Abstract
The possible involvement of the protein kinase C (PKC) pathway in transcriptional regulation of the human insulin-like growth factor-I (IGF-I) gene has been suggested. In this study, we sought to determine whether a PKC-dependent pathway is implicated in the transcriptional control, and if it is, how this occurs. Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) caused an increase in the activity of the human IGF-I gene major promoter in HepG2 cells. A CCAAT/enhancer-binding protein (C/EBP) binding site located at +22 to +30 was bound by C/EBP beta in a TPA-dependent manner and was solely responsible for the TPA responsiveness. This increase in C/EBP beta activity occurs through transcriptional and posttranslational regulation, and the latter is mediated by activation of p90 ribosomal S6 kinase (RSK): co-expression of dominant negative RSK abolished the TPA-responsive and C/EBP beta-dependent transactivation. Also, TPA-responsive activation of GAL4-C/EBP beta chimera required the Ser residue known as the RSK target. In SK-N-MC cells, which display constitutive, high expression of IGF-I on use of the major promoter, a large amount of C/EBP beta binding was observed with the C/EBP site in the basal state. Treatment with PKC inhibitors substantially reduced the promoter activity and mRNA amounts of IGF-I, with the binding of C/EBP beta to the C/EBP site also being reduced. When the C/EBP site was disrupted, the basal promoter activity was reduced, but the reduction by the PKC inhibitor was no longer observed. These observations suggest that the increase of C/EBP beta binding to the C/EBP site, which is in part mediated via activation of RSK, can primarily explain the TPA responsiveness of the IGF-I gene promoter. The intrinsic PKC activity in SK-N-MC cells should play a major role in the constitutive, high expression of IGF-I and may therefore contribute in part to the maintenance of the tumor phenotype of the cells.
Highlights
Insulin-like growth factor I (IGF-I)1, a 70-residue single-chain growth-promoting polypeptide, is produced in many organs and tissues and plays a major role in somatic growth, cell survival, tissue differentiation and intermediary metabolism [1,2,3]
Human IGF-I gene promoter-1 is activated by phorbol ester — First we examined whether the major promoter of the human IGF-I gene is a target of protein kinase-C (PKC) activation
Each reporter gene plasmid contained various lengths of IGF-I gene 5’-flanking sequences and 197 bp of the exon 1 untranslated region linked to the firefly luciferase reporter, because this portion of exon 1 untranslated region appeared to be important for basal promoter activity of promoter-1 in SK-N-MC cells [20]
Summary
Insulin-like growth factor I (IGF-I), a 70-residue single-chain growth-promoting polypeptide, is produced in many organs and tissues and plays a major role in somatic growth, cell survival, tissue differentiation and intermediary metabolism [1,2,3]. The result of a nuclear run-on assay indicated that treatment of human macrophage-like cells with 12-o-tetradecanoylphorbol 13acetate (TPA) increased the transcription rate of the IGF-I gene four- to fivefold [7], suggesting that the human IGF-I gene regulatory sequences contain something that responds to PKC. Support for this comes from our recent observations with the chicken IGF-I gene, i.e., that the gene promoter can be activated by TPA through an AP-1 binding site located in it [8]. It is unknown whether the mammalian IGF-I genes are activated by PKC, and if they are, how this occurs
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