Cloning of the mouse insulin receptor substrate-3 (mIRS-3) promoter, and its regulation by p53.
The insulin receptor susbtrate-3 (IRS-3) is a member of a family of intermediate adapter proteins that function as major intracellular targets for phosphorylation by the activated insulin and IGF-I receptors. Among the four IRS proteins identified so far, IRS-3 exhibits a rather peculiar expression pattern during both the embryonic development and adult life, suggesting a different mechanism of regulation of its expression. In this study, we cloned the 5' flanking region of the mIRS-3 gene and analyzed its promoter activity. The mIRS-3 promoter is inhibited by wild-type p53, and this effect is completely abolished by cotransfection of a dominant negative p53. Tumor-derived p53 mutants show variable, but lower suppressing capability than wt p53. In addition, treatment with doxorubicin inhibits endogenous expression of mIRS-3 mRNA in C2C12 and 3T3-L1 cells. The DNA region spanning from nucleotides -287 and -178 in the mIRS-3 promoter is responsible for a 32.2% reduction of the mouse double minute 2 (MDM2) promoter activity, suggesting its involvement in the p53-mediated inhibitory effect. In conclusion, our study demonstrates that the mIRS-3 promoter is regulated by p53 at the transcriptional level. The inhibition of mIRS-3 promoter by wild-type p53, and its de-repression by tumor-derived p53 mutants, appears to be similar to that previously reported for the IGF-I receptor promoter, suggesting a common role of these two genes in p53-mediated cell growth and differentiation.
Sciacchitano
Salvatore
S
Chair of Endocrinology, Second Faculty of Medicine, Università La Sapienza di Roma, Centro Ricerca Ospedale S. Pietro Fatebenefratelli, 00189 Roma, Italy. salvatore.sciacchitano@uniroma1.it
Orecchio
Andrea
A
Lavra
Luca
L
Misiti
Silvia
S
Giacchini
Anna
A
Zani
Massimo
M
Danese
Daniele
D
Gurtner
Aymone
A
Soddu
Silvia
S
Di Mario
Umberto
U
Andreoli
Mario
M
D024506:5' Flanking Region; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004317:Doxorubicin / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D007668:Kidney / Q000166:cytology / Q000196:embryology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D010750:Phosphoproteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; C491585:IRS3P protein, human; D055504:Insulin Receptor Substrate Proteins; C108872:Irs3 protein, mouse; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D004317:Doxorubicin; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2