Insulin-like Growth Factor-binding Protein-1 Transcription Research Articles

Abstract 1797: Loss of FoxO1 expression is associated with decreased IGF-1R levels and alters IGFBP-1 induction in tamoxifen-resistant breast cancer cells

Abstract Breast cancer is one of the leading causes of cancer-related deaths in the world. The majority of breast cancers express estrogen receptor alpha (ERα) and tamoxifen, a selective estrogen receptor modulator, is commonly prescribed as adjuvant therapy for ERα-positive breast cancer patients. The development of chemoresistance is a common occurrence in treated patients, however, the molecular mechanisms associated with acquired tamoxifen resistance are not well defined. Previous work from our lab identified a novel mechanism of tamoxifen action that involved the accumulation of extracellular Insulin-like growth factor binding protein-1 (IGFBP-1) and modulates IGF-1-dependent signaling transduction. Tamoxifen-induced IGFBP-1 induction in this model was mediated by G protein-coupled estrogen receptor (GPER)-dependent CREB activation as shown by phosphorylation on serine 133. To determine IGFBP-1 induction is altered during the development of chemoresistance, this mechanism of action was studied in tamoxifen resistant MCF-7 (TamR) cells. Whereas CREB was constitutively phosphorylated in TamR cells, IGFBP-1 levels were not elevated when compared to parental cells and IGFBP-1 was not induced by tamoxifen treatment in TamR cells. These data suggest that another transcription factor(s) are also necessary for the induction of IGFBP-1 in breast cancer cells. FoxO1 is a known regulator of IGFBP-1 transcription and we tested whether FoxO1 contributes to tamoxifen-induced IGFBP-1 transcription. Our results indicate that FoxO1 expression levels were significantly reduced in TamR cells compared with parental cells. Furthermore, FoxO1 knockdown experiments demonstrated this transcription factor mediates tamoxifen-induced IGFBP-1 induction in MCF-7 cells. Consistent with previously published observations, IGF-1R expression was reduced in TamR cells compared to parental MCF-7 cells. To determine if loss of IGF-1R expression in involved in the loss of FoxO1 expression in TamR cells, IGF-1R was exogenously expressed and FoxO1 expression was measured. IGF-1R expression increases FoxO1 levels and partially restores tamoxifen-induced IGFBP-1 induction in TamR cells. These results suggest that FoxO1-mediated IGFBP-1 accumulation is altered during the development of tamoxifen resistance and is associated with decreased IGF-1R expression. Citation Format: Ali Vaziri-Gohar, Kevin D. Houston. Loss of FoxO1 expression is associated with decreased IGF-1R levels and alters IGFBP-1 induction in tamoxifen-resistant breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1797.

Abstract 4421: GPER-mediated activation of the cAMP response element-binding protein (CREB) and subsequent IGFBP-1 expression is altered during the development of tamoxifen resistance

Abstract Tamoxifen is a commonly prescribed anti-estrogen treatment for breast cancer patients; this selective estrogen receptor modulator functions as an ERα-antagonist in breast tissue and inhibits cell proliferation and survival of breast cancer cells. More recently, the active metabolite of tamoxifen, 4-OHT (Tam), has been shown to activate the G protein-coupled estrogen receptor (GPER). Previous studies from our laboratory identified a novel, GPER-dependent mechanism of Tam action in breast cancer cells that requires the extracellular accumulation of the insulin-like growth factor binding protein-1 (IGFBP-1). The molecular mechanisms of Tam-induced IGFBP-1 production mediated by GPER have not been determined. GPER activation has been shown to stimulate cAMP production and PKA activation in several cell types. We hypothesized the cAMP response element-binding protein (CREB) activation is required for Tam-induced IGFBP-1 production in breast caner cells. To determine if CREB activation may be involved in IGFBP-1 transcription, CREB phosphorylation (S133) status and nuclear accumulation were determined in MCF-7 cells after Tam treatment. The phosphorylation and nuclear accumulation of CREB were observed in Tam-treated MCF-7 cells suggesting that CREB activation may be required for Tam-induced IGFBP-1 transcription. Increased CREB phosphorylation and IGFBP-1 transcription were inhibited by pretreatment with the PKA inhibitor H-89 supporting a role for CREB in Tam-induced IGFBP-1 expression. H-89 pretreatment also significantly decreased the previously reported inhibition of IGF-1-stimulated signaling by Tam treatment in breast cancer cells. We have previously reported that Tam-induced IGFBP-1 expression is dysregulated in Tam-resistant MCF-7 cells (MCF-7TamR). To determine if aberrant CREB signaling results in the observed dysregulation, similar experiments were performed in MCF-7TamR cells. Our results show that CREB phosphorylation and nuclear accumulation are decreased in Tam-resistant cells compared to Tam-sensitive cells. Taken together, these results suggest that activation of GPER results in CREB-dependent IGFBP-1 production and subsequent inhibition of IGF-1-stimulated signaling in breast cancer cells treated with Tam. Furthermore, dysregulation of GPER-mediated CREB activation may be involved in the development of Tam resistance in breast cancer. Citation Format: Ali Vaziri-Gohar, Kevin D. Houston. GPER-mediated activation of the cAMP response element-binding protein (CREB) and subsequent IGFBP-1 expression is altered during the development of tamoxifen resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4421. doi:10.1158/1538-7445.AM2015-4421

