Ceramide decreases surfactant protein B gene expression via downregulation of TTF-1 DNA binding activity.
Ceramide, a sphingolipid, is an important signaling molecule in the inflammatory response. Mediators of acute lung injury such as TNF-alpha, platelet-activating factor, and Fas/Apo ligand stimulate sphingomyelin hydrolysis to increase intracellular ceramide levels. Surfactant protein B (SP-B), a hydrophobic protein of pulmonary surfactant, is essential for surfactant function and lung stability. In this study we investigated the effects of ceramide on SP-B gene expression in H441 lung epithelial cells. Ceramide decreased SP-B mRNA levels in control and dexamethasone-treated cells after 24-h incubation and inhibition of SP-B mRNA was associated with inhibition of immunoreactive SP-B. In transient transfections assays, ceramide inhibited SP-B promoter activity, indicating that the inhibitory effects are exerted at the transcriptional level. Deletion mapping experiments showed that the ceramide-responsive region is located within the -233/-80-bp region of human SP-B promoter. Electrophoretic mobility shift and reporter assays showed that ceramide reduced the DNA binding activity and transactivation capability of thyroid transcription factor 1 (TTF-1/Nkx2.1), a key factor for SP-B promoter activity. Collectively these data showed that ceramide inhibits SP-B gene expression by reducing the DNA biding activity of TTF-1/Nkx2.1 transcription factor. Protein kinase C inhibitor bisindolylmaleimide and the protein tyrosine kinase inhibitor genistein partially reversed ceramide inhibition, indicating that protein kinases play important roles in the ceramide inhibition of SP-B gene expression. Chemical inhibitors of de novo ceramide synthesis and sphingomyelin hydrolysis had no effect on TNF-alpha inhibition of SP-B promoter activity and mRNA levels, suggesting that ceramide does not play a role in the inhibition.
Sparkman
Loretta
L
Dept. of Molecular Biology, University of Texas Health Center at Tyler, TX 75708-3154, USA.
Chandru
Hemakumar
H
Boggaram
Vijayakumar
V
D004247:DNA / Q000378:metabolism*; D015536:Down-Regulation; D005786:Gene Expression Regulation; D051541:Hepatocyte Nuclear Factor 3-alpha / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms; D008246:Lysophospholipids / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011494:Protein Kinases / Q000502:physiology; D037701:Pulmonary Surfactant-Associated Protein B / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000187:drug effects; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000502:physiology; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D051541:Hepatocyte Nuclear Factor 3-alpha; D008246:Lysophospholipids; C064769:N-acetylsphingosine; C000614412:NKX2-1 protein, human; D009687:Nuclear Proteins; D037701:Pulmonary Surfactant-Associated Protein B; D012333:RNA, Messenger; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; C060506:sphingosine 1-phosphate; D004247:DNA; D011494:Protein Kinases; D013110:Sphingosine