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Differential expression of mu-opioid receptor gene in CXBK and B6 mice by Sp1
Po-Wei Lee1, Sally Wu, Yu-May Lee
1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Abstract:
It is well known that there are individual differences in the sensitivity to analgesics. The CXBK mice are characterized by reduced sensitivity to morphine and by partial deficiency in mu-opioid receptor (MOR) expression. The sequences of MOR genes in CXBK and B6 mice are identical in their coding regions but differ at 5'-untranslated region (UTR) nucleotide -202 (C nucleotide in CXBK, but A nucleotide in B6). In this report, we identified an Sp1 element (-211 to -204) immediately before the polymorphic nucleotide. In electrophoretic mobility shift assay, nuclear protein binding to the B6-Sp1 sequence was more efficient than to the CXBK-Sp1 sequence, and anti-Sp1 but not anti-CREB antibody interfered with the formation of the DNA-protein complex. In MOR-expressing cell lines SH-SY5Y, P19, and PC12, B6 MOR promoter possessed high transcription activity than the CXBK promoter, and Sp1 inhibitor PDTC reduced the promoter activities. In SL2 cells that lack endogenous Sp1 expression, B6 and CXBK MOR promoters demonstrated equal activity, whereas overexpression of Sp1 in SL2 cells enhanced B6 MOR promoter activity better than the CXBK promoter. Together, the A-to-C change at MOR 5'-UTR decreases Sp1 binding and MOR gene transcription, which could underlie the reduced morphine expression in CXBK mice.
Insights
Individual differences in morphine sensitivity may stem from genetic variations. A specific DNA change in CXBK mice reduces mu-opioid receptor (MOR) gene transcription by affecting Sp1 protein binding.
Area of Science:
- Genetics
- Pharmacology
- Molecular Biology
Background:
- Individual differences in analgesic sensitivity are well-documented.
- CXBK mice exhibit reduced morphine sensitivity and lower mu-opioid receptor (MOR) expression.
- MOR gene coding regions are identical in CXBK and B6 mice, but a 5'-UTR nucleotide difference exists.
Purpose of the Study:
- To investigate the functional impact of a specific nucleotide variation in the MOR 5'-UTR on gene transcription.
- To determine the role of Sp1 transcription factor in mediating MOR gene expression differences between CXBK and B6 mice.
Main Methods:
- Electrophoretic mobility shift assays (EMSA) to assess protein binding to MOR promoter sequences.
- Reporter gene assays in various cell lines (SH-SY5Y, P19, PC12, SL2) to measure MOR promoter activity.
- Use of Sp1 inhibitor (PDTC) and Sp1 overexpression in SL2 cells.
Main Results:
- Nuclear protein binding to the B6 MOR promoter sequence was more efficient than to the CXBK sequence.
- Sp1, but not CREB, antibody interfered with DNA-protein complex formation.
- B6 MOR promoter showed higher transcription activity than CXBK promoter in MOR-expressing cells.
- Sp1 inhibition reduced MOR promoter activity; Sp1 overexpression differentially enhanced promoter activity in SL2 cells.
Conclusions:
- The A-to-C nucleotide change at -202 in the MOR 5'-UTR of CXBK mice decreases Sp1 binding.
- This reduced Sp1 binding leads to decreased MOR gene transcription.
- The identified mechanism may explain the reduced morphine sensitivity observed in CXBK mice.