RTEF-1 Attenuates Blood Glucose Levels by Regulating Insulin-Like Growth Factor Binding Protein-1 in the Endothelium

Related transcriptional enhancer factor-1 (RTEF-1) plays an important role in endothelial cell function by regulating angiogenesis; however, the mechanism underlying the role of RTEF-1 in the endothelium in vivo is not well defined. We investigated the biological functions of RTEF-1 by disrupting the gene that encodes it in mice endothelium -specific RTEF-1-deficient transgenic mice (RTEF-1(-/-)). RTEF-1(-/-) mice showed significantly increased blood glucose levels and insulin resistance, accompanied by decreased levels of insulin-like growth factor binding protein-1 (IGFBP-1) mRNA in the endothelium and decreased serum IGFBP-1 levels. Additionally, the RTEF-1(-/-) phenotype was exacerbated when the mice were fed a high-fat diet, which correlated with decreased IGFBP-1 levels. In contrast, vascular endothelial cadherin/RTEF-1-overexpressing(1) transgenic mice (VE-Cad/RTEF1) demonstrated improved glucose clearance and insulin sensitivity in response to a high-fat diet. Furthermore, we demonstrated that RTEF-1 upregulates IGFBP-1 through selective binding and promotion of transcription from the insulin response element site. Insulin prevented RTEF-1 expression and significantly inhibited IGFBP-1 transcription in endothelial cells in a dose-dependent fashion. To the best of our knowledge, this is the first report demonstrating that RTEF-1 stimulates promoter activity through an insulin response element and also mediates the effects of insulin on gene expression. These results show that RTEF-1-stimulated IGFBP-1 expression may be central to the mechanism by which RTEF-1 attenuates blood glucose levels. These findings provide the basis for novel insights into the transcriptional regulation of IGFBP-1 and contribute to our understanding of the role of vascular endothelial cells in metabolism.

Ligand activated relaxin receptor increases the transcription of IGFBP-1 and prolactin in human decidual and endometrial stromal cells

The aim of this study was to investigate relaxin (RLX) receptor-mediated gene activation in human endometrium. We determined the promoter activities of insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin (PRL) and identified sequence(s) that mediate RLX activated transcription in human decidual cells and endometrial stromal cells. In human decidual cells, the promoter activity of IGFBP-1 was increased significantly in cells incubated with RLX. In endometrial stromal cells, the RLX mediated activation was enhanced only when stromal cells were co-transfected with RLX-receptor (LGR7) expression vector and RLX alone had little effect (Mazella et al., 2004). Deletion and mutation analysis showed that the cAMP regulatory element (CRE, -263 to -259 bp) in the IGFBP-1 promoter was essential for the activation. In addition, RLX increased the phosphorylation of CRE binding protein (CREB to p-CREB) and p-CREB resided in the nucleus, indicating that RLX activates the protein kinase (PKA) system in decidual cells. Gel shift assay showed that nuclear extracts prepared from RLX treated decidual cells increased the binding to the CRE site of the IGFBP-1 promoter. RLX increased the PRL promoter activity mediated through the region containing multiple CCAAT/enhancer-binding proteins (C/EBP) binding sites that have been shown to mediate the PRL gene activation by cAMP analogue (Pohnke et al., 1999). RLX enhanced IGFBP-1 promoter activity was inhibited by cAMP dependent PKA inhibitor, H-89. PRL promoter activity was inhibited by both H-89 and U0126 indicating multiple signalling pathways are activated by RLX in endometrial cells for different target gene activation.

