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Mechanism of E1A-induced transforming growth factor-beta (TGF-beta) resistance in mouse keratinocytes involves
1Laboratory of Chemoprevention, NCI, National Institutes of Health, Bethesda, Maryland 20892-5055, USA.
Abstract:
Cellular transformation driven by the E1A oncogene is associated with the development of cellular resistance to the growth inhibitory effects of transforming growth factor-beta (TGF-beta). We demonstrate that development of resistance occurs simultaneously with decreased expression of TGF-beta type II receptor (TGF-beta RII) mRNA and protein. To determine whether changes in transcriptional regulation are responsible for the decreased receptor expression in E1A-transformed cells, a series of mobility shift assays was performed utilizing nuclear extracts from E1A-transformed and untransformed murine keratinocytes using radiolabeled positive regulatory elements (PRE1 and PRE2) of the TGF-beta RII promoter. The results from these assays suggest that E1A-transformed cells express markedly lower levels of nuclear proteins that bind specifically to PRE1 and PRE2. Transfection of both E1A-transformed and untransformed cell lines with a series of mutant promoter constructs confirmed that both PREs contribute significantly to basal expression of TGF-beta RII and that inactivation of either element leads to markedly reduced promoter activity. We conclude that development of TGF-beta resistance in E1A-transformed cells is achieved in part through transcriptional down-regulation of the TGF-beta RII gene and that this down-regulation is the result of decreased expression of unidentified transcription factor complexes that interact with PRE1 and PRE2.
Insights
Cellular transformation by the E1A oncogene causes resistance to transforming growth factor-beta (TGF-beta). This resistance is linked to reduced expression of the TGF-beta type II receptor (TGF-beta RII) due to transcriptional down-regulation.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Cellular transformation via E1A oncogene induces resistance to growth inhibitory signals of TGF-beta.
- This resistance correlates with diminished expression of TGF-beta type II receptor (TGF-beta RII).
Purpose of the Study:
- To investigate the transcriptional regulation mechanisms underlying decreased TGF-beta RII expression in E1A-transformed cells.
- To determine if reduced binding of nuclear proteins to the TGF-beta RII promoter contributes to this phenomenon.
Main Methods:
- Mobility shift assays using nuclear extracts from E1A-transformed and untransformed murine keratinocytes.
- Analysis of radiolabeled positive regulatory elements (PRE1 and PRE2) of the TGF-beta RII promoter.
- Transfection studies with mutant promoter constructs to assess the role of PRE1 and PRE2.
Main Results:
- E1A-transformed cells exhibit significantly lower levels of nuclear proteins binding to PRE1 and PRE2.
- Both PRE1 and PRE2 are crucial for basal TGF-beta RII expression, with inactivation leading to reduced promoter activity.
- Decreased expression of transcription factor complexes binding to PRE1 and PRE2 was observed.
Conclusions:
- TGF-beta resistance in E1A-transformed cells is partly mediated by transcriptional down-regulation of the TGF-beta RII gene.
- This down-regulation results from reduced expression of specific transcription factor complexes interacting with the TGF-beta RII promoter's regulatory elements.