TNF-alpha increases transcription of G alpha(i-2) in human airway smooth muscle cells

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that has an important role in the regulation of airway smooth muscle tone and re activity. We have shown previously that TNF-alpha upregulates the expressio n of Gai-2 protein without significantly increasing G(s)alpha protein and e nhances adenylyl cyclase inhibition by carbachol in cultured human airway s mooth muscle cells (Hotta K, Emala CW, and Hirshman CA. Am J Physiol Lung C ell Mol Physiol 276: L405-L411, 1999). The present study was designed to in vestigate the molecular mechanisms by which TNF-alpha upregulates G alpha(i -2) protein in these cells. TNF-alpha pretreatment for 48 h increased the e xpression of G alpha(i-2) protein without significantly altering the G alph a(i-2) protein half-life (41.0 +/- 8.2 h for control and 46.8 +/- 5.2 h for TNF-alpha-treated cells). Inhibition of new protein synthesis by cyclohexi mide blocked the increase in G alpha(i-2) protein induced by TNF-alpha. Fur thermore, TNF-alpha treatment for 12-24 h increased the steady-state level of G alpha(i-2) mRNA without significantly altering G alpha(i-2) mRNA half- life (9.0 +/- 0.75 h for control and 8.9 +/- 1.1 h for TNF-alpha-treated ce lls). The transcription inhibitor actinomycin D blocked the increase in G a lpha(i-2) mRNA induced by TNF-alpha. These observations indicate that the i ncrease in G alpha(i-2) protein induced by TNF-alpha is due to an increased rate of G alpha(i-2) protein synthesis, most likely as a consequence of th e transcriptional increase in the steady-state levels of its mRNA.