Elevated insulin, proinsulin and insulin-like growth factor-binding protein-1 in liver disease

Insulin-like growth factor-binding protein-1 (IGFBP-1) is one of six soluble binding proteins that regulate the actions of the insulin-like growth factors (IGFs). Liver is the major source of IGFBP-1 in non-pregnant humans. In normal physiology, IGFBP-1 transcription is potently inhibited by insulin and serum levels are limited by a rapid clearance rate. Elevated levels of IGFBP-1 in liver disease have been attributed to insulin resistance; however, the relationships between these analytes have not been defined. We studied insulin, proinsulin and IGFBP-1 in normal subjects (NL, N=47, 43+/-12 yr), cirrhosis (CIR, N=29, 54+/-14 yr), hepatocellular carcinoma (HCC, N=42, 61+/-11 yr), and other liver tumors (TUM, N=8, 60+/-17 yr). All three analytes were significantly increased in liver disease (mean+/-SEM; p-values relative to normals): IGFBP-1 (NL 24+/-4 ng/ml; CIR 235+/-53, p<0.0001; HCC 505+/-105, p<0.0001; TUM 118+/-36, p<0.0001), insulin (NL 72+/-4 pM; CIR 261+/-62, p<0.0002; HCC 180+/-25, p<0.0001; TUM 189+/-58, p<0.0001), proinsulin (NL 6.5+/-0.7 pM; CIR 36.8+/-7.7, p<0.0001; HCC 26.2+/-3.8, p<0.0001; TUM 32.1+/-9.7, p<0.0001). The ratio of proinsulin to insulin was also significantly elevated in liver disease. A typical curvilinear inverse relationship of insulin and IGFBP-1 was observed, but was shifted several fold higher for the liver disease groups. Our results demonstrate that insulin and proinsulin are elevated in liver disease. However, these elevations are paradoxically accompanied by elevated IGFBP-1 levels, indicating disruption of normal regulatory mechanisms. IGFBP-1 is postulated to play a dynamic role in metabolic substrate utilization via regulation of free IGF. Therefore, inappropriate elevation of IGFBP-1 could play an important role in the metabolic disturbances associated with liver disease.

Hox proteins activate the IGFBP-1 promoter and suppress the function of hPR in human endometrial cells.

Previous studies have shown that progestin activates the transcription of IGFBP-1 (insulin-like growth factor binding protein-1). Four regions in the IGFBP-1 promotor have been identified to enhance the transcription. Two of the regions, located at -73 to -65 bp and -319 to -311 bp formed identical DNA-protein complexes with the nuclear extracts of endometrial stromal/decidual cells. To identify the binding protein(s) in endometrial cells that interact with these two regions, we have used the TGTCAATTA repeats (-319 to -11 bp of the IGFBP-1 promoter) to screen the human decidual cDNA library by yeast one-hybrid system. We found that Hox A10, HoxA11, HoxB2, HoxB4, and HoxD11 interacted with the TGTCAATTA repeats in yeast cells. Among these hox genes, the full-length coding region of HoxA10, HoxA11, and HoxB4 were used for functional analysis in three types of endometrial cells, undifferentiated endometrial stromal cells, decidual cells (differentiated stromal cells) and endometrial adenocarcinoma cell line (HEC1-B). All these endometrial cells produce IGFBP-1. Transient transfection assay showed that HoxA10 expression vector increased the promoter activity (the IGFBP-1 proximal promoter containing TGC/TCAATTA and two functional PRE sites) in endometrial stromal cells and in HEC-1B cells, but not in decidual cells. HoxB4 enhanced the promoter activity only in decidual cells, while HoxA11 had no apparent effect in all three types of cells. To evaluate whether Hox proteins would interact with progesterone receptor (hPR), cells were transfected with the promoter construct, Hox and hPR expression vectors. hPR alone activated the IGFBP-1 promoter activity, but expression of Hox gene suppressed the activation. Hox proteins also suppressed the hPR enhanced promoter activities of MMTV (containing consensus-PRE sites) and glycodelin (GdA, containing Sp1 site which mediates the hPR function). These data showed that Hox genes selectively activate the transcription of the IGFBP-1 and GdA genes in different types of endometrial cells. Hox genes, however, suppress the hPR enhanced activities. In addition, we found that HoxB4 expression was induced by estrogen and progestin. Other investigators have shown that HoxA10 and 11 were stimulated by progestin. These findings show that Hox proteins are molecular mediators of the steroid hormones during endometrial cell development.

Gene- and Activation-specific Mechanisms for Insulin Inhibition of Basal and Glucocorticoid-induced Insulin-like Growth Factor Binding Protein-1 and Phosphoenolpyruvate Carboxykinase Transcription

