Abstract
Ceramide, a product of sphingomyelin hydrolysis, is now recognized as an intracellular lipid messenger, which mediates the effects of extracellular agents on cellular growth, differentiation and apoptosis. Recently, ceramide has been implicated in the regulation of phospholipase D (PLD). In this study, we examined the effects of ceramide on the activity and mRNA level of PLD during apoptotic process in FRTL-5 thyroid cells. C2-ceramide (N-acetyl sphingosine) induced apoptosis in FRTL-5 thyroid cells. Fluorescent staining showed that ceramide induced the typical features of apoptosis including condensed or fragmented nuclei. DNA fragmentation was also observed by agarose gel electrophoresis. Flow cytometric cell cycle analysis showed more clearly that ceramide induced apoptotic cell death in FRTL-5 thyroid cells. The treatment of FRTL-5 thyroid cells with thyroid-stimulating hormone (TSH) resulted in an increased PLD activity in a dose- and time-dependent manner. However, the TSH-induced increase in PLD activity was down-regulated within 2 h after ceramide treatment. Furthermore, the levels of PLD mRNA were found to be decreased throughout apoptotic process as inferred by reverse transcription-polymerase chain reaction. However, the decreases in PLD mRNA levels were not correlated with those in PLD activities after ceramide treatment. Taken together, these data suggest that ceramide inhibits the PLD activity in an early apoptotic phase and down-regulation of the levels of PLD mRNA may be implicated in apoptotic process in FRTL-5 thyroid cells.
Highlights
Signal transduction system generating phospholipid metabolites constitutes one of the most important systems for the generation of second messenger to regulate cell growth
Ceramide induced apoptotic cell death in FRTL-5 thyroid cells To ascertain whether ceramide initiates an apoptotic response in FRTL-5 thyroid cells, experiment was carried out using the synthetic ceramide analogue C2ceramide (N-acetyl sphingosine)
Changes in mRNA expression of Phospholipase D (PLD) Because thyroid-stimulating hormone (TSH)-induced increase of PLD activity was decreased in an early phase of ceramide-induced apoptosis, we examined the changes of gene expression for PLD by RT-PCR throughout the apoptotic process
Summary
Signal transduction system generating phospholipid metabolites constitutes one of the most important systems for the generation of second messenger to regulate cell growth. Phospholipase D (PLD) plays a key role in signal transduction and its activation occurs in a wide range of cell types in response to hormones and growth factors and is implicated in the regulation of inflammatory and immune response, cellular trafficking, and cell growth (Exton, 1994). PLD catalyzes the hydrolysis of cell phospholipid, mainly phosphatidylcholine (PC), resulting in the formation of phosphatidic acid (PA), which acts by itself as cellular messenger (Exton, 1990) or can be transformed by PA phosphohydrolase into diacylglycerol (DAG), which is essential for the activation of protein kinase C (PKC) (Nishizuka, 1995). Recent investigations indicate that signaling events following cellular exposure to tumor necrosis factor α (TNFα), Fas ligand, glucocorticoids, irradiation and other DNAdamaging agents seem to converge in intracellular signals that result in apoptosis (Arends and Wyllie, 1991; Williams and Smith, 1993; Hoffman and Liebermann, 1994; Wyllie, 1995)
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