FUNCTIONAL-ANALYSIS OF GLUCOCORTICOID AND INSULIN-RESPONSE SEQUENCES IN THE RAT INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-1 PROMOTER

Insulin-like growth factor-binding protein-1 (IGFBP-1) is produced by the liver and regulated by glucocorticoids and insulin at the level of gene transcription. To identify DNA sequences mediating the effects o f glucocorticoids and insulin on IGFBP-1 promoter activity we created luciferase reporter gene constructs and performed transfection studies in H4IIE hepatoma cells. Initial studies confirmed that the IGFBP-1 p romoter is functional when inserted in the correct orientation, but no t in the reverse orientation. Dexamethasone (DEX) increased promoter a ctivity 10-fold, and insulin reversed this effect of DEX by 85% at 8 h . The effects of DEX were abolished when constructs were truncated to 89 bases from the RNA cap site, and DNase footprinting with the DNA-bi nding domain of the human glucocorticoid receptor identified an imperf ect palindrome containing two receptor-binding sites separated by thre e nucleotides typical of a glucocorticoid response element (GRE) at th is location. Mutation of either binding site (or half-site) disrupted the effects of DEX, confirming that this sequence functions as a GRE. Two other regions of the promoter also footprinted with the glucocorti coid receptor protein and contained sequences consistent with glucocor ticoid receptor-binding sites; however, neither of these footprints co ntained the full structure expected of a functional GRE, and neither m utation nor deletion of these other sequences altered the effects of D EX on promoter activity. To identify the DNA sequences required for th e effects of insulin on glucocorticoid-stimulated promoter activity, w e created internal deletions throughout the IGFBP-1 promoter region. D eletion of the 22-basepair (bp) sequence immediately 5' from the GRE d isrupted the effect of insulin and appeared to increase basal promoter activity at least a-fold in each of eight experiments (P<0.001 vs. in tact promoter). This region of the IGFBP-1 promoter contains a 19-bp p alindrome (CAAAACAAACTTATTTTG) that overlaps the 5'-end of the GRE and is fully conserved in the human IGFBP-1 promoter. Each half of this p alindrome resembles previously identified insulin response sequences, and deletion/mutation analysis suggests that each half may contribute to the effects of insulin on promoter activity. Gel shift studies conf irmed that this palindrome binds H4IIE nuclear proteins. In summary, w e have identified a GRE in the 5'-promoter region of the rat IGFBP-1 g ene approximately 90 bp up-stream from the RNA cap site as well as a c ontiguous 22-bp region that plays a critical role in mediating the eff ects of insulin on glucocorticoid-stimulated promoter activity. Intera ctions between cis- and trans-acting factors in this region of the IGF BP-1 promoter appear to play a critical role in mediating effects of g lucocorticoids and insulin on hepatic expression of IGFBP-1 and may th ereby contribute to the modulation of IGF bioactivity in metabolic dis ease.