Title: Intracellular Signaling in Rat Cultured Vascular Smooth Muscle Cells: Roles of Nuclear Factor- B and p38 Mitogen-Activated Protein Kinase on Tumor Necrosis Factor- Production
Abstract: Lipopolysaccharide (LPS) is responsible for initiating host responses leading to septic shock, and tumor necrosis factor-α (TNFα) is thought to be its primary mediator. In addition, TNFα is one of the major components of the pathogenesis of insulin resistance in various conditions. It has been shown that LPS induced TNFα production in rat vascular smooth muscle cells (VSMC). However, little is known about the signaling pathway by which VSMC in culture produce TNFα. We investigated the possible signaling components involved in this pathway. LPS elicited phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) and p38 MAPK, degradation of inhibitor of κB (IκB), and an increase in nuclear binding activity of activating protein-1 and nuclear factor-κB (NF-κB). Different types of NF-κB inhibitors, pyrrolidine dithiocarbamate and MG132, which specifically abolished IκB degradation and subsequent NF-κB activation by LPS, suppressed TNFα secretion from VSMC. Although PD98059, a specific MAPK kinase inhibitor and SB203580, a specific p38 MAPK inhibitor, had no effect on NF-κB activity, SB203580 suppressed TNFα secretion; however, PD98059 did not. A cotransfection assay showed that transfection of dominant negative IκB or pretreatment with SB203580 suppressed the TNFα gene promotor-dependent transcription. TNFα messenger RNA expression induced by LPS was inhibited by pyrrolidine dithiocarbamate, MG132, and SB203580, but not by PD98059. These observations indicate that TNFα production in VSMC is stimulated by LPS, and its transcription and translation are dependent on NF-κB activation through proteasome-mediated IκB degradation. It is likely that p38 MAPK may play a critical role in regulating transcription of the TNFα gene in VSMC, unlike in other cell lines.