CLONING AND CHARACTERIZATION OF THE PROMOTER FOR THE RAT INSULIN-LIKEGROWTH FACTOR-BINDING PROTEIN-3 GENE

Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFB P in the adult circulation, is produced by a wide range of cell and ti ssue types. IGFBP-3 appears to be regulated by transcriptional and/or posttranslational mechanisms in a species-, cell-, and development-spe cific manner. In vitro and in vivo studies suggest that a number of fa ctors (e.g. cAMP, GH, insulin-like growth factor-I, epidermal growth f actor, TSH, and FSH) can act as transcriptional regulators of IGFBP-3 in particular cell types. To address the mechanistic basis for these o bservations, we isolated the rat IGFBP-3 gene and began characterizati on and analysis of the hormonal regulation of its promoter. The rat IG FBP-3 gene is located within 2 adjacent EcoRI fragments spanning about 10 kilobases. Southern analysis indicated a single copy gene. A 1.18- kilobase fragment 5' to the translation initiation codon has been sequ enced and showed 65% homology with the corresponding human IGFBP-3 seq uence. The region between - 100 and -1 bp relative to the transcriptio n start site showed 85% homology. The transcription start site was 118 basepairs (bp) up-stream of the initiation codon, and a TATA box cons ensus was located 27 bp 5' to this CAP site. No CAAT box was present, but a CpG island was identified. Consensus sequences for a number of p utative response elements (e.g. activating protein-a, insulin, TSH/ins ulin-like growth factor, and GH) were present within -700 bp of the CA P site. A series of 5'-truncated chloramphenicol acetyltransferase rep orter constructs has been transfected into both COS-1 cells and the ra t thyroid cell line FRTL-5. Both basal and hormonally responsive (TSH and phorbol ester) promoter activities have been localized within the first 472 bases of the promoter region. These data indicate that suita ble transfected cell systems can be established in which additional in vestigations can be undertaken into the mechanisms of cell- and specie s-specific hormonal regulation of IGFBP-3 gene expression.