The insulin response sequence (IRS) of the phosphoenolpyruvate carboxykinase (PEPCK) promoter, located within the glucocorticoid response unit, was first characterized by its ability to mediate insulin inhibition when inserted into a thymidine kinase promoter. The IRSs of the PEPCK and insulin-like growth factor binding protein-1 (IGFBP-1) promoters have been proposed to contribute to regulation by glucocorticoids and insulin. Forkhead (FKHR) recognizes IRS sequences, is phosphorylated in response to insulin, and mediates insulin inhibition of basal IGFBP-1 transcription in an IRS-dependent manner. Here, we investigate the contributions of FKHR and IRSs to insulin inhibition of basal and glucocorticoid-induced transcription of PEPCK and IGFBP-1. Expression of T/S/S, in which three putative protein kinase B (PKB) sites in FKHR are mutated, reduced insulin inhibition of basal expression of IGFBP-1 but not PEPCK. Mutation of the IGFBP-1 IRSs abolished insulin inhibition in the presence of T/S/S. Mutation of the PEPCK IRS had no effect on insulin inhibition in the presence of T/S/S, indicating that insulin inhibits PEPCK transcription independently of the IRS or of the putative PKB phosphorylation sites in FKHR. Mutations in the IRS or FKHR had no effect on insulin inhibition of glucocorticoid-induced transcription of either the PEPCK or IGFBP-1 gene. Thus, insulin uses gene- and activation-specific mechanisms to regulate the basal and glucocorticoid-induced activity of these genes.

Growth hormone suppression of insulin-like growth factor binding protein-1 promoter activity

Hepatic transcription of insulin-like growth factor binding protein-1 (IGFBP-1) is rapidly downregulated by growth hormone (GH) which is also known to induce expression of c-fos and c-jun. Co-expression of c-fos or c-jun in rat hepatocytes, individually or together, suppresses IGFBP-1 promoter activity by ≈60%. When hepatic nuclear extracts from sham-operated, hypophysectomized (hypox) and GH-treated hypox rats were analyzed by DNase-1 footprinting, differences in the protection pattern were identified in three regions of the IGFBP-1 promoter. F1 corresponding to −660 to −640 bp showed acute changes in response to GH administration. In additional regions, F2 and F3, representing −758 to −748 bp and −477 to −447 bp, respectively, differences were apparent between nuclear extracts from the hypox and sham-operated rats. When F1 and F2 were removed by deletion of the region from −824 to−557 bp, the GH response was lost but suppression by co-expression of c-fos and c-jun was preserved. A putative AP-1 binding site was present in the F3 footprint region, however removal of F3 did not affect the GH responsiveness. These data indicate that several distinct sequences, other than the putative AP-1 site are involved in mediating the GH effects on IGFBP-1 transcription.

Insulin-like growth factor binding protein-1: recent findings and new directions.

In 1988, insulin-like growth factor-binding protein-1 (IGFBP-1) became the first characterized member of a group of structurally related soluble proteins which specifically bind and modulate the actions of the IGFs. Since then, a wealth of information has accumulated regarding the physiology of this dynamic serum protein. In this review, we update our 1993 summary (Lee PDK et al. Proc Soc Exp Biol Med 204:4-29) of the status of IGFBP-1 research. The IGFBP-1 protein sequence contains 12 N-terminal and 6 C-terminal cysteine residues which are conserved in other mammalian IGFBP-1 sequences and amongst other IGFBPs; both of the cysteine-rich regions are required for optimal IGF binding. The nonconserved IGFBP-1 midregion may act as both a hinge which defines ligand binding characteristics and as a specific target for protease activity. Integrin-binding and phosphorylation sites within the IGFBP-1 sequence have functional significance in vitro, but their physiologic relevance in vivo have not been defined. The human IGFBP-1 and IGFBP-3 genes are contiguous and located in close proximity to the homeobox A (HOXA) gene cluster on chromosome 7. The other IGFBP genes, located on chromosomes 2, 12, and 17, are also associated with HOX clusters, suggesting evolutionary linkage of the IGFBP and HOX gene families. Similarities between the hIGFBP-1 and phosphoenolpyruvate kinase (PEPCK) promoters, including regions conferring insulin, glucocorticoid, and cyclic adenosine-monophosphate responses, are consistent with our previous hypothesis that IGFBP-1 is involved in regulation of glucose metabolism. The tissue-specific patterns of IGFBP-1 gene expression in liver, kidney, decidua, and ovary may be due to stimulation of IGFBP-1 transcription by hepatic nuclear factor 1 (HNF1) proteins. Clinical and basic studies of IGFBP-1 physiology have been aided by several recently developed assay methods. Numerous investigations have confirmed that insulin, via inhibition of IGFBP-1 transcription, is the primary determinant of IGFBP-1 expression both in vitro and in vivo. IGF-I and IGF-II also have specific inhibitory effects on IGFBP-1 expression. Glucocorticoids and cAMP stimulate IGFBP-1 transcription, but these effects are observed only in conditions of low or absent insulin effect. Other stimulants of IGFBP-1 expression include thyroid hormones and epidermal growth factor. Phorbol ester stimulation of IGFBP-1 expression can supersede the effects of insulin in vitro;however, the mechanism and in vivo correlates of this effect have not been determined. Cytokines and, perhaps, growth hormones may affect IGFBP-1 expression, perhaps by altering the regulatory actions of insulin; this effect may have important clinical relevance. IGFBP-1 expression is upregulated in liver and (nonhuman) kidney during postinjury regeneration. The IGF-inhibitory actions of IGFBP-1 has been confirmed by numerous in vitro studies and several in vivo animal investigations, including administration of recombinant IGFBP-1 and IGFBP-1 transgenic models. IGFBP-1 has been shown to inhibit somatic linear growth, weight gain, tissue growth, and glucose metabolism. Moreover, IGFBP-1 appears to be a primary determinant of free IGF-I levels in serum. Excess levels of IGFBP-1 may contribute to growth failure in intrauterine growth restriction and in pediatric chronic renal failure, while low IGFBP-1 levels are associated with obesity and with cardiovascular risk factors in insulin resistance syndromes. Serum IGFBP-1 measurements may be useful biochemical marker in these pathologic conditions. IGFBP-1 is expressed in decidualized stromal cells of the uterine endometrium and in ovarian granulosa cells. IGFBP-1, together with IGFs, insulin, ovarian steroids, cytokines, and other factors, is involved in a complex system which regulates menstrual cycles, ovulation, decidualization, blastocyst implantation, and fetal growth. (ABSTRACT TRUNCATED)

