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Characterisation of functional inhibition of the glucocorticoid receptor by Fos/Jun
1University of Bern, Department of Clinical and Experimental Cancer Research, Switzerland.
Abstract:
We have studied the effects of Fos and Fos/Jun on glucocorticoid induction of hormone-sensitive gene expression. In NIH3T3 cells overexpression of Fos or Fos/Jun by transfection of pSV2-fos and pSV2-jun inhibited glucocorticoid-dependent expression of MMTV LTR-CAT. Expression of p39v-mos had a similar effect on glucocorticoid-dependent reporter gene expression which is most likely mediated by simulation of endogenous Fos. In both cases, this inhibition could be overcome by overexpression of the glucocorticoid receptor (GR) from a transiently transfected expression vector. In receptor deficient CV-1 cells glucocorticoid-dependent reporter gene expression was induced by a range of functional GR truncation mutants. It was established that the C/D domain of the receptor was a sufficient target for inhibition by Fos and Fos/Jun. The C/D domain encompasses the DNA-binding domain, a dimerisation domain and a weak transactivational domain of the GR. When present simultaneously in the cell nucleus Fos and Jun were shown to form a specific and stable protein/protein complex with the glucocorticoid receptor. Finally, it was demonstrated that the GR interacts physically with both Fos and Jun when cotranslated simultaneously in vitro. We propose that this interaction may be the mechanism by which Fos or Fos/Jun bring about inhibition of GR function.
Insights
Fos and Fos/Jun proteins inhibit glucocorticoid receptor (GR) function by forming stable complexes with the GR's C/D domain. This interaction blocks hormone-sensitive gene expression but can be overcome by increasing GR levels.
Area of Science:
- Molecular Biology
- Cellular Biology
- Endocrinology
Background:
- Glucocorticoids regulate gene expression via the glucocorticoid receptor (GR).
- Fos and Fos/Jun are transcription factors that can modulate gene expression.
- The interplay between Fos/Jun and GR in regulating hormone-sensitive genes is not fully understood.
Purpose of the Study:
- To investigate the effects of Fos and Fos/Jun on glucocorticoid-induced gene expression.
- To identify the specific domain of the GR targeted by Fos/Jun.
- To elucidate the molecular mechanism underlying the interaction between Fos/Jun and GR.
Main Methods:
- Overexpression of Fos, Fos/Jun, and GR in NIH3T3 and CV-1 cells.
- Use of MMTV LTR-CAT reporter gene assays to measure gene expression.
- Analysis of GR truncation mutants to map interaction domains.
- In vitro co-translation and co-immunoprecipitation assays to assess protein-protein interactions.
Main Results:
- Overexpression of Fos or Fos/Jun inhibited glucocorticoid-dependent reporter gene expression.
- This inhibition was overcome by overexpression of the glucocorticoid receptor.
- The C/D domain of the GR was identified as the target for inhibition by Fos and Fos/Jun.
- Fos and Jun formed stable complexes with the GR in the nucleus and physically interacted with GR in vitro.
Conclusions:
- Fos and Fos/Jun inhibit GR-mediated gene expression by directly interacting with the GR's C/D domain.
- This physical interaction likely disrupts GR function, leading to reduced hormone-sensitive gene expression.
- The findings provide a molecular mechanism for Fos/Jun-mediated repression of glucocorticoid signaling.