The insulin-like growth factor binding protein-1 (IGFBP-1) gene is highly expressed in fetal, perinatal, and regenerating liver. Up-regulation is transcriptionally mediated in regenerating liver and occurs in the first few minutes to hours after partial hepatectomy. In transgenic mice a 970-bp region from ุ776 to ุ151 of the IGFBP-1 promoter was sufficient for tissue-specific and induced expression of the gene in fetal and hepatectomized livers. However weak and/or poorly regulated expression in some transgenic lines suggested the existence of other regulatory regions. Here, genomic clones containing large regions 5ะ of the mouse IGFBP-1 gene sequence were isolated, subcloned, and sequenced. Deoxyribonuclease I (DNaseI) hypersensitivity analyses identified clusters of tissue-specific nucleasesensitive sites in the promoter region, ุ100 to ุ300, ุ2,300, ุ3,100, and ุ5,000 along with other weak sites. After partial hepatectomy, enhanced sensitivity and/or novel sites were detected in the ุ100/ุ300, ุ5,000, and ุ3,100 regions, the promoter region remaining the most hypersensitive. A subset of these sites was present in fetal and perinatal livers. Novel tissue-specific sites that interacted with C/EBP and hepatic nuclear factor 3 (HNF3) transcription factors were identified in the ุ3,100 region. A hepatectomy-induced DNA binding complex containing the transcription factor USF1 was identified within the ุ100 to ุ300 region of the promoter. These results suggested that a complex array of tissue-specific and hepatic proliferationinduced transcription factors combine to regulate both the proximal promoter and more distal regulatory elements of the IGFBP-1 gene. (HEPATOLOGY 1999;30:1187-1197.)
The 6 secreted members of the insulin-like growth factor binding protein (IGFBP) family bind and modulate the bioavailability and activity of insulin-like growth factors I and II (IGF-I and IGF-II). 1,2 Although most of the serum IGF is bound in a tertiary complex with IGFBP-3 and an acid-labile subunit, 3,4 IGFBP-1 appears to be the major regulator of the small but significant free IGF fraction. [5][6][7][8] Hepatocytes are the major source of serum IGFBP-1, 9-12 but expression is also seen in the rodent kidney, [13][14][15] human decidualized endometrium, [16][17][18] and human ovarian granulosa cells. 19 As recently reviewed, the majority of studies indicate that IGFBP-1 inhibits IGF growth and metabolic functions by preventing receptor binding, although under certain experimental conditions IGFBP-1 seems to enhance mitogenic activities. 20 Its negative transcriptional regulation by insulin in response to changes in host nutritional status, 20-25 mediated via similar promoter elements in the gluconeogenic gene PEPCK, 25,26 support a role for IGFBP-1 in glucose counterregulation. However, during abdominal surgery, severe infection, trauma, and burn injury increased hepatic IGFBP-1 expression, and serum levels appear to be unrelated to insulin levels [27][28][29][30] and may be influenced instead by cytokines such as interleukin 6 (IL-6). 31 Likewise, despite high insulin levels, IGFBP-1 expression is also high during the perinatal period. 14,32,33 IGFBP-1 is one of the most highly induced immediate early genes transcribed in the remnant liver in the absence of protein synthesis by preexisting factors after partial hepatectomy. 14,34 The increase in expression after partial hepatectomy is primarily transcriptionally mediated. 14 The IGFBP-1 promoter lacks known growth-regulated binding sites present in the promoters of other immediate early genes suggesting that induction during liver regeneration may involve a novel mechanism. The first 300 bp of the promoter region contains several cross species conserved functional DNA binding sites. The hepatic nuclear factor 1 (HNF1) site is required for basal expression of the IGFBP-1 gene and most likely confers tissue specificity. [35][36][37][38] Insulin acting through the insulin response element/HNF3 binding site 39,40 is a major negative regulator of IGFBP-1, although this correlation is uncoupled during the perinatal period and in various injury models as discussed previously. Insulin levels drop after partial hepatectomy allowing potential stimulation of IGFBP-1 expression by other factors. The insulin response element and the glucocorticoid response elements are required for maximal stimulation of IGFBP-1 expression by glucocorticoids. 41 Cortisol, glucagon, and other activators of cAMP increase IGFBP-1 gene expression through an identified