Mu-opioid receptor (MOPr) mediates several functions including pain transmission, respiration, cardiac and gastrointestinal function, and immune responses. Human mu-opioid receptor gene (OPRM1) expression is tightly modulated by several cytokines and neurotrophic factors in both neuronal and immune cells; particularly, OPRM1 expression is increased during neurogenesis, regulates the survival of maturing neurons and is implicated in ischemia-induced neuronal death. The Repressor Element 1 Silencing Transcription factor (REST), a regulator of a subset of genes in differentiating and post mitotic neurons, is implicated in OPRM1 transcriptional repression as well and it has been shown to counteract IGF-I-mediated up-regulation of its expression. Albeit several studies have been performed to characterize OPRM1 transcriptional regulation, extracellular signaling molecules and mechanisms involved in such processes are still not fully understood. In this study, both REST-expressing and REST-non expressing neuronal cells were employed to investigate the role of protein kinase C (PKC) on OPRM1 transcription, in the context of the potential influence of REST. OPRM1 endogenous mRNA levels were evaluated by Real Time PCR whereas OPRM1 promoter transcriptional activity was assessed by employing specific Luciferase/OPRM1 promoter reporter vectors. We observed that in native SH-SY5Y neuroblastoma cells, which endogenously express REST, PKC activation with phorbol 12-myristate 13-acetate (PMA, 16 nM; 24 h) significantly down regulates OPRM1 transcription and concomitantly elevates the REST binding activity to its repressor element 1 on the OPRM1 promoter. On the contrary, when REST expression is knocked-down by an antisense strategy or by retinoic acid-induced cell differentiation, PMA significantly up-regulates OPRM1 gene transcription. REST therefore seems to be crucial in determining the differential effect of PMA on OPRM1 transcription, as its presence in native SH-SY5Y cells is enough to favor OPRM1 down-regulation whereas its absence in antisense-treated or retinoic acid differentiated SH-SY5Y cells allows PKC-dependent up-regulation of OPRM1 transcription. PMA acts through a PKC-dependent pathway requiring downstream extracellular-signal-regulated kinase 1/2 and the transcription factor AP-1, as it was demonstrated by co-administration of PMA and specific PKC, JNK and MAPKK inhibitors. PKC inhibitor GF109203 prevented both PMA-induced down-regulation of OPRM1 transcription in native SH-SY5Y cells and PMA-mediated OPRM1 up-regulation in retinoic acid- differentiated SH-SY5Y cells. Furthermore, JNK and MAPKK inhibitors, SP600125 and PD98059 respectively, but not p38 and PI3K inhibitors, SB203580 and LY294002 respectively, prevented OPRM1 up-regulation in differentiated SH-SY5Y cells, thus suggesting that PKC modulates OPRM1 transcription through the activation of the transcription factor AP-1. This observation was also confirmed by employing decoys oligonucleotide specific for AP-1 inhibition. Experiments were also carried out in SH-SY5Y cells and PC-12 cells transfected with promoter/Luciferase reporter vectors containing different OPRM1 promoter fragments. In these assays we observed that OPRM1 promoter transcriptional activity is up-regulated by PMA-dependent PKC activation in PC12 cells, which lack of REST expression, as well as in SH-SY5Y cells either transfected with OPRM1 promoter constructs deficient in RE1 (the REST DNA binding element) or when REST is down-regulated in retinoic acid-differentiated cells, thus confirming the role played by REST in the differential modulation of OPRM1 transcription by PKC. The finding that PMA may up-regulate OPRM1 expression in differentiating neuroblastoma cells and that this effect depends on REST expression is new and is relevant in elucidating MOPr role in neurogenesis and in post-mitotic neurons. Several studies have demonstrated that OPRM1 gene expression is seen very early on in the embryonic rat brain with an increase observed during the critical period of neurogenesis, neuronal migration, and synaptogenesis, suggesting a role of this opioid receptor in brain developmental processes. Recently, it has been shown that MOPr regulates the survival of maturing neurons in adult hippocampal neurogenesis, therefore, MOPr could be included among the target genes regulated by PKC in a time-dependent manner during neurogenesis. Our results provide new insights into how OPRM1 transcription is regulated and may help to better understand its contribution to epigenetic modifications and reprogramming of neuronal cells exposed to PKC-activating agents in differentiated cells. These findings are relevant considering the potential involvement of such processes in chronic pain treatment.
Bedini A., Baiula M., Spampinato S. (2009). Protein Kinase C-dependent up-regulation of human mu-opioid receptor gene (OPRM1) transcription is negatively influenced by the transcription factor REST. s.l : s.n.