Hepatic nuclear factor 3 and high mobility group I/Y proteins bind the insulin response element of the insulin-like growth factor-binding protein-1 promoter.

The insulin response element (IRE) of the human insulin-like growth factor-binding protein-1 (IGFBP-1) promoter contains a palindrome of the T(A/G)TTT sequence crucial to hormonal regulation of many genes. In initial studies of how this IRE participates in hormonal regulation, the electromobility shift assay was used under a variety of conditions to identify IRE-binding proteins. An exhaustive search identified five proteins that specifically bind this IRE; purified proteins were used to show that all five are related to either the high mobility group I/Y (HMGI/Y) or hepatic nuclear factor 3 (HNF3) protein families. Further studies used purified HNF3 and HMGI proteins to show: 1) eah protects the IGFBP-1 IRE from deoxyribonuclease I (DNaseI) digestion; and 2) HNF3 but not HMGI/Y binds to the related phosphoenolpyruvate carboxykinase and Apo CIII IREs. A series of IRE mutants with variable responsiveness to insulin were used to show that the presence of a TGTTT sequence in the mutants did parallel, but HMGI/Y and HNF3 binding to the mutants did not parallel, the ability of the mutants to confer the inhibitory effect of insulin. In contrast, HNF3 binding to these IRE mutants roughly correlates with response of the mutants to glucocorticoids. The way by which HNF3 and/or other as yet unidentified IRE-binding proteins confer insulin inhibition to IGFBP-1 transcription and the role of HMGI/Y in IRE function have yet to be established.

Growth hormone modulates insulin regulation of hepatic insulin-like growth factor binding protein-1 transcription.

Hepatic transcription of insulin-like growth factor-binding protein-1 (IGFBP-1) is enhanced in hypophysectomized (hypox) rats and can be rapidly down-regulated by GH administration. Here we examined the effect of insulin on IGFBP-1 messenger RNA abundance in hypox rats and the effects of insulin and GH on IGFBP-1/chloramphenicol acetyltransferase (CAT) reporter plasmids transiently transfected into isolated hepatocytes from pituitary-intact and hypox rats. Unlike GH, administration of insulin to hypox rats in doses of 10 or 50 micrograms/100 g BW had no effect on hepatic IGFBP-1 messenger RNA abundance. Insulin at 10(-7) M resulted in a 42.1 +/- 9.8% suppression of CAT activity in hepatocytes from pituitary-intact animals transfected with a CAT reporter plasmid containing 1671 bp of the 5'-flanking region of the rat IGFBP-1 gene. In the same assay, GH at a concentration of 2.3 x 10(-8) M significantly reduced CAT activity. In contrast, insulin had no effect on CAT activity in hepatocytes from hypox rats, whereas GH resulted in comparable suppression of CAT activity in hepatocytes from hypox rats and pituitary-intact rats, 13.6 +/- 2.3% vs. 18.2 +/- 3.2%. Deletional analysis and mobility shift assays were used to identify the GH-responsive regions in the IGFBP-1 gene. GH suppression of CAT activity was lost when the IGFBP-1 5'-flanking region was deleted down to -277 bp, whereas insulin suppression was retained for all but the smallest fragment of the IGFBP-1 gene. Mobility shift assays were used to compare nuclear extracts from sham-operated, hypox, and GH-treated hypox rats. When hepatic nuclear extracts from hypox rats were incubated with the -277 to -82 and the -556 to -368 bp fragments, retarded bands were apparent that were not present in the extracts from sham-operated rats. GH treatment of hypox rats 15 or 30 min before death completely normalized the retardation pattern seen with the -277 to -82 bp fragment, but did not affect the pattern seen with the -556 to -368 bp fragment. A 20-bp fragment corresponding to the previously identified insulin response element, -108 to -89 bp, was also analyzed. An additional retarded band, not seen with nuclear extracts from sham-operated rats, was apparent when nuclear extracts of hypox rats or GH-treated hypox rats were used. These data provide the first in vitro evidence that GH directly regulates transcription of IGFBP-1 expression. In addition, our findings suggest that GH modulates insulin regulation of IGFBP-1 transcription, possibly by altering the milieu of trans-acting factors that interact with both the insulin response element and distinct upstream sites.

