Abstract
The mu opioid receptor (MOR) is expressed in the central nervous system and specific cell lines with varying expression levels perhaps playing important roles. One of the neuronal-specific transcription regulators, neuron-restrictive silencer factor (NRSF), has been shown to repress the expression of neuron-specific genes in non-neuronal cells. However, we showed here that the neuron-restrictive silencer element (NRSE) of MOR functions as a critical regulator to repress the MOR gene expression in specific neuronal cells depending on NRSF expression level. Using co-transfection studies, we showed that the NRSE of the MOR promoter is functional in NRSF-positive cells (NS20Y and HeLa) but not in NRSF-negative cells (PC12). NRSF binds to the NRSE of the MOR gene in a sequence-specific manner confirmed by supershift and chromatin immunoprecipitation assays, respectively. The suppression of NRSF activity with either trichostatin A or a dominant-negative NRSF induced MOR promoter activity and transcription of the MOR gene. When the NRSF was disrupted in NS20Y and HeLa cells using small interfering RNA, the transcription of the endogenous target MOR gene increased significantly. This provides direct evidence the role of NRSF in the cells and also indicates that NRSF expression is regulated by post-translational modification in neuronal NMB cells. Our data suggested that NRSF can function as a repressor of MOR transcription in specific cells, via a mechanism dependent on the MOR NRSE.
Highlights
Three major types of opioid receptors, , ␦, and , have been cloned and shown to belong to the G-protein-coupled receptor superfamily [1]
The proximal promoter of the mouse MOR gene is known to be regulated via various cis-acting elements and trans-acting factors, all of which are important for the proximal promoter activity [12,13,14]
It has a high homology with the mouse MOR neuron-restrictive silencer element (NRSE), which is located from Ϫ9 to ϩ12 bp of the MOR promoter, as well as with the rat MOR NRSE
Summary
Three major types of opioid receptors, , ␦, and , have been cloned and shown to belong to the G-protein-coupled receptor superfamily [1]. opioid binding sites are present in the developing rat brain by the 14th day of gestation, whereas mRNA can be detected as early as E11.5 with in situ hybridization These observations suggest that the MOR is actively involved in the developing mammalian nervous system. The repression effected by NRSF required the interaction of NRSF with the histone deacetylase (HDAC), which induces hypoacetylation of histone [16] This transcription factor has been proposed to play an important role in establishing and maintaining expression of neuron-specific genes. Previous study [18] has shown that the activity of the NRSF promoter functioned in neuronal and non-neuronal cells This result indicated that the post-translational modification of NRSF is important to regulate its target gene
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