Effect of ceramide on apoptosis and phospholipase D activity in FRTL-5 thyroid cells

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. C 2 -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.

Protein kinase C and phospholipase D: intimate interactions in intracellular signaling

“…Around the same time, a partial inhibition of phorbol ester-induced PLD activity by C 2 -ceramide was reported to occur in human neutrophils [101], as well as a partial inhibition of PLD activity by C 2 -and C 6 -ceramide in fibroblasts stimulated with phorbol esters and thrombin [102]. These results have been substantiated by numerous other studies that showed inhibition of PLD by C 2 -ceramide or by C 6 -ceramide and ceramide generated by exogenous SMases [103][104][105][106][107][108][109][110][111][112][113][114][115][116]. Stimulation of cells with agonists that activate PLD has been shown to correlate with the translocation or association of PLD activators to membrane surfaces.…”

D609-sensitive tyrosine phosphorylation is involved in Fas-mediated phospholipase D activation

“…PLD activity was determined by the formation of phosphatidylethanol (PEt) as described elsewhere (Park et al, 1999). Briefly, A20 cells cultured on 6-well plates were metabolically labeled with 1 µCi/ml of [ 3 H]-palmitic acid in serum free medium for 18 h. The cells were then pretreated with 1%(v/v) ethanol for 15 min before stimulation with anti-Fas mAb.…”

Dexamethasone enhances phospholipase D activity in M-1 cells

“…Assay of PLD activity was based upon the unique ability of this enzyme to stimulate transphosphatidylation in the presence of ethanol to form the corresponding PEth (Kang et al, 1998;Park et al, 1999). M-1 cells were labeled with [ 3 H]myristic acid (1 µCi/ml) for 16 h. The cells were washed twice by phosphate buffered saline (PBS) and resuspended in assay buffer (20 mM Hepes, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 3 mM MgCl 2 , 2 mM CaCl 2 , 2 mM EGTA, and 1 mg/ml bovine serum albumin).…”