The heat shock response inhibits RANTES gene expression in cultured human lung epithelium

The chemokine RANTES is thought to be involved in the pathophysiology of inflammation-associated acute lung injury. Although much is known regarding signals that induce RANTES gene expression, relatively few data exist regarding signals that inhibit RANTES gene expression. The heat shock response, a highly conserved cellular defense mechanism, has been demonstrated to inhibit a variety of lung proinflammatory responses. We tested the hypothesis that induction of the heat shock response inhibits RANTES gene expression. Treatment of A549 cells with TNF-alpha induced RANTES gene expression in a concentration-dependent manner. Induction of the heat shock response inhibited subsequent TNF-alpha-mediated RANTES mRNA expression and secretion of immunoreactive RANTES. Transient transfection assays involving a RANTES promoter-luciferase reporter plasmid demonstrated that the heat shock response inhibited TNF-alpha-mediated activation of the RANTES promoter. Inhibition of NF-kappaB nuclear translocation with isohelenin inhibited TNF-alpha-mediated RANTES mRNA expression, indicating that RANTES gene expression is NF-kappaB dependent in A549 cells. Induction of the heat shock response inhibited degradation of the NF-kappaB inhibitory protein, I-kappaBalpha but did not significantly inhibit phosphorylation of I-kappaBalpha. We conclude that the heat shock response inhibits RANTES gene expression by a mechanism involving inhibition of NF-kappaB nuclear translocation and subsequent inhibition of RANTES promoter activation. The mechanism by which the heat shock response inhibits NF-kappaB nuclear translocation involves stabilization of I-kappaBalpha, without significantly affecting phosphorylation of I-kappaBalpha.     

Involvement of double-stranded RNA-activated protein kinase in the synergistic activation of nuclear factor-kappaB by tumor necrosis factor-alpha and gamma-interferon in preneuronal cells

Tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (IFN-gamma) cooperate during a variety of biological responses and ultimately synergistically enhance the expression of genes involved in immune and inflammatory responses. Recently, we demonstrated that IFN-gamma can significantly potentiate TNF-alpha-induced nuclear factor (NF)-kappaB nuclear translocation in neuronal derived and endothelial cell lines. The mechanism by which these two cytokines exert their synergistic effect on NF-kappaB involves the de novo degradation of the NF-kappaB inhibitor, IkappaBbeta. The double-stranded RNA-dependent kinase PKR is IFN-inducible and has been implicated in the activation of NF-kappaB; therefore, we examined the possibility that PKR may play a role in the synergistic activation of NF-kappaB during TNF-alpha/IFN-gamma cotreatment. The PKR inhibitor 2-aminopurine (2-AP) inhibited TNF-alpha/IFN-gamma-induced NF-kappaB nuclear translocation in neuronal derived cells but not in endothelial cells. The induced degradation of IkappaBbeta, which is normally observed upon TNF-alpha/IFN-gamma cotreatment, was blocked completely by 2-AP in neuronal derived cells. Also, 2-AP treatment or overexpression of a catalytically inactive PKR inhibited the TNF-alpha/IFN-gamma-induced synergistic activation of kappaB-dependent gene expression. Our results suggest that the signal generated by IFN-gamma during TNF-alpha/IFN-gamma cotreatment may require PKR to elicit enhanced NF-kappaB activity, and this signal may affect the stability of the IkappaBbeta protein.