Intracellular signaling in rat cultured vascular smooth muscle cells: Roles of nuclear factor-kappa B and p38 mitogen-activated protein kinase on tumor necrosis factor-alpha production

Lipopolysaccharide (LPS) is responsible for initiating host responses leadi ng to septic shock, and tumor necrosis factor-alpha (TNF alpha) is thought to be its primary mediator. In addition, TNF alpha is one of the major comp onents of the pathogenesis of insulin resistance in various conditions. It has been shown that LPS induced TNF alpha production in rat vascular smooth muscle cells (VSMC). However, little is known about the signaling pathway by which VSMC in culture produce TNF alpha. We investigated the possible si gnaling components involved in this pathway. LPS elicited phosphorylation o f p42/44 mitogen-activated protein kinase (MAPK) and p38 MAPK, degradation of inhibitor of kappa B (I kappa B), and an increase in nuclear binding act ivity of activating protein-1 and nuclear factor-kappa B (NF-kappa B). Diff erent types of NF-kappa B inhibitors, pyrrolidine dithiocarbamate and MG132 , which specifically abolished I kappa B degradation and subsequent NF-kapp a B activation by LPS, suppressed TNF alpha secretion from VSMC. Although P D98059, a specific MAPK kinase inhibitor and SB203580, a specific p38 MAPK inhibitor, had no effect on NF-kappa B activity, SB203580 suppressed TNF al pha secretion; however, PD98059 did not. A cotransfection assay showed that transfection of dominant negative I kappa B or pretreatment with SB203580 suppressed the TNF alpha gene promotor-dependent transcription. TNF alpha m essenger RNA expression induced by LPS was inhibited by pyrrolidine dithioc arbamate, MG132, and SB203580, but not by PD98059. These observations indic ate that TNF alpha production in VSMC is stimulated by LPS, and its transcr iption and translation are dependent on NF-kappa B activation through prote asome-mediated I kappa B degradation. It is likely that p38 MAPK may play a critical role in regulating transcription of the TNF alpha gene in VSMC, u nlike in other cell lines.