TGF-? signaling in A549 lung carcinoma cells: lipid second messengers

Abstract

Transforming growth factor‐β (TGF‐β) is a potent inducer of numerous extracellular matrix components, largely through a transcriptional mechanism. To define the postreceptor signaling pathways used by TGF‐β in the induction of extracellular matrix gene expression, we have utilized the human lung carcinoma cell line, A549, in transfection experiments with the TGF‐β 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‐β signaling. Here we provide evidence that TGF‐β induces a rapid and transient increase in diacylglycerol (DAG) production. When cells transfected with the p3TP‐Lux reporter plasmid are simultaneously treated with TGF‐β and a DAG kinase inhibitor, we observed a higher level of luciferase than with TGF‐β alone. We also find elevated levels of phosphocholine in cells following TGF‐β 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‐β indicating that the bacterial PC‐PLC can mimic the TGF‐β 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‐β. Furthermore, buffering intracellular calcium with the calcium chelating agent BAPTA/AM failed to block TGF‐β induction of the p3TP‐Lux reporter. Thus the TGF‐β signaling pathway appears to involve the production of diacylglycerol but is independent of calcium. J. Cell. Biochem. 78:588–594, 2000.