UV-B stress induces 14-3-3 epsilon mRNA increase and delocalisation in Paracentrotus lividus sea urchin embryos.

UV-Bstressinduces14-3-3epsilonmRNAincreaseand delocalisationin Paracentrotus lividus seaurchinembryos Roberta Russo, Francesca Zito, Caterina Costa, Rosa Bonaventura, Valeria Matranga Consiglio Nazionale delle Ricerche, Istituto di Biomedicina e Immunologia Molecolare ''Alberto Monroy'', Palermo, Italy Members of the 14-3-3 protein family are involved in many important cellular events regulating the equilibrium state between cell survival and apoptosis. They take part in complex mechanisms involved in signal transduction, transcription, mitochondrial pathways and others (Morrison 2008). The 14-3-3 genes are highly conserved, from plants to humans, and some are responsive to UV radiation. We have previously shown that Paracentrotus lividus (Pl) sea urchin embryos exposed at cleavage stages to different doses of UV-B, ranging from 10 to 800 J/ m2, develop with dose-dependent morphological abnormalities, including major defects in skeleton and archenteron (primitive intestine) elongation, and increased hsp70 protein levels (Bonaventura et al. 2006). It was of interest to investigate the expression levels and spatial localization of the 14-3-3 gene product (Pl14-3-3 epsilon) in response to UV-B radiation in sea urchin embryos. To this purpose we isolated the complete cDNA encoding for the 14-3-3 epsilon isoform from embryos exposed to UV-B at the dose of 800 J/m2. By QPCR we analyzed the expression levels of the Pl14-3-3 epsilon mRNA during development and found a stage-dependent proportional increase of the transcript from mesenchyme blastula to prism. When cleavage stage embryos (32 cells) were exposed to UV-B, we found a 2.3-fold (400 J/m2) and 2.7-fold (800 J/m2) increase in Pl14-3-3 epsilon mRNA levels. In addition, we examined the spatial expression of 14-3-3 epsilon mRNA by in situ hybridization in both control and UV-B exposed embryos harvested at late developmental stages (pluteus). The transcripts were found restricted to the archenteron in gastrula stage control embryos, whereas they were widely delocalised in all germ layers in UV-B exposed embryo. These results paralleled the failure of archenteron elongation, observed microscopically, and the abnormal tissue differentiation, detected by specific endoderm markers. In conclusion, this is the first report describing the complete cDNA coding for the 14-3-3 epsilon isoform in P. lividus sea urchin embryos and the first evidence of a transcriptional regulation of the gene in response to UV-B exposure. This work has been supported in part by: EU - UV-TOX Project, Contract N. EVK3-CT-1999-00005, ASI MoMA Project Contract N?1/014/06/0, EU- ITN Biomintec Project, Contract N.215507. Bonaventura R, Poma V, Russo R, Zito F, Matranga V (2006) Effects of UV-B radiation on development and hsp70 expression in sea urchin cleavage embryos. Marine Biology 149: 79-86. Morrison DK (2008) The 14-3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development. Trends in Cell Biology 19: 16-23.