Insulin-like growth factor-I regulates transcription of the elastin gene

Abstract

Neonatal rat aortic smooth muscle cell cultures were used to investigate the mechanisms by which insulin-like growth factor-I (IGF-I) up-regulates aortic elastogenesis. The addition of IGF-I (50 ng/ml) to quiescent smooth muscle cell cultures resulted in a 5-fold increase in the steady-state levels of tropoelastin mRNA beginning between 2 and 4 h and reaching maximal levels at 8 h. Addition of cycloheximide blocked the effect of IGF-I. Nuclear run-on transcription analyses of nuclei isolated from IGF-I-treated cells showed increased synthesis of new tropoelastin transcripts indicating that transcriptional activation is a major component of IGF-I up-regulation. Transient transfections with deletion constructs containing different portions of the elastin 5'-upstream 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 extracted from control and IGF-I-treated cells demonstrated that IGF-I treatment 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.