INSULIN-LIKE GROWTH FACTOR-I REGULATES TRANSCRIPTION OF THE ELASTIN GENE

Neonatal rat aortic smooth muscle cell cultures were used to investiga te the mechanisms by which insulin-like growth factor-I (IGF-I) up-reg ulates aortic elastogenesis. The addition of IGF-I (50 ng/ml) to quies cent smooth muscle cell cultures resulted in a 5-fold increase in the steady-state levels of tropoelastin mRNA beginning between 2 and 4 h a nd reaching maximal levels at 8 h. Addition of cycloheximide blocked t he effect of IGF-I. Nuclear run-on transcription analyses of nuclei is olated from IGF-I-treated cells showed increased synthesis of new trop oelastin transcripts indicating that transcriptional activation is a m ajor component of IGF-I up-regulation. Transient transfections with de letion constructs containing different portions of the elastin 5'-upst ream region localized the IGF-I-responsive area to sequences between - 195 and -136 base pairs and further showed that this region contains a negative element. Gel retardation assays using nuclear proteins extra cted from control and IGF-I-treated cells demonstrated that IGF-I trea tment results in the loss of binding complexes. Footprint analyses of specific binding complexes affected by IGF-I show the deprotection of two closely positioned sequences spanning positions -165 to -137 base pairs. These results suggest that IGF-I up-regulation of elastogenesis involves the abrogation of a negative element functionality.