INSULIN STIMULATES RAT CALMODULIN-I GENE-TRANSCRIPTION THROUGH ACTIVATION OF SP1

We have shown previously that insulin positively regulates transcripti on of the rat calmodulin (CaM) I gene. This activation occurs concomit antly with the activation of the low-Km adenosine 3':5'-cyclic phospha te phosphodiesterase (PDE), which appears to be coregulated with CaM. Rat hepatoma H-411E cells were transfected with plasmids containing va rious lengths of the putative CaM promoter coupled to a luciferase rep orter and were challenged with insulin. We demonstrate that insulin-st imulated transcription of CaM I gene is mediated by a 392-bp 5'-flanki ng region of the CaM I gene, encompassing 185 bp downstream and 207 bp upstream of the start site of transcription. The CaM I promoter conta ins three potential Spl sites, located at -114 through -109 [(3), +], -77 through -72 [(2), -] and at +53 through +58 [(1), +]. The gel mobi lity shift assays demonstrated that nuclear protein(s) associate with all three Spl sites. We present data demonstrating the relative import ance of the three Spl 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 1 139, 2.6X, Delta 182; prCaM I 130, 2.1X, Delta 194; and prCaM I 1463, negligible activity. In sum mary, the maximal insulin stimulation of CaM gene expression is seen w hen the promoter region contains at least two Spl sites.