c-myc and pRB: role in TGF-beta 1 inhibition of keratinocyte proliferation
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C S Murphy1, J A Pietenpol, K Münger
1Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Abstract:
The TGF-beta s are potent inhibitors of proliferation of most cell types in culture and in vivo. Previous studies have demonstrated that TGF-beta inhibition of skin keratinocyte proliferation involves suppression of c-myc transcription. Evidence derived from use of expression plasmids for certain DNA viral oncoproteins has suggested that the retinoblastoma gene (RB) may be involved in this process. Transient expression of pRB, like TGF-beta 1, in skin keratinocytes represses expression of a human c-myc reporter plasmid, and the same c-myc promoter region (TCE) is required for repression by either TGF-beta 1 or pRB. We showed here that proliferation and c-myc expression in a cell line lacking normal pRB (DU145 human prostate adenocarcinoma cells) are not inhibited by TGF-beta 1. Oligonucleotides containing the TCE were found to bind to a cellular protein of approximately 106 kD (termed p106) in Southwestern assays, utilizing extracts from both the skin keratinocytes and DU145 cells. TCE binding to p106 was diminished by TGF-beta in TGF-beta-sensitive skin keratinocytes but not in TGF-beta-insensitive SV40-transformed keratinocytes. These data support the hypothesis that pRB is required for TGF-beta 1 suppression of c-myc transcription and suggest the involvement of a cellular factor(s) in addition to pRB in the TGF-beta 1 pathway for inhibition of c-myc transcription and growth inhibition.
Insights
Transforming growth factor-beta (TGF-β) inhibits cell proliferation by suppressing c-myc transcription. The retinoblastoma protein (pRB) is essential for this TGF-β-mediated growth inhibition, acting via a specific c-myc promoter element.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Transforming growth factor-beta (TGF-β) is a potent inhibitor of cell proliferation.
- TGF-β's inhibition of keratinocyte proliferation involves the suppression of c-myc transcription.
- The retinoblastoma gene (RB) product, pRB, has been implicated in this process.
Purpose of the Study:
- To investigate the role of pRB in TGF-β-mediated suppression of c-myc transcription and cell proliferation.
- To identify cellular factors involved in the TGF-β signaling pathway.
Main Methods:
- Transient expression of pRB and TGF-β 1 in skin keratinocytes.
- Analysis of c-myc reporter plasmid expression.
- Southwestern assays using oligonucleotides corresponding to the c-myc TCE region.
Main Results:
- Skin keratinocytes lacking normal pRB (DU145 cells) were insensitive to TGF-β 1-induced inhibition of proliferation and c-myc expression.
- A ~106 kD protein (p106) bound to the c-myc TCE region.
- TGF-β diminished TCE binding to p106 in TGF-β-sensitive keratinocytes but not in resistant cells.
Conclusions:
- pRB is required for TGF-β 1-mediated suppression of c-myc transcription.
- A cellular factor(s) in addition to pRB is involved in the TGF-β 1 pathway for growth inhibition.