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Regulation of constitutive expression of mouse PTEN by the 5'-untranslated region
Baoguang Han1, Zizheng Dong, Yang Liu
1Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Abstract:
PTEN tumor suppressor serves as a major negative regulator of survival signaling mediated by PI3 kinase/AKT/protein kinase B pathway, and is inactivated in various human tumors. Elucidation of mechanisms responsible for PTEN expression is important for providing insight into strategies to control the loss of PTEN expression in human cancers. Although recent studies suggested that p53 and Egr-1 can modulate induced PTEN expression, the mechanism responsible for ubiquitous constitutive expression of PTEN remains elusive. PTEN mRNA contains a highly conserved and GC-rich 5'-untranslated region (5'-UTR). Recently, it has been shown that the long 5'-UTR sequences of several growth-regulated mRNAs contain promoters that can generate mRNAs with shorter 5'-UTRs. In this paper, we tested whether the 5'-UTR sequence of mouse PTEN contains a promoter that is responsible for constitutive expression of PTEN. We found that the long 5'-UTR sequence of mouse PTEN severely inhibits translation of PTEN and a heterologous gene firefly luciferase. Deletion of the most 5'-UTR sequence would enhance translation efficiency 100-fold. We also showed that the 5'-UTR sequence of mouse PTEN does not have an internal ribosome entry site (IRES) that can mediate cap-independent initiation of translation. Instead, we found that the 5'-UTR sequence of mouse PTEN contains a strong promoter that drives the production of a transcript with shorter 5'-UTRs, which can be translated with higher efficiency. This promoter was mapped to the region between -551 and -220 bases upstream of the translation start codon. Cotransfection analysis using Drosophila SL2 cells showed that Sp1 is one of the major transcription factors that can constitutively activate this promoter. Two endogenous PTEN transcripts with 5'-UTRs of 193 and 109 bases were found in DU145 and H226 cell lines. Based on these observations, we conclude that the PTEN expression may be regulated at both transcriptional and translational levels, and that the 5'-UTR sequence of PTEN contains a promoter that is responsible for constitutive PTEN expression.
Insights
The PTEN gene
Area of Science:
- Molecular Biology
- Cancer Biology
- Gene Regulation
Background:
- PTEN tumor suppressor is crucial for regulating PI3K/AKT signaling and is often lost in cancers.
- Understanding PTEN expression mechanisms is vital for developing cancer therapies.
- Previous studies hinted at p53 and Egr-1 roles, but constitutive PTEN expression mechanisms remained unclear.
Purpose of the Study:
- To investigate if the 5'-untranslated region (5'-UTR) of mouse PTEN contains a promoter driving its constitutive expression.
- To determine the role of the PTEN 5'-UTR in regulating PTEN translation.
Main Methods:
- Deletion analysis of the PTEN 5'-UTR to assess its impact on translation efficiency.
- Testing for internal ribosome entry site (IRES) activity within the PTEN 5'-UTR.
- Mapping the promoter region within the 5'-UTR using deletion constructs.
- Identifying transcription factors that activate the PTEN 5'-UTR promoter via cotransfection assays.
Main Results:
- The long PTEN 5'-UTR significantly inhibited translation of both PTEN and a reporter gene.
- Deletion of the 5'-UTR enhanced translation efficiency by 100-fold.
- No IRES activity was detected in the PTEN 5'-UTR.
- A promoter was identified within the PTEN 5'-UTR (-551 to -220 bp upstream of the start codon) driving shorter, more efficiently translated transcripts.
- Sp1 was identified as a key transcription factor activating this promoter.
- Endogenous PTEN transcripts with shorter 5'-UTRs (193 and 109 bases) were detected in human cancer cell lines.
Conclusions:
- PTEN expression is regulated at both transcriptional and translational levels.
- A promoter within the PTEN 5'-UTR drives the production of shorter, more efficiently translated PTEN transcripts, contributing to constitutive PTEN expression.
- This finding offers new insights into PTEN regulation in cancer.
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