Transcription of macrophage IGF-I exon 1 is positively regulated by the 5'-untranslated region and negatively regulated by the 5'-flanking region.
Idiopathic pulmonary fibrosis (IPF) is an insidious lung disease with no known cure or effective therapy. Macrophage-derived insulin-like growth factor-I (IGF-I) is thought to play a role in the pathogenesis of IPF; however, little is known about the control of IGF-I expression in macrophages. In this report we investigated the cis-regulatory elements that control basal expression using luciferase reporter constructs in RAW 264.7 macrophages. We show that the +95 to +329 region contains elements necessary to direct maximal promoter activity, whereas the +251 to +329 region contains the minimal promoter. Mapping transcriptional start sites for endogenous IGF-I in primary macrophages revealed that the major transcriptional start site is centered at +150, whereas the most 3'-transcriptional start site is centered at +255. Nuclear proteins from primary and RAW 264.7 macrophages bind specifically to the region required for maximal promoter activity (+134 to +173) and to the region required for minimal promoter activity (+267 to +299). Antibody supershift assays indicate that Sp3 bound to the +267 to +299 region. Moreover, mutation of the putative binding site reduced Sp3 binding in EMSAs and increased promoter activity in luciferase reporter gene assays. We also found that the regions from -1711 to -855 and -855 to -337 contain putative macrophage-specific suppressor elements that do not function in HeLa or COS-7 epithelial cell lines. These data support the view that macrophage IGF-I expression is positively regulated by elements located in the 5'-untranslated region and negatively regulated by elements in the 5'-flanking region of the IGF-I gene.
Wynes
Murry W
MW
Program in Cell Biology, Dept. of Pediatrics, Neustadt Rm. D405, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206, USA.
Riches
David W H
DW
D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; C497122:Sp3 protein, mouse; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D007334:Insulin-Like Growth Factor I; D008156:Luciferases