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Insulin-like growth factor binding protein-3 (IGFBP-3), the major IGFBP in the circulation, is synthesized by the vascular endothelium in vivo and has been shown to be an important modulator of the physiological effects of IGF. IGFBP-3 is regulated by a number of growth factors/cytokines to which the vascular endothelium is exposed, including IGF-I stimulation and TGF-beta1 inhibition of IGFBP-3 in cultured endothelial cells. To understand the mechanisms of transcriptional regulation of IGFBP-3, we have cloned the bovine IGFBP-3 gene and begun the functional analysis of its promoter. Southern analysis indicated a single copy gene. The gene spanned approximately 10 kb and was divided into five exons, the fifth containing the 3' untranslated region. The transcription start site was 137 bp upstream of the initiation codon and a TATA 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 transfection studies with a series of 5' truncated luciferase reporter constructs were conducted in primary cultures of bovine aorta endothelial cells. Results of the transfection studies indicated that 1) nearly 80% of the maximal basal promoter activity was retained within the first 130 bp of the 5' flanking sequence; 2) this region responded to IGF-I, despite lacking the TTF-1/TTF-2 (thyroid specific transcription factors) binding elements that are required for IGF-I stimulation of thyroglobulin synthesis. These binding elements have also been suggested to be involved in IGF-I regulation of IGFBP-3 transcription, thus, implying the existence of novel cis-acting elements that mediate the IGF-I stimulation of bovine endothelial cell IGFBP-3 mRNA synthesis; 3) deletion of the GC rich sequence element resulted in a 60% reduction in basal promoter activity as well as loss of the IGF-1 stimulatory effect; 4) the TGF-beta1 mediated inhibition of IGFBP-3 transcription required sequence element(s) beyond 1.5 kb of its promoter.

Bovine Insulin-Like Growth Factor Binding Protein-3: Organization of the Chromosomal Gene and Functional Analysis of Its Promoter