PKC modulation of mu-opioid receptor gene (OPRM1) transcription is influenced by the transcription factor REST

Andrea Bedini, Monica Baiula and Santi Spampinato. Department of Pharmacology, University of Bologna, Irnerio 48 – 40126, Italy. RE1 Silencing Transcription Factor (REST) represses transcription of neuronal genes in non-neuronal mature cells and in neuronal stem cells, where it is expressed at high levels. REST plays a pivotal role in regulating neural cell fate determination by restricting neuronal gene expression to post-mitotic neurons1 . Protein kinase C (PKC) induces different signaling pathways, some of which are related to opioid receptors: PKC activation, in fact, down-regulates endogenous human mu-opioid receptor (MOPr) mRNA in SH-SY5Y neuroblastoma cells2 whereas enhances mu-opioid receptor gene (OPRM1) transcription in the same cells when OPRM1 promoter fragment from nucleotide -2624 to nucleotide -165 is considered3 . These observations arise the hypothesis that there might be transcription factors binding the OPRM1 promoter region closest to ATG and down-regulating OPRM1 transcription: as REST has been shown to bind human OPRM1 promoter at nucleotides from -9 to +12 and we have previously observed that phorbol 12-myristate 13- acetate (PMA) induces REST, we wondered whether REST is involved in PKC-mediated MOPr downregulation. Therefore, three reporter plasmids bearing OPRM1 promoter fragments -1672/+64, -1672/- 10 and -1672/-254, respectively, have been cloned and transfected in SH-SY5Y cells, which express REST, and in PC-12, which lack of REST expression4 . In SH-SY5Y cells, PMA-induced PKC determined transcriptional activation of the OPRM1 promoter fragments lacking the RE1 site for REST (-1672/-10 and -1672/-254) and transcriptional repression of the OPRM1 promoter fragment bearing the REST target sequence (-1672/+64). In PC-12 cells PMA-induced PKC determined the transcriptional activation of all the OPRM1 promoter fragments. Then endogenous MOPr transcripts were evaluated by real-time PCR: in SH-SY5Y cells, PMA down-regulated MOPr mRNA levels whereas REST knockdown by specific antisense oligonucleotide prevented OPRM1 transcription down-regulation by PMA. Similarly, retinoic acid differentiation of SH-SY5Y cells down-regulated REST expression, thus preventing PMA-induced MOPr down-regulation. Taken together our results show for the first time that PMA down-regulation of OPRM1 transcription in neurons involves REST, which can be the critical switch that determines different PMA-mediated effects according to its expression levels. Ref.: (1) Di Toro et al., European journal of neuroscience, 2005, 21, 46-58 ; (2) Gies et al., Anestesiology, 1997, 87, 1127-38; (3) Börner et al., Molecular Pharmacology, 2002, 61, 800-5 ; (4) Bedini et al., Journal of Neurochemistry, 2008