Cloning of the mouse insulin receptor substrate-3 (mIRS-3) promoter, and its regulation by p53 | JoVE Visualize

Cloning of the mouse insulin receptor substrate-3 (mIRS-3) promoter, and its regulation by p53

Salvatore Sciacchitano¹, Andrea Orecchio, Luca Lavra

¹Chair of Endocrinology, Second Faculty of Medicine, Università La Sapienza di Roma, Centro Ricerca Ospedale S. Pietro Fatebenefratelli, 00189 Roma, Italy. salvatore.sciacchitano@uniroma1.it

Molecular Endocrinology (Baltimore, Md.)|June 29, 2002

View abstract on PubMed

Summary

The p53 tumor suppressor regulates insulin receptor substrate-3 (IRS-3) gene expression transcriptionally. Wild-type p53 inhibits IRS-3 promoter activity, while tumor mutants show reduced suppression, impacting cell growth and differentiation.

Area of Science:

Molecular Biology
Gene Regulation
Cancer Biology

Background:

Insulin receptor substrate-3 (IRS-3) is an adapter protein involved in insulin and IGF-I signaling pathways.
IRS-3 displays a unique expression pattern, suggesting distinct regulatory mechanisms.
The p53 tumor suppressor plays a critical role in cell cycle control and apoptosis.

Purpose of the Study:

To investigate the transcriptional regulation of the mouse IRS-3 (mIRS-3) gene promoter.
To determine the role of wild-type and mutant p53 in regulating mIRS-3 gene expression.
To identify the specific DNA region within the mIRS-3 promoter involved in p53-mediated regulation.

Main Methods:

Cloning and analysis of the 5' flanking region of the mIRS-3 gene.
Reporter gene assays to assess promoter activity.
Transfection with wild-type p53, dominant-negative p53, and tumor-derived p53 mutants.
Doxorubicin treatment to assess endogenous mIRS-3 mRNA expression.
Analysis of mIRS-3 promoter activity in conjunction with the MDM2 promoter.

Main Results:

The mIRS-3 promoter activity is suppressed by wild-type p53.
Dominant-negative p53 abolishes the inhibitory effect of wild-type p53.
Tumor-derived p53 mutants exhibit reduced suppression of mIRS-3 promoter activity compared to wild-type p53.
Doxorubicin treatment reduces endogenous mIRS-3 mRNA levels.
A specific region (-287 to -178 bp) of the mIRS-3 promoter reduces MDM2 promoter activity, implicating it in p53-mediated inhibition.

Conclusions:

The mIRS-3 promoter is regulated by p53 at the transcriptional level.
p53-mediated inhibition of mIRS-3, and its de-repression by mutant p53, parallels regulation of the IGF-I receptor promoter.
These findings suggest a shared role for IRS-3 and IGF-I receptor in p53-mediated control of cell growth and differentiation.