Human insulin-like growth factor binding protein-4 and -6 : gene structure and transcription regulation

Abstract: Insulin-like growth factors (IGF-I and IGF-II) are among the more abundant growth factors isolated from bone tissue and are synthesized by various bone cells including osteoblasts. The activity of IGFs is controlled by a family of binding proteins (IGFBPs) divided into subfamilies depending on their individual affinity for IGFs. IGFBP-4 to -6 have been found in bone, and suggested to participate in regulation of bone matrix. This thesis is based on findings concerning structure, regulation, and chromosomal localization of the genes for human IGFBP-4 and -6. The human IGFBP-4 gene had been located previously to chromosome l7q 12-2 1. 1, but a new microsatellite within the IGFBP-4 gene enabled high-resolution genetic mapping to the vicinity of BRCA1. The human IGFBP-6 gene was mapped to chromosome 12q 13 and characterized: IGFBP6 is spanning 4.7 kb and consist of four exons separated by three introns with sizes 2661, 182, and 884. Three mRNA CAP sites were located to 10 1, 100, and 96 bp upstream of the translation initiation site, but no TATA or CAAT consensus sequences were found 5' of these sites. The IGFBP-4 gene is contained within 15.3 kb, with its transcription initiation site located 28 bp downstream of a TATA box sequence and 286 bp 5' of the translation initiation codon. The gene is composed of four exons separated by three introns with sizes 8903, 787, and 2386 bp. Basal promoter activity of IGFBP4 decreased 60% between - 135 to - 103, and was completely repressed by deletions from -60 to -41. A 3-hour treatment with prostaglandin E2 augmented IGFBP4 transcription two-fold, whereas a 24-hours treatment with interferon-[gamma] decreased IGFBP4 transcription to 40%. EMSA showed that prostaglandin E2 activated Sp I, which was capable to bind to a GC-box located from -46 to -43, whereas interferon-[gamma] stimulated an inhibitory nuclear protein with an affinity for the NF-[kappa]B recognition sequence located from -47 to -44. The IGFBP4 promoter had a CpG/GpC ratio of 1.08 and was shown to be influenced by its methylation status. A partially methylated promoter, constructed by HpaII in vitro methylation, had 50% activity after 24 hours, and this activity decreased to 34% after this time point. Complete methylation of the promoter by the Sssl-methylase abolished transcription. Treatment with a deacetylase inhibitor increased IGFBP4 transcription activity to 750% in cells transfected with non-methylated and partially methylated constructs, but was not able to affect IGFBP4 transcription activity in cells transfected with the completely methylated promoter.