d-609 and 4-bromophenacyl-bromide

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.

Topics: Acetophenones; Arachidonic Acid; Bridged-Ring Compounds; Calcium; Chelating Agents; Chromatography, Thin Layer; Diglycerides; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Inhibitors; Extracellular Matrix; Humans; Hydrolysis; Lipids; Lung Neoplasms; Norbornanes; Phosphatidylcholines; Phosphodiesterase Inhibitors; Plasmids; Protein Isoforms; Second Messenger Systems; Sphingosine; Thiocarbamates; Thiones; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Type C Phospholipases