Basal and induced sphingosine kinase 1 activity in A549 carcinoma cells: function in cell survival and IL-1beta and TNF-alpha induced production of inflammatory mediators.
Sphingosine-1-phosphate, a lipid mediator produced by sphingosine kinases, regulates diverse cellular processes, ranging from cell growth and survival to effector functions, such as proinflammatory mediator synthesis. Using human A549 epithelial lung carcinoma cells as a model system, we observed transient upregulation of sphingosine kinase type 1 (SPHK1) enzyme activity upon stimulation with both TNF-alpha or IL-1beta. This transient activation of SPHK1 was found to be required for cytokine-induced COX-2 transcription and PGE2 production, since not only specific siRNA (abolishing both basal and induced SPHK1 enzyme activity), but also a dominant-negative SPHK1 mutant (suppressing induced SPHK1 activity only) both reduced COX-2 and PGE2. Furthermore, TNF-alpha- or IL-1beta-induced transcription of selected cytokines, chemokines, and adhesion molecules (IL-6, RANTES, MCP-1, and VCAM-1) was found to require SPHK1 activation. Suppression of SPHK1 activation led to reduction of cytokine-induced IkappaBalpha phosphorylation and consequently diminished NFkappaB activity due to reduced nuclear translocation of RelA (p65), explaining the dependence of inflammatory mediator production on SPHK1 activation. Inhibition of basal SPHK1 activity by N,N-dimethylsphingosine or by downregulation of its expression using siRNA induced spontaneous apoptosis in A549 cells, an effect that can be explained through interference with constitutive NFkappaB activity in this cell type. In contrast, expression of the dominant-negative mutant did not induce apoptosis. Taken together, these findings demonstrate a role of SPHK1 activation in proinflammatory signalling and of SPHK1 basal activity in survival of A549 lung carcinoma cells.
Billich
Andreas
A
Novartis Institutes for BioMedical Research, Brunnerstrasse 59, Vienna A-1235, Austria. andreas.billich@pharma.novartis.com
Bornancin
Frédéric
F
Mechtcheriakova
Diana
D
Natt
François
F
Huesken
Dieter
D
Baumruker
Thomas
T
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D016718:Arachidonic Acid / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D018946:Chemokine CCL5 / Q000235:genetics / Q000378:metabolism; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D018836:Inflammation Mediators / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology*; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D008565:Membrane Proteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D010766:Phosphorylation / Q000187:drug effects; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics; D011498:Protein Precursors / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D051996:Transcription Factor RelA; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics; D000099157:Sphingosine Kinase
D016718:Arachidonic Acid; D018932:Chemokine CCL2; D018946:Chemokine CCL5; D051546:Cyclooxygenase 2; D015232:Dinoprostone; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D018836:Inflammation Mediators; D007375:Interleukin-1; D015850:Interleukin-6; D015759:Ionomycin; D008565:Membrane Proteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D017853:Phosphotransferases (Alcohol Group Acceptor); D011451:Prostaglandin-Endoperoxide Synthases; D011498:Protein Precursors; D034741:RNA, Small Interfering; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D000099157:Sphingosine Kinase; C111835:CCL2 protein, human; C000606204:NFKBIA protein, human; C016658:phorbolol myristate acetate; C496540:PTGS2 protein, human; C066324:ceramide kinase; C061800:N,N-dimethylsphingosine