The elevated expression of prostate apoptosis response-4 (PAR-4) induces apoptosis in differentiating mouse embryonic stem (ES) cells. In embryoid body (EB) cells and the E15.5 stage of embryonic mouse brain, PAR-4 is expressed as two isoforms (38 and 33 kDa). Using mouse EB-derived RNA as a template we have cloned and characterized a novel isoform of PAR-4 (PAR-4/p33) that lacks exon 3 and shows a bona fide splice junction of exons 2 and 4. The molecular mass for PAR-4/p33 is estimated to be 33 kDa, corresponding to the short form found in the EB cells and E15.5 mouse brain. The fluorescent fusion protein of PAR-4/p33 is mainly found in the cytosol and is co-distributed with F-actin filaments, while that of the 38 kDa full length PAR-4/p38 is predominantly translocated to the nucleus. In contrast to the full length PAR-4 (PAR-4/p38), ectopic expression of PAR-4/p33 does not result in the activation of caspase 3 and the induction of apoptosis. PAR-4/p33 forms a complex with PAR-4/p38, which inhibits its nuclear translocation and the induction of apoptosis. PAR-4/p33 is suggested to be a dominant negative isoform of PAR-4/p38 and may regulate PAR-4-dependent apoptosis.
To study regulatory mechanisms of ganglioside biosynthesis, genes for sialyltransferase II (ST2) or acetylgalactosaminyltransferase I (GalNAcT) were transfected into different cell lines of a substrain of murine neuroblastoma F‐11 cells (F‐11A). While complex β‐series ganglioside synthesis is negligible in the parental line, cells transfected with genes for either enzyme express complex gangliosides requiring both enzyme activities. We have used primers specific for each of the two genes to determine the level of gene expression (1) in the parental cells where endogenous ST2 and GalNAcT activities are very low, and (2) in each of the transfected cell lines where both enzyme activities are present and complex gangliosides are synthesized. Transcripts for both ST2 and GalNAcT were amplified from cDNA that was reverse‐transcribed from the parental as well as both transfected cell lines. While some differences in transcript number and size were noted between parental, ST2‐transfected, and GalNAcT‐transfected cells, suggesting some degree of transcriptional control, all the cell lines clearly expressed transcripts for both genes. These results support the view that ST2 and GalNAcT act in a coordinated fashion, perhaps as a multienzyme complex, to catalyze the synthesis of complex gangliosides, and that synthesis of complex β‐series gangliosides in these cells is regulated primarily post‐transcriptionally. Acknowledgements: Supported by NIH R15‐DC05179 to L.N.I, NIH NS‐11853 to R.K.Y, and NIH‐NCRR(RCMI) G12‐RRO8124 to the Border Biomedical Research Center at the University of Texas at El Paso.
Lipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of β‐series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F‐11) cells with sialyltransferase‐II (ST2) resulted in a 70% reduction of apoptosis that was induced with the novel ceramide analog (NCA) N‐oleoyl serinol (S18). This was most likely due to an 80% reduced expression of prostate apoptotis response‐4 (PAR‐4). PAR‐4 expression and apoptosis were restored by preincubation of ST2‐transfected cells with N‐butyl deoxinojirimycin (NB‐DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 MAPK‐kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR‐4. Apoptosis and PAR‐4 expression, however, were elevated in the ventricular zone, which only weakly stained for complex gangliosides. Complex gangliosides may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide‐induced apoptosis by up‐regulation of MAPK and reduction of PAR‐4 expression. NCAs may be useful to analyse the molecular mechanisms that underlie or counteract ceramide‐induced apoptosis during neuronal development. Acknowledgements: Supported by grants MH61934‐04 (to E.B.) and NS11853 (to R.K.Y.).
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