Insulin stimulates rat calmodulin I gene transcription through activation of Sp1.
We have shown previously that insulin positively regulates transcription of the rat calmodulin (CaM) I gene. This activation occurs concomitantly with the activation of the low-Km adenosine 3':5'-cyclic phosphate phosphodiesterase (PDE), which appears to be coregulated with CaM. Rat hepatoma H-411E cells were transfected with plasmids containing various lengths of the putative CaM promoter coupled to a luciferease reporter and were challenged with insulin. We demonstrate that insulin-stimulated transcription of CaM I gene is mediated by a 392-bp 5'-flanking region of the CaM I gene, encompassing 185 bp downstream and 207 bp upstream of the start site of transcription. The CaM I promoter contains three potential Sp1 sites, located at -114 through -109 [(3), +], -77 through -72 [(2), -] and at +53 through +58 [(1), +]. The gel mobility shift assays demonstrated that nuclear protein(s) associate with all three sp1 sites. We present data demonstrating the relative importance of the three Sp1 sites for the insulin effect: prCaM I 1835, 3.8x, delta 1081; prCaM I 392, 5.3x, delta 1055; prCaM I 180, 3.7x, delta 462; prCaM I 237, 1.6x, delta 478; prCaM I 139, 2.6x, delta 182; prCaM I 130, 2.1x, delta 194; and prCaM I 1463, negligible activity. In summary, the maximal insulin stimulation of CaM gene expression is seen when the promoter region contains at least two Sp1 sites.
Solomon
S S
SS
Research Service, Veterans Administration Medical Center, Memphis, Tennessee 38104, USA.
Palazzolo
M R
MR
Takahashi
T
T
Raghow
R
R
D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007328:Insulin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D002147:Calmodulin; D007328:Insulin; D016329:Sp1 Transcription Factor; D015105:3',5'-Cyclic-AMP Phosphodiesterases