Identification of cis-elements mediating the stimulation of rat insulin-like growth factor-binding protein-1 promoter activity by dexamethasone, cyclic adenosine 3',5'-monophosphate, and phorbol esters, and inhibition by insulin.

Insulin-like growth factor-binding protein-1 (IGFBP-1) modulates the action of IGFs on target cells. IGFBP-1 transcription is highly regulated by hormonal and metabolic factors. In rat H4-II-E hepatoma cells, IGFBP-1 messenger RNA is stimulated by dexamethasone, cAMP, and phorbol esters, and dominantly inhibited by insulin. To identify the cis-elements that determine transcriptional regulation by these agents, we have coupled rat IGFBP-1 promoter fragments to a luciferase reporter gene and transfected H4-II-E cells using the cationic liposome procedure. Promoter fragments whose 5'-end was at nucleotide (nt) -925 or -327 (with respect to the transcription initiation site, 1) conferred positive regulation of promoter activity by dexamethasone, cAMP, and phorbol esters. Insulin inhibited promoter activity in the presence of any of the three stimulatory agents. Stimulation by cAMP or phorbol esters was abolished when the region between nt -327 and -235 was deleted. Although this region contains potential activating protein-2 and activating protein-1 sites, the sites responsible for this regulation have not yet been identified. By contrast, stimulation by dexamethasone was retained in deletion constructs whose 5'-end was at nt -92, but was abolished by site mutagenesis of either the left or right half-sites of a potential glucocorticoid response element (GRE) located between nt -91 and -77. Surprisingly, substitution mutations in an up-stream region, -108 to -99 (M4), also decreased dexamethasone-stimulated promoter activity despite the presence of an intact GRE. We postulate that a positive factor that binds to the wild-type M4 region neutralizes factors that inhibit interaction of the glucocorticoid receptor with the GRE. The M4 region also is involved in inhibition by insulin. Insulin inhibition of dexamethasone-stimulated promoter activity was lost after deletion of nt -135 to -92 or mutation of the region between nt -108 and -99. This insulin response element is conserved in the human IGFBP-1 promoter and is homologous to the insulin response element of the phosphoenolpyruvate carboxykinase gene, which also is rapidly inhibited by insulin in H4-II-E cells. The rat IGFBP-1 promoter provides a valuable model system for studying the multihormonal regulation of transcription.

Insulin-like growth factor (IGF) suppression of IGFBP-1 production: evidence for mediation by the type I IGF receptor.

The regulation of insulin-like growth factor binding protein-1 (IGFBP-1) by its ligands, IGF-I and IGF-II, was studied in continuous cultures of HepG2 human hepatoma cells. Both IGF-I and IGF-II in concentrations as low as 1-10 nmol/l caused significant suppression of IGFBP-I protein levels. This suppression was accompanied by decreased IGFBP-1 mRNA levels occurring within 2-4 h of exposure to IGF-I or IGF-II, and by a significant decrease in IGFBP-1 promoter activity. IGF-I and IGF-II were equipotent in suppressing basal levels of IGFBP-1 protein, mRNA and promoter activity. IGF-I, IGF-II, and IGF-analogs with low IGFBP-1 affinity, (des 1-3)IGF-I and long R3IGF-I, all potently suppressed the previously characterized increase in IGFBP-1 protein levels and promoter activity induced by cAMP and theophylline. In contrast, [Leu-27]IGF-II, which interacts with the type II but not type I IGF receptor, had no effect on IGFBP-1 protein levels or promoter activity. Our data indicate that IGFBP-1 production is inhibited by its ligands, IGF-I and IGF-II, and that this effect is probably mediated at the transcriptional level. The effects of IGF-I and IGF-II apparently occur as a result of binding to the type I IGF receptor, and are similar to the previously characterized suppressive effects of insulin on IGFBP-1 transcription mediated through the insulin receptor. When considered with previous data regarding expression of IGFBP-1 and the type I IGF receptor, our results suggest that IGF regulation of IGFBP-1 may play an as yet undefined role in fetal development and postnatal hepatic regeneration.

