Effects Of Ceramide On MCLR-induced Protein Phosphatase2A Subunits And Its Substrates Changes In A Human Liver Cell Line-HL7702

Background:Microcystin-LR (MCLR) is one of the most common as well as the most toxic member of microcystins (MCs) produced by bluegreen alga. MCLR has been recognized as the strong inhibitor of protein phoshatase2A (PP2A), which may be the major reason of the implicated intoxication mechanism of MCLR. Ceramide is an essential secondary messenger in cells and regulates various cellular mechanisms. Furthermore, ceramide upregulates PP2A/B55a and PP2A/B56a subunits. Our previous studies have shown that MCLR can induce ceramide generation in human embryonic kidney cells-HEK293and mice kidney. The present study is to investigate the role of ceramide on MCLR-induced change of protein levels of PP2A subunits and phosphorylation of its substrates in human liver cells-HL7702.Methods:The HL7702cells were selected to expose MCLR cotreated with or without desipramine (DESI). The ceramide level is determined by immunofluorescence. Immunoblotting is used to study the protein levels of PP2A and phosphorylation level of its substrates. Cell apoptosis is investigated by using hoechst33342stain.Results:1. MCLR induces the generation of ceramide in HL7702cells.2. Using DESI to inhibit the synthsis of ceramide can alleviate the upregulation of PP2A/B55a and PP2A/B56a on protein level with MCLR exposure.3. Hyperphosphorylation of c-myc、 Akt、 bcl-2、 Hsp27can alleviate when MCLR and DESI were cotreated in HL7702cells.4. MCLR may not induce cell apoptosis with or without DESI in HL7702cells.Conclusion:MCLR can induce ceramide elevation in HL7702cells by the way of synthesis. Ceramide may mediate MCLR-induced upregulation of protein levels of PP2A/B55α and PP2A/B56α as well as hyperphosphorylation of c-myc, Akt, bcl-2and Hsp27. It is indicated that ceramide is likely involved in MCLR intoxication mechanism in HL7702cells, but the exact mechanism remains to be further study.