TNF-alpha increases transcription of Galpha(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 reactivity. We have shown previously that TNF-alpha upregulates the expression of Galpha(i-2) protein without significantly increasing G(s)alpha 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-alpha upregulates Galpha(i-2) protein in these cells. TNF-alpha pretreatment for 48 h increased the expression of Galpha(i-2) protein without significantly altering the Galpha(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 cycloheximide blocked the increase in Galpha(i-2) protein induced by TNF-alpha. Furthermore, TNF-alpha treatment for 12-24 h increased the steady-state level of Galpha(i-2) mRNA without significantly altering Galpha(i-2) mRNA half-life (9.0 +/- 0.75 h for control and 8.9 +/- 1.1 h for TNF-alpha-treated cells). The transcription inhibitor actinomycin D blocked the increase in Galpha(i-2) mRNA induced by TNF-alpha. These observations indicate that the increase in Galpha(i-2) protein induced by TNF-alpha is due to an increased rate of Galpha(i-2) protein synthesis, most likely as a consequence of the transcriptional increase in the steady-state levels of its mRNA.
Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
K
K
Hotta
C A
CA
Hirshman
C W
CW
Emala
D002452:Cell Count / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D005786:Gene Expression Regulation / Q000187:drug effects; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D014132:Trachea / Q000166:cytology / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
D019384:Nucleic Acid Synthesis Inhibitors; D011500:Protein Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin; D003513:Cycloheximide; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins