title: TGF-beta signaling in A549 lung carcinoma cells: lipid second messengers.
abstract: Transforming growth factor-beta (TGF-beta) is a potent inducer of numerous extracellular matrix components, largely through a transcriptional mechanism. To define the postreceptor signaling pathways used by TGF-beta in the induction of extracellular matrix gene expression, we have utilized the human lung carcinoma cell line, A549, in transfection experiments with the TGF-beta inducible reporter construct, p3TP-Lux. Previous work from this laboratory using pharmacologic agents suggested that a phosphatidylcholine-specific phospholipase C and protein kinase C may be involved in early aspects of TGF-beta signaling. Here we provide evidence that TGF-beta induces a rapid and transient increase in diacylglycerol (DAG) production. When cells transfected with the p3TP-Lux reporter plasmid are simultaneously treated with TGF-beta and a DAG kinase inhibitor, we observed a higher level of luciferase than with TGF-beta alone. We also find elevated levels of phosphocholine in cells following TGF-beta treatment. Further, exogenously added bacterial phosphatidylcholine phospholipase C (PC-PLC) is capable of inducing expression of the p3TP-Lux reporter to the same extent as TGF-beta indicating that the bacterial PC-PLC can mimic the TGF-beta effect. In contrast, neither hexanoyl sphingosine (a ceramide analogue) nor arachadonic acid induce expression of the p3TP-Lux reporter. Measurements with the fluorescent, calcium-sensitive dye, FURA2, indicated that there was no change in intracellular calcium in response to TGF-beta. Furthermore, buffering intracellular calcium with the calcium chelating agent BAPTA/AM failed to block TGF-beta induction of the p3TP-Lux reporter. Thus the TGF-beta signaling pathway appears to involve the production of diacylglycerol but is independent of calcium.
authors.affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts, 01655, USA. Ronald.Ignotz@umassmed.edu
authors.forename: R A
authors.initials: RA
authors.lastname: Ignotz
authors.forename: T
authors.initials: T
authors.lastname: Honeyman
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D016718:Arachidonic Acid / Q000494:pharmacology; D001952:Bridged-Ring Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D004075:Diglycerides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008055:Lipids / Q000502:physiology*; D008175:Lung Neoplasms / Q000378:metabolism*; D009636:Norbornanes; D010713:Phosphatidylcholines / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D015290:Second Messenger Systems / Q000502:physiology*; D013110:Sphingosine / Q000494:pharmacology; D013859:Thiocarbamates; D013871:Thiones / Q000494:pharmacology; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism
chemical_list: D000098:Acetophenones; D001952:Bridged-Ring Compounds; D002614:Chelating Agents; D004075:Diglycerides; D004791:Enzyme Inhibitors; D008055:Lipids; D009636:Norbornanes; D010713:Phosphatidylcholines; D010726:Phosphodiesterase Inhibitors; D020033:Protein Isoforms; D013859:Thiocarbamates; D013871:Thiones; D016212:Transforming Growth Factor beta; D016718:Arachidonic Acid; D004533:Egtazic Acid; C046498:tricyclodecane-9-yl-xanthogenate; D010738:Type C Phospholipases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D013110:Sphingosine; C006821:4-bromophenacyl bromide; D002118:Calcium