The glucocorticoid response element II is functionally homologous in rat and human insulin-like growth factor-binding protein-1 promoters

In vivo, insulin-like growth factor-binding protein-1 (IGFBP-1) modulates t he IGFs' bioavailability and may contribute to their delivery to peripheral tissues. In rat and human hepatocytes, glucocorticoids stimulate IG-FBP-1 gene transcription through homologous glucocorticoid response units (GRU). Transfection experiments have shown that one of these, GRUB (nucleotide (nt ) -121 to -85 and nt -111 to -74 in human and rat promoters, respectively), was on its own able to mediate the glucocorticoid response in rat but not in human species (Suwanichkul, A., Allander, S., Morris, S. L. & Powell, D. R. (1994) J. Biol. Chem. 269, 30835-30841, Goswami, a, Lacson, R., Yang, E ,, Sam, R. & Unterman, T. (1994) Endocrinology 134, 736-743, and Sub, D. S. , Ooi, G. T, & Rechler, M. M. (1994) Mob Endocrinol. 8, 794-805), A close c omparison of GRUB sequences has pointed out a C to A transition in the unde rlying GREII, which creates a GATC tetranucleotide in rat species. This tet ranucleotide is submitted to adenosyl methylation (dam methylation) in most Escherichia coil bacterial strains, but not in eucaryotic cells. We showed (i) that on its own, the unmethylated rat GRUB (propagated in dam E. coli strains) was inactive, as is the case for its human counterpart (nonsignifi cant glucocorticoid inductions, 1.48 +/- 0.23 and 1.7 +/- 0.35-fold in Chin ese hamster ovary cells, respectively) and (ii) that its adenosyl methylati on in standard dam(+) bacterial strains yielded a functional GRU (6.5 +/- 1 .1 and 13.1 +/- 3.9-fold glucocorticoid inductions in Chinese hamster ovary and HepG2 cells, respectively). Transient transfection in HepG2 hepatoma c ells clearly showed that the interaction of liver-enriched trans-acting fac tor(s) with the 5'-overlapping insulin response element does not enable the unmethylated rat GRU2 or the human GRUB to become responsive to glucocorti coids (nonsignificant 2.21 +/- 0.48 and 1.20 +/- 0.06-fold induction, respe ctively). Furthermore, we have correlated these functional data with in vit ro DNA-protein interaction studies: the dam methylated rat GREII displayed a 2.8-fold higher affinity for the glucocorticoid receptor than its unmethy lated counterpart.