Cycloheximide stabilizes insulin-like growth factor-binding protein-1 (IGFBP-1) mRNA and inhibits IGFBP-1 transcription in H4-II-E rat hepatoma cells

The insulin-like growth factor-binding proteins (IGFBPs) are a family of six proteins that modulate the biological activity of IGF-I and IGF-II and determine their bioavailability to tissues. One of the IGFBPs, IGFBP-1, is distinctive in the dynamic response of its levels in human plasma to metabolic changes. Parallel changes occur in IGFBP-1 mRNA and IGFBP-1 transcription in rat liver. Using the well differentiated H4-II-E rat hepatoma cell line as a model system, we demonstrated previously that IGFBP-1 transcription is positively regulated by dexamethasone and negatively regulated by insulin. We now examine the effect of the protein synthesis inhibitor, cycloheximide, on the hormonal regulation of IGFBP-1 gene expression. Preincubation of H4-II-E cells with 10.7 microM cycloheximide for 1.5 h did not prevent the induction of IGFBP-1 mRNA and IGFBP-1 transcription (determined in nuclear run-on assays) by dexamethasone. By contrast, cycloheximide treatment abolished the decrease in IGFBP-1 mRNA induced by insulin. Insulin rapidly decreased IGFBP-1 transcription in the absence of cycloheximide (> 50% inhibition in 20 min) and caused a similar decrease in cells pretreated with cycloheximide. Cycloheximide alone also decreased IGFBP-1 transcription. Similar results were observed with a second protein synthesis inhibitor, anisomycin, which also prevented the insulin-induced decrease in IGFBP-1 mRNA without abolishing the insulin-induced inhibition of IGFBP-1 transcription. These results suggest that although insulin decreases IGFBP-1 gene transcription in the presence of protein synthesis inhibitors, IGFBP-1 mRNA levels are maintained because of stabilization of the mRNA. Stabilization was demonstrated directly in actinomycin D-treated cells, where the t1/2 of IGFBP-1 mRNA increased from approximately 2 to approximately 20 h in the presence of cycloheximide; insulin did not affect IGFBP-1 mRNA turnover. Thus, cycloheximide-sensitive labile proteins contribute to the maintenance of basal IGFBP-1 promoter activity and the rapid turnover of IGFBP-1 mRNA, which determine the dynamic regulation of IGFBP-1 gene expression.

HNF1 activates transcription of the human gene for insulin-like growth factor binding protein-1.

Insulin-like growth factor binding protein-1 (IGFBP-1) is expressed primarily in the liver, kidney, and uterus. Basal IGFBP-1 promoter activity in human HEP G2 hepatoma cells is dependent upon a proximal promoter element that binds hepatic nuclear factor 1 (HNF1), a protein that is likely to be an important factor regulating the expression of many genes in liver and kidney. To test whether HNF1 activates IGFBP-1 transcription, HEP G2 cells and HeLa cells were cotransfected transiently with HNF1 expression vectors and with IGFBP-1 promoter/chloramphenicol acetyltransferase reporter gene constructs. HNF1 increased IGFBP-1 promoter activity in both HEP G2 and HeLa cells. Gel mobility-shift assays and additional transfections in HeLa cells showed that expressed full-length and carboxy-terminal truncated forms of HNF1 could each bind the HNF1 cis element of the IGFBP-1 promoter; however, significant trans-activation only occurred in the presence of the full-length HNF1 protein, similar to past experience with these two HNF1 forms and the albumin promoter. Further studies showed that IGFBP-1 promoter constructs containing mutations with high or low affinity for HNF1 responded to HNF1 expression with increased or decreased activity, respectively, relative to the native promoter. These studies suggest that HNF1 and/or related proteins play a role in hepatic, and perhaps also renal, expression of IGFBP-1.

Insulin rapidly decreases insulin-like growth factor-binding protein-1 gene transcription in streptozotocin-diabetic rats.

