TNF-α increases transcription of Gαi-2 in human airway smooth muscle cells

Abstract

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that has an important role in the regulation of airway smooth muscle tone and reactivity. We have shown previously that TNF-α upregulates the expression of Gαi-2 protein without significantly increasing Gsα protein and enhances adenylyl cyclase inhibition by carbachol in cultured human airway smooth muscle cells (Hotta K, Emala CW, and Hirshman CA. Am J Physiol Lung Cell Mol Physiol 276: L405–L411, 1999). The present study was designed to investigate the molecular mechanisms by which TNF-α upregulates Gαi-2 protein in these cells. TNF-α pretreatment for 48 h increased the expression of Gαi-2 protein without significantly altering the Gαi-2 protein half-life (41.0 ± 8.2 h for control and 46.8 ± 5.2 h for TNF-α-treated cells). Inhibition of new protein synthesis by cycloheximide blocked the increase in Gαi-2 protein induced by TNF-α. Furthermore, TNF-α treatment for 12–24 h increased the steady-state level of Gαi-2 mRNA without significantly altering Gαi-2 mRNA half-life (9.0 ± 0.75 h for control and 8.9 ± 1.1 h for TNF-α-treated cells). The transcription inhibitor actinomycin D blocked the increase in Gαi-2 mRNA induced by TNF-α. These observations indicate that the increase in Gαi-2 protein induced by TNF-α is due to an increased rate of Gαi-2 protein synthesis, most likely as a consequence of the transcriptional increase in the steady-state levels of its mRNA.