BOVINE INSULIN-LIKE-GROWTH-FACTOR BINDING PROTEIN-3 - ORGANIZATION OFTHE CHROMOSOMAL GENE AND FUNCTIONAL-ANALYSIS OF ITS PROMOTER

Insulin-like growth factor binding protein-3 (IGFBP-3), the major IGFB P in the circulation, is synthesized by the vascular endothelium in vi vo and has been shown to be an important modulator of the physiologica l effects of IGF. IGFBP-3 is regulated by a number of growth factors/c ytokines to which the vascular endothelium is exposed, including IGF-I stimulation and TGF-beta 1 inhibition of IGFBP-3 in cultured endothel ial cells. To understand the mechanisms of transcriptional regulation of IGFBP-3, we have cloned the bovine IGFBP-3 gene and begun the funct ional analysis of its promoter. Southern analysis indicated a single c opy gene. The gene spanned approximately 10 kb and was divided into fi ve exons, the fifth containing the 3' untranslated region. The transcr iption start site was 137 bp upstream of the initiation codon and a TA TA box was located 26 bp 5' to this CAP site. No CAAT box was present but a GC rich sequence element, containing two overlapping putative AP -2 binding elements, was located 5' to the TATA box. Transient transfe ction studies with a series of 5' truncated luciferase reporter constr ucts were conducted in primary cultures of bovine aorta endothelial ce lls. Results of the transfection studies indicated that 1) nearly 80% of the maximal basal promoter activity was retained within the first 1 30 bp of the 5' flanking sequence; 2) this: region responded to IGF-I, despite lacking the TTF-1/TTF-2 (thyroid specific transcription facto rs) binding elements that are required for IGF-I stimulation of thyrog lobulin synthesis. These binding elements have also been suggested to be involved in IGF-I regulation of IGFBP-3 transcription, thus, implyi ng the existence of novel cis-acting elements that mediate the IGF-I s timulation of bovine endothelial cell IGFBP-3 mRNA synthesis; 3) delet ion of the CC rich sequence element resulted in a GOR reduction in bas al promoter activity as well as loss of the IGF-1 stimulatory effect; 4) the TSF-beta 1 mediated inhibition of IGFBP-3 transcription require d sequence element(s) beyond 1.5 kb of its promoter.