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. 1991 Mar;11(3):1647–1653. doi: 10.1128/mcb.11.3.1647

Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

M C Pepin 1, N Barden 1
PMCID: PMC369463  PMID: 1996114

Abstract

Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Becker P. B., Gloss B., Schmid W., Strähle U., Schütz G. In vivo protein-DNA interactions in a glucocorticoid response element require the presence of the hormone. Nature. 1986 Dec 18;324(6098):686–688. doi: 10.1038/324686a0. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Carroll, Curtis G. C., Mendels J. Neuroendocrine regulation in depression. I. Limbic system-adrenocortical dysfunction. Arch Gen Psychiatry. 1976 Sep;33(9):1039–1044. doi: 10.1001/archpsyc.1976.01770090029002. [DOI] [PubMed] [Google Scholar]
  4. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  5. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Greden J. F., Gardner R., King D., Grunhaus L., Carroll B. J., Kronfol Z. Dexamethasone suppression tests in antidepressant treatment of melancholia. The process of normalization and test-retest reproducibility. Arch Gen Psychiatry. 1983 May;40(5):493–500. doi: 10.1001/archpsyc.1983.01790050019002. [DOI] [PubMed] [Google Scholar]
  7. Izant J. G., Weintraub H. Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA. Science. 1985 Jul 26;229(4711):345–352. doi: 10.1126/science.2990048. [DOI] [PubMed] [Google Scholar]
  8. Izant J. G., Weintraub H. Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis. Cell. 1984 Apr;36(4):1007–1015. doi: 10.1016/0092-8674(84)90050-3. [DOI] [PubMed] [Google Scholar]
  9. Julien J. P., Tretjakoff I., Beaudet L., Peterson A. Expression and assembly of a human neurofilament protein in transgenic mice provide a novel neuronal marking system. Genes Dev. 1987 Dec;1(10):1085–1095. doi: 10.1101/gad.1.10.1085. [DOI] [PubMed] [Google Scholar]
  10. Kim S. K., Wold B. J. Stable reduction of thymidine kinase activity in cells expressing high levels of anti-sense RNA. Cell. 1985 Aug;42(1):129–138. doi: 10.1016/s0092-8674(85)80108-2. [DOI] [PubMed] [Google Scholar]
  11. Linkowski P., Mendlewicz J., Leclercq R., Brasseur M., Hubain P., Golstein J., Copinschi G., Van Cauter E. The 24-hour profile of adrenocorticotropin and cortisol in major depressive illness. J Clin Endocrinol Metab. 1985 Sep;61(3):429–438. doi: 10.1210/jcem-61-3-429. [DOI] [PubMed] [Google Scholar]
  12. McGarry T. J., Lindquist S. Inhibition of heat shock protein synthesis by heat-inducible antisense RNA. Proc Natl Acad Sci U S A. 1986 Jan;83(2):399–403. doi: 10.1073/pnas.83.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Melton D. A. Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc Natl Acad Sci U S A. 1985 Jan;82(1):144–148. doi: 10.1073/pnas.82.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Miesfeld R., Okret S., Wikström A. C., Wrange O., Gustafsson J. A., Yamamoto K. R. Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature. 1984 Dec 20;312(5996):779–781. doi: 10.1038/312779a0. [DOI] [PubMed] [Google Scholar]
  15. Payvar F., DeFranco D., Firestone G. L., Edgar B., Wrange O., Okret S., Gustafsson J. A., Yamamoto K. R. Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region. Cell. 1983 Dec;35(2 Pt 1):381–392. doi: 10.1016/0092-8674(83)90171-x. [DOI] [PubMed] [Google Scholar]
  16. Pepin M. C., Beaulieu S., Barden N. Antidepressants regulate glucocorticoid receptor messenger RNA concentrations in primary neuronal cultures. Brain Res Mol Brain Res. 1989 Jul;6(1):77–83. doi: 10.1016/0169-328x(89)90031-4. [DOI] [PubMed] [Google Scholar]
  17. Scheidereit C., Geisse S., Westphal H. M., Beato M. The glucocorticoid receptor binds to defined nucleotide sequences near the promoter of mouse mammary tumour virus. Nature. 1983 Aug 25;304(5928):749–752. doi: 10.1038/304749a0. [DOI] [PubMed] [Google Scholar]
  18. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  19. van der Eb A. J., Graham F. L. Assay of transforming activity of tumor virus DNA. Methods Enzymol. 1980;65(1):826–839. doi: 10.1016/s0076-6879(80)65077-0. [DOI] [PubMed] [Google Scholar]

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