Characterization and regulation of the mouse insulin receptor substrate gene promoter.
To evaluate the potential for regulation of the insulin receptor substrate IRS-1, we have cloned the mouse IRS-1 gene, identified its promoter, and analyzed promoter activity in the basal state and in response to stimulation. The 5'-region of the mouse IRS-1 gene lacks typical CAAT and TATA boxes but contains nine potential Sp1 binding sites consistent with a housekeeping gene. The 5'-region of the IRS-1 gene also has significant regions of homology with the promoters of the progesterone receptor gene, the insulin-like growth factor I receptor gene, and the androgen receptor gene. Multiple transcription start sites were identified 0.4-1.2 kilobases (kb) upstream from the start codon. Using a chloramphenicol acetyl transferase assay in Chinese hamster ovary (CHO) cells, basal promoter activity was present in the 3.2 kb 5'-flanking region of IRS-1 gene. Within this region, there were 184-base pair and 60-base pair negative regulatory elements at -3.2 kb and -1.6 kb surrounded by positive elements. By gel shift assay, a nuclear factor was identified in CHO cells which binds to -1606 and -1586 sequence in the negative regulatory element and appears to be distinct from C/EBP, CREB, and AP-1. In 3T3-F442A adipocytes dexamethasone treatment significantly decreased IRS-1 mRNA and IRS-1 protein. This was due to a decrease in the half-life of IRS-1 mRNA, with no change in IRS-1 promoter-chloramphenicol acetyl transferase activity. Insulin also decreased IRS-1 protein by approximately 60% within 9 h but did so without altering IRS-1 mRNA levels or chloramphenicol acetyl transferase activity. Thus, both insulin and dexamethasone down-regulate IRS-1 expression at the posttranscriptional level; with insulin this is probably due to an effect on protein half-life, whereas with dexamethasone the effect is due to a change in the half-life of IRS-1 mRNA.
Araki
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Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA.
Haag
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Matsuda
K
K
Shichiri
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M
Kahn
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CR
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*
D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D010750:Phosphoproteins