Protein Kinase C-dependent up-regulation of human mu-opioid receptor gene (OPRM1) transcription is negatively influenced by the transcription factor REST
BEDINI, ANDREA;BAIULA, MONICA;SPAMPINATO, SANTI MARIO
2009
Abstract
Mu-opioid receptor (MOPr) mediates several functions including pain transmission, respiration, cardiac and gastrointestinal function, and immune responses. Human mu-opioid receptor gene (OPRM1) expression is tightly modulated by several cytokines and neurotrophic factors in both neuronal and immune cells; particularly, OPRM1 expression is increased during neurogenesis, regulates the survival of maturing neurons and is implicated in ischemia-induced neuronal death. The Repressor Element 1 Silencing Transcription factor (REST), a regulator of a subset of genes in differentiating and post mitotic neurons, is implicated in OPRM1 transcriptional repression as well and it has been shown to counteract IGF-I-mediated up-regulation of its expression. Albeit several studies have been performed to characterize OPRM1 transcriptional regulation, extracellular signaling molecules and mechanisms involved in such processes are still not fully understood. In this study, both REST-expressing and REST-non expressing neuronal cells were employed to investigate the role of protein kinase C (PKC) on OPRM1 transcription, in the context of the potential influence of REST. OPRM1 endogenous mRNA levels were evaluated by Real Time PCR whereas OPRM1 promoter transcriptional activity was assessed by employing specific Luciferase/OPRM1 promoter reporter vectors. We observed that in native SH-SY5Y neuroblastoma cells, which endogenously express REST, PKC activation with phorbol 12-myristate 13-acetate (PMA, 16 nM; 24 h) significantly down regulates OPRM1 transcription and concomitantly elevates the REST binding activity to its repressor element 1 on the OPRM1 promoter. On the contrary, when REST expression is knocked-down by an antisense strategy or by retinoic acid-induced cell differentiation, PMA significantly up-regulates OPRM1 gene transcription. REST therefore seems to be crucial in determining the differential effect of PMA on OPRM1 transcription, as its presence in native SH-SY5Y cells is enough to favor OPRM1 down-regulation whereas its absence in antisense-treated or retinoic acid differentiated SH-SY5Y cells allows PKC-dependent up-regulation of OPRM1 transcription. PMA acts through a PKC-dependent pathway requiring downstream extracellular-signal-regulated kinase 1/2 and the transcription factor AP-1, as it was demonstrated by co-administration of PMA and specific PKC, JNK and MAPKK inhibitors. PKC inhibitor GF109203 prevented both PMA-induced down-regulation of OPRM1 transcription in native SH-SY5Y cells and PMA-mediated OPRM1 up-regulation in retinoic acid- differentiated SH-SY5Y cells. Furthermore, JNK and MAPKK inhibitors, SP600125 and PD98059 respectively, but not p38 and PI3K inhibitors, SB203580 and LY294002 respectively, prevented OPRM1 up-regulation in differentiated SH-SY5Y cells, thus suggesting that PKC modulates OPRM1 transcription through the activation of the transcription factor AP-1. This observation was also confirmed by employing decoys oligonucleotide specific for AP-1 inhibition. Experiments were also carried out in SH-SY5Y cells and PC-12 cells transfected with promoter/Luciferase reporter vectors containing different OPRM1 promoter fragments. In these assays we observed that OPRM1 promoter transcriptional activity is up-regulated by PMA-dependent PKC activation in PC12 cells, which lack of REST expression, as well as in SH-SY5Y cells either transfected with OPRM1 promoter constructs deficient in RE1 (the REST DNA binding element) or when REST is down-regulated in retinoic acid-differentiated cells, thus confirming the role played by REST in the differential modulation of OPRM1 transcription by PKC. The finding that PMA may up-regulate OPRM1 expression in differentiating neuroblastoma cells and that this effect depends on REST expression is new and is relevant in elucidating MOPr role in neurogenesis and in post-mitotic neurons. Several studies have demonstrated that OPRM1 gene expression is seen very early on in the embryonic rat brain with an increase observed during the critical period of neurogenesis, neuronal migration, and synaptogenesis, suggesting a role of this opioid receptor in brain developmental processes. Recently, it has been shown that MOPr regulates the survival of maturing neurons in adult hippocampal neurogenesis, therefore, MOPr could be included among the target genes regulated by PKC in a time-dependent manner during neurogenesis. Our results provide new insights into how OPRM1 transcription is regulated and may help to better understand its contribution to epigenetic modifications and reprogramming of neuronal cells exposed to PKC-activating agents in differentiated cells. These findings are relevant considering the potential involvement of such processes in chronic pain treatment.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
