The rise and fall of ceramide and 1,2-diacylglycerol (DAG): Modulation by transforming growth factor-β1 (TGFβ1) and by epidermal growth factor (EGF)

Transforming growth factor β1 (TGFβ1) increases the phosphorylation of the epidermal growth factor (EGF) receptor and inhibits the growth of A431 cells, but the mechanism of TGFβ1 signaling is unknown. Recent studies from this and other laboratories suggest a novel sphingomyelin signal transduction pathway (1-4). Ceramide, which is generated by sphingomyelinase action, can be deacylated to sphingoid bases, which are potential inhibitors of protein C (PKC). Ceramide appears to have bioeffector properties. Cell-permeable ceramide analogs stimulate monocytic differentiation of human leukemia (HL60) cells (1), as well as the phosphorylation of the EGF receptor at Thr⁶⁶⁹ in A431 human epidermoid carcinoma cells (2). Further studies (3,4) demonstrate the existence of a ceramide-activated protein kinase (CAPK) that may mediate some of these aspects. The present studies aim to investigate the mechanism of TGFβ1 signaling and to explore whether TGFβ1's pathway involves activation of PKC 1,2-Diacylglycerol (DAG) and/or stimulation of a CAPK by ceramide. Ceramide and DAG levels of A431 cells are determined by thin layer chromatography (TLC) after treatment with either TGFβ1 or with EGF. 100pM TGFβ1 treatment for 1 hr increases the cellular contents of DAG 2-fold. 20nM EGF treatment for 15 min decreases it 0.5-fold. Ceramide levels are reduced 2-fold by TGFβ1 and almost unaffected by EGF. To evaluate the involvement of other components of signal transduction, the effects of TGFβ1 and EGF on PKC activity are studied. 20 nM EGF decreases membrane PKC activity to 0.5-fold of controls, whereas 100pM TGFβ1 treatment of A431 cells increases this activity 4-fold. Modulation of PKC activity is paralled by translocation of the enzyme between the cytosol and the membrane as determined by Western immunoblot analysis. These studies suggest that TGFβ1 and EGF may have regulatory effects on both sphingolipid and phospholipid metabolisms which could transmodulate both the CAPK and the PKC mediated signal tranduction pathways.