Insulin-like growth factor-binding protein-1 (IGFBP-1) can inhibit or potentiate IGF action. The biological activity of IGFBP-1 is determined by many factors, including its abundance in tissues and plasma, posttranslational modifications, and localization. IGFBP-1 levels in human plasma are highly regulated. They are increased after acute fasting and in diabetes, and are rapidly reversed by refeeding and insulin treatment, respectively. Similarly, IGFBP-1 mRNA is increased in the liver of severely diabetic and ketotic rats and decreased after 4 days of insulin treatment. Insulin rapidly decreases IGFBP-1 mRNA and IGFBP-1 transcription in rat hepatoma cells. The present study asks whether the increase in IGFBP-1 mRNA in diabetic rat liver reflects increased gene transcription, whether insulin decreases IGFBP-1 mRNA through a transcriptional or posttranscriptional mechanism, and whether this decrease is sufficiently rapid to account for the dynamic fluctuations in plasma IGFBP-1. Rats were injected ip with 100 mg/kg streptozotocin and used 7 days later when they were hyperglycemic and failed to gain weight, but were not ketotic. Hepatic IGFBP-1 mRNA levels were 13.6 +/- 5.3-fold greater in diabetic than control liver and decreased to the low levels in nondiabetic controls within 1 h after insulin treatment. In run-on transcription assays, IGFBP-1 transcription was 12.6 +/- 1.5-fold greater in nuclei from diabetic than control liver and decreased to low control levels by 1 h after insulin injection. Normalization of hepatic IGFBP-1 mRNA in insulin-treated diabetic animals did not require restoration of euglycemia. IGFBP-1 mRNA and IGFBP-1 gene transcription also were increased in the kidney of diabetic ketotic rats. We propose that the dynamic regulation of IGFBP-1 gene transcription in diabetes and after insulin treatment, by determining the availability of IGFBP-1 in tissues and plasma, may be a critical factor in the modulation of IGF action.

Expression of insulin-like growth factor binding protein-1 mRNA in human fetal kidney.

Expression of insulin-like growth factor binding protein-1 (IGFBP-1) messenger RNA (mRNA) was studied in tissues of human fetuses from 15 to 23 weeks of gestation. Northern blot analysis revealed IGFBP-1 mRNA in the fetal liver and kidney but not in other fetal tissues, including the brain, heart, lung, skeletal muscle and spleen. Studies by in situ hybridization histochemistry showed that, in all fetal kidneys tested, the IGFBP-1 mRNA was localized preferentially to the epithelial cells of the collecting ducts, as well as to the cells of developing glomeruli and in the subcapsular nephrogenic mesenchyme. Less intense labeling for IGFBP-1 mRNA was seen in the connective tissue stroma of the medullary pyramids. A weak signal was detected in the mature glomeruli, and in the cells of the medullary mesenchyme and capsular connective tissue. IGFBP-1 protein was detected by immunoperoxidase staining mostly around small blood vessels but not in the respective endothelium. The protein was also present in many epithelial cells of the collecting ducts and in stromal connective tissue. These results show that the predominant sites of IGFBP-1 transcription in the developing kidney are those with most active differentiation.

Dexamethasone Increases Hepatic Insulin-Like Growth Factor Binding Protein-1 (IGFBP-1) mRNA and Serum IGFBP-1 Concentrations in the Rat*

Glucocorticoid excess is associated with growth retardation in both man and experimental animals. We have previously reported that dexamethasone (DXM) inhibits growth hormone induction of insulin-like growth factor-I (IGF-I) mRNA in the hypophysectomized rat and reduces steady-state IGF-I mRNA levels in the intact rat. However, in these experiments, DXM had surprisingly little effect on serum IGF-I concentrations. Here, we have examined the effect of DXM on hepatic insulin-like growth factor binding protein-1 (IGFBP-1) mRNA levels and on serum IGFBP-1 concentrations. After a single ip injection of DXM, 6 micrograms/100 g body wt, IGFBP-1 mRNA increased 2.24 +/- 0.25-fold, P less than 0.05 at 1 h and declined to normal levels in 6-12 h. A dose-dependent increase in increased hepatic IGFBP-1 mRNA abundance was seen in rats killed 1 h after an ip injection of DXM, 0.1 to 60 micrograms/100g body wt. As little as 1 microgram/100g body wt, significantly enhanced hepatic IGFBP-1 mRNA levels; 2.02- +/- 0.38-fold, P less than 0.05. This effect appeared to be post-transcriptional, since IGFBP-1 transcription in hepatic nuclei from rats treated with DXM was not significantly different from untreated rats. When DXM, 1 microgram/rat, was administered daily for 6 days a significant increase in IGFBP-1 mRNA was detected; 2.02 +/- 0.39-fold, P less than 0.05. A more marked increase was seen with 6 and 60 micrograms/rat of DXM; 4.47 +/- 1.08- and 10.61 +/- 0.31-fold, respectively. Serum was analyzed by sodium dodecyl sulfate-plyacrylamide gel electrophoresis and immunoblotting using antisera raised against a synthetic peptide derived from the predicted sequence of rat IGFBP-1. The antisera recognized a doublet of approximately 30 kDa. A dose-dependent increase in the abundance of these BPs were seen in the serum from rats treated chronically with DXM. The observations reported here clearly demonstrate that DXM increases hepatic IGFBP-1 mRNA and serum IGFBP-1 concentrations. If IGFBP-1 functions to decrease the bioavailability of IGF-1 in vivo the enhanced expression of IGFBP-1 may be an additional mechanism whereby glucocorticoid excess results in growth retardation.