The pharmacological effect of morphine as a painkiller is mediated mainly via the mu opioid receptor (MOR) and is dependent on the number of MORs in the cell surface membrane. While several studies have reported that the MOR gene is regulated by various cis-and trans-acting factors, many questions remain unanswered regarding in vivo regulation. The present study shows that epigenetic silencing and activation of the MOR gene are achieved through coordinated regulation at both the histone and DNA levels. In P19 mouse embryonal carcinoma cells, expression of the MOR was greatly increased after neuronal differentiation. MOR expression could also be induced by a demethylating agent (5-aza-2-deoxycytidine) or histone deacetylase inhibitors in the P19 cells, suggesting involvement of DNA methylation and histone deacetylation for MOR gene silencing. Analysis of CpG DNA methylation revealed that the proximal promoter region was unmethylated in differentiated cells compared to its hypermethylation in undifferentiated cells. In contrast, the methylation of other regions was not changed in either cell type. Similar methylation patterns were observed in the mouse brain. In vitro methylation of the MOR promoters suppressed promoter activity in the reporter assay. Upon differentiation, the in vivo interaction of MeCP2 was reduced in the MOR promoter region, coincident with histone modifications that are relevant to active transcription. When MeCP2 was disrupted using MeCP2 small interfering RNA, the endogenous MOR gene was increased. These data suggest that DNA methylation is closely linked to the MeCP2-mediated chromatin structure of the MOR gene. Here, we propose that an epigenetic mechanism consisting of DNA methylation and chromatin modification underlies the cell stage-specific mechanism of MOR gene expression.Opioids exert their pharmacological and physiological effects through binding to their endogenous receptors. Three types of opioid receptors, mu (), delta (␦), and kappa (), all belonging to the G-protein-coupled receptor superfamily, have been cloned. Upon agonist binding, these receptors couple to G proteins and affect several signal transduction pathways thought to mediate a broad range of functions and pharmacological effects of endogenous and exogenous opioids (51). Previous studies suggested that the opioid receptor (MOR) plays a key role in mediating the major clinical effects of analgesics, such as morphine, as well as the development of tolerance and physical dependence upon prolonged administration (39). MOR is mainly expressed in the central nervous system, with densities varying greatly in different regions, which can display different functional roles (55). During mouse embryonic development, the MOR message was specifically observed as early as embryonic day 8.5 (E8.5) using the reverse transcription (RT)-PCR method (44). In contrast, MOR transcripts were detected only beginning at E12 using the radioligand binding method (70) and at E10.5 by in situ hybridization (85). Transcript levels gradually incr...

Amino acids are essential to support protein synthesis, which is required for cell growth and proliferation. Amino acid uptake into the cell is mediated by amino acid transporters located on the plasma membrane. 1,2)The system L amino acid transporters are an amino acid transport system that transports neutral amino acids, including several essential amino acids, in a Na ϩ -independent manner.1,3) It is a major route for providing living cells, including cancer cells, with amino acids. 1,3)We isolated LAT1 (L-type amino acid transporter 1), the first isoform of a system L amino acid transporter, from C6 rat glioma cells.4) Subsequently, we and other researchers cloned LAT2 (L-type amino acid transporter 2), the second isoform of a system L amino acid transporter.5-7) The structure and function of LAT1 are very similar to those of LAT2. Both isoforms are predicted to be 12-membrane-spanning proteins.4-7) They require an additional single-membranespanning protein, the heavy chain of 4F2 antigen (4F2hc), for their functional expression in the plasma membrane. [4][5][6][7][8][9][10][11][12] Both LAT1 and LAT2 form a heterodimeric complex via a disulfide bond with 4F2hc. [4][5][6][7][8][9][10][11][12] However, LAT2 is more ubiquitously expressed than LAT1, and it transports not only large neutral amino acids, but also small neutral amino acids in a fashion that appears to have broader substrate selectivity than LAT1. [4][5][6][7][9][10][11][12][13][14] In addition, LAT1 is highly expressed in malignant tumors, presumably to support their continuous growth and proliferation. 4,8,15,16) 2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) is a model compound for the study of amino acid transporters, as it is a system L selective inhibitor.1,17) Because system L amino acid transporters transport neutral amino acids, including several essential amino acids, if they are blocked in living cells by a specific inhibitor such as BCH, the cells would be damaged; damage would be caused specifically by the deprivation of amino acids necessary for protein synthesis, cell growth and proliferation. In addition, several reports have demonstrated that amino acid uptake increased during the proliferation process of cancer cells to support DNA and protein biosynthesis. [18][19][20] It is proposed that the manipulation of system L activity, particularly that of LAT1, would have therapeutic implications. The inhibition of LAT1 activity in cancer cells could be effective in the suppression of cancer cell growth by depriving cancer cells of essential amino acids.17,21) However, the mechanism by which inhibition of LAT1 can cause cancer cell growth suppression or cytotoxicity of cancer cells is not entirely clear.In this study, we examined the effect of BCH on cell growth and its mechanism of cell growth suppression in cancer cells. We used cells that highly expressed only LAT1 among all the system L transporters. [22][23][24] Our results show that the system L selective inhibitor BCH, at suitable concentrations, can induce the suppressio...

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The pharmacological action of morphine as a pain medication is mediated primarily through the μ-opioid receptor (MOR). With few exceptions, MOR is expressed in brain regions where opioid actions take place. The basis for this unique spatial expression of MOR remains undetermined. Recently, we reported that DNA methylation of the MOR promoter plays an important role in regulating MOR in P19 cells. In this study, we show that the differential expression of MOR in microdissected mouse brain regions coincides with DNA methylation and histone modifications. MOR expression could be induced by a demethylating agent or a histone deacetylase inhibitor in MOR-negative cells, suggesting that the MOR gene can be silenced under epigenetic control. Increases in the in vivo interaction of methyl-CpG-binding protein 2 (MeCP2) were observed in the cerebellum, in which the MOR promoter was hypermethylated and MOR expression was the lowest among all brain regions tested. MeCP2 is associated closely with Rett syndrome, a neurodevelopmental disorder. We also established novel evidence for a functional role for MeCP2’s association with the chromatin-remodelling factor Brg1 and DNA methyltransferase Dnmt1, suggesting a possible role for MeCP2 in chromatin remodelling during MOR gene regulation. We conclude that MOR gene expression is epigenetically programmed in various brain regions and that MeCP2 assists the epigenetic program during DNA methylation and chromatin remodelling of the MOR promoter.