecting phospho-c-Jun levels. In contrast, curcumin, an activating protein (AP)-1 inhibitor, attenuated TNF-alpha-stimulated phospho-c-Jun levels and fractalkine expression without discernible effects on TNF-alpha-induced degradation of I-kappaBalpha or NF-kappaB nuclear translocation. Neither PD 98059 nor calphostin C affected TNF-alpha-induced degradation of I-kappaBalpha or NF-kappaB nuclear translocation. Additional experiments examining the role of cAMP on MC fractalkine expression showed that the incubation of MCs with TNF-alpha and either db-cAMP or forskolin attenuated TNF-alpha-stimulated fractalkine mRNA and protein expression, preceded by attenuation of TNF-alpha-activated phosphorylation of p42/44 MAPK, and c-Jun, but not phosphorylation of PKCzeta/iota or nuclear translocation of NF-kappaB. Conclusion. The present data indicate that TNF-alpha activation of PKCzeta/iota, p42/44 MAPK, c-Jun/AP-1, and p65/NF-kappaB are involved in TNF-alpha-stimulated MC fractalkine expression, with the soluble fractalkine mediating in part the TNF-alpha-induced monocyte transmigration in vitro. Uncoupling of p42/44 MAPK or c-Jun/AP-1 signals may contribute to cAMP inhibition of MC fractalkine expression activated by TNF-alpha.