Mithramycin A up-regulates the endogenous MOR transcription
Previously, we reported that the MOR transcription is suppressed by NRSE in the mouse MOR promoter through binding of NRSF (11). In the current study, we have studied the role of G/C box sequences (GGGGGCGGGGC), identified in mouse, rat and human in adjacent to NRSE, which is a consensus nucleotide-binding sites for the Sp transcription factors (Figure 1A). To assess the functionality of the putative Sp factor binding site on the expression of the MOR genes, NS20Y cells were treated with mithramycin A, a DNA-binding blocking drug that inhibits the binding of transcription factor to G/C-specific regions of DNA. Mithramycin A is a cell-permeable agent that binds GC-rich DNA sequences and is frequently used to explore the sequence dependency of DNA-binding factors (20). As shown in Figure 1B and D, RT–PCR and real-time RT–PCR revealed that the mRNA levels of the MOR were increased by mithramycin A treatment in a dose-dependent manner in the NRSF positive NS20Y cells. However, in the NRSF negative PC12 cells, MOR transcript level was not affected by mithramycin A treatment (Figure 1C). In accordance with other studies, mithramycin A did not change mRNA levels of β-actin (21). These results indicated that the putative Sp-binding site may mainly act as a negative element for the expression of MOR gene in NS20Y cells.