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Biomedical subjects

D R Powell

Publications and source records attributed to D R Powell.

At least 55 records · Page 3Linked to original sources
PubMed1997

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.

Base Sequence↗
PubMed1997

Osteogenic protein-1 stimulates production of insulin-like growth factor binding protein-3 nuclear transcripts in human osteosarcoma cells.

To begin delineating molecular mechanisms by which osteogenic protein-1 (OP-1) modulates its effect on the insulin-like growth factor (IGF) system in human skeletal cells, we evaluated time-course effects of OP-1 on the expression of IGFBP-3 messenger RNA (mRNA) in human SaOS-2 osteosarcoma cells and found that 100 ng/ml of OP-1 increased (maximum 10.7-fold at 24 h; P < 0.01) the level of IGFBP-3 mRNA in a time-dependent manner (from 3-36 h; treatment x time interaction, P < 0.001). The stimulatory effect of OP-1 on IGFBP-3 mRNA was not promoted by transcript stabilization; actually, OP-1 treatment selectively increased the decay of mRNA for IGFBP-3 (T1/2 = 5 h vs. 24 h for OP-1 and controls), but not for IGFBP-4 or beta-actin. Conversely, OP-1 acutely increased IGFBP-3 nuclear transcript abundance in total RNA samples ranging between 1-24 h of treatment. After 6 h of treatment, OP-1 produced an average 4-fold increase (P < 0.02; n = 4 experiments) in the level of IGFBP-3 nuclear transcripts vs. a 3-fold increase (P < 0.01; n = 2 experiments) in mRNA abundance. The OP-1 stimulated induction of IGFBP-3 nuclear transcript and mRNA expression was dependent on de novo protein synthesis. Transient transfection experiments were undertaken to isolate putative OP-1 stimulatory cis-elements within 1.8-kb of the IGFBP-3 5'-flanking region in SaOS-2 and TE-85 osteosarcoma cells. In these experiments, OP-1 did not stimulate IGFBP-3 proximal promoter activity in either cell line, thus suggesting that OP-1 reactive domains may be located either beyond the currently established 5'-flanking region, or within internal exon/intron regions of the IGFBP-3 gene. In conclusion, OP-1 treatment stimulates IGFBP-3 expression in human osteoblastic cells by a mechanism that largely promotes the production of IGFBP-3 nuclear transcripts, a process that requires de novo protein synthesis, and overrides an OP-1-induced targeted degradation of IGFBP-3 steady-state mRNA.

Actins↗
PubMed1997

Decreased hepatic insulin-like growth factor (IGF)-I and increased IGF binding protein-1 and -2 gene expression in experimental uremia.

The imbalance between normal insulin-like growth factor-I (IGF-I) and markedly increased IGF binding protein (IGFBP) plasma levels plays a pathogenic role for growth retardation and catabolism in children with chronic renal failure. To investigate the mechanism of these alterations, experiments were performed in an experimental model of uremia in rats (5/6 nephrectomy) and in pair-fed and ad libitum-fed sham-operated controls Using a specific solution hybridization/RNase protection assay, we observed a marked reduction of hepatic IGF-I messenger RNA (mRNA) abundance at steady state in uremic animals (37 +/- 5% of control) compared both with pair-fed (65 +/- 10%) and ad libitum-fed controls (100 +/- 11%) (P < 0.001). Reduced IGF-I gene expression was clearly organ-specific; it was most pronounced in liver (significant vs., pair-fed controls) and lung and muscle tissue (significant vs., ad libitum-fed controls); no change was observed in kidney and heart tissue. To determine a potential mechanism of reduced hepatic IGF-I gene expression in uremia, the hepatic GH receptor gene expression in the same experimental animals was analyzed by specific solution hybridization/RNase protection assay. Uremic animals had a 20-30% reduction of hepatic GH receptor mRNA abundance compared with controls. Hepatic GHBP expression in uremia was decreased in parallel. Despite the reduction of hepatic IGF-I mRNA abundance, plasma IGF-I levels in uremia were not different from ad libitum-fed controls. This discrepancy is explained by an increased concentration of IGFBPs in uremic plasma. By RIA, plasma IGFBP-1 levels in uremia were increased 4-fold; by Western immunoblot, plasma IGFBP-2 levels were increased 7-fold and plasma IGFBP-4 levels were increased 2-fold compared with both control groups. Intact IGFBP-3 (M(r), approximately 48 kDa) and low molecular IGFBP-3 fragments were not significantly different among the three groups. By Northern blot analysis, hepatic IGFBP-1 mRNA levels in uremia were 2-fold higher than in controls. IGFBP-2 mRNA abundance in liver tissue was increased 4-fold, whereas in kidney there was a significant reduction of IGFBP-2 mRNA (30% of control). IGFBP-4 mRNA was increased by 50% in kidney but not in liver. Plasma insulin and corticosterone levels were not different among the groups. Our study shows that hepatic IGF-I gene expression was specifically reduced in uremia, partially as the consequence of a reduced hepatic GH receptor gene expression. One of the mechanisms contributing to increased IGFBP levels in uremia is increased hepatic gene expression of IGFBP-1 and IGFBP-2. The imbalance between reduced hepatic IGF-I production and increased hepatic IGFBP-1 and 2 production is likely to play a pathogenic role for catabolism and growth failure in CRF.

Animal Nutritional Physiological Phenomena↗
PubMed1997

Kinetics of insulin-like growth factor (IGF) and IGF-binding protein responses to a single dose of growth hormone.

The in vivo physiological relationships among GH, the insulin-like growth factors (IGFs), and the IGF-binding proteins (IGFBPs) are not completely defined, and single random measurements of these serum proteins do not completely reveal their dynamic relationships. We report the kinetic responses of the IGFs and IGF-binding proteins to exogenous GH in 23 subjects with untreated GH deficiency [5 women and 18 men; age, 15.0 +/- 6.2 yr (+/- s.d.), height z-score = -4.4 +/- 2.2 (+/- s.d.); body mass index = 19.3 +/- 2.4 kg/m2]. After an overnight fast, subjects were given a sc dose of recombinant human GH (2.85 i.u./m2), and blood was sampled from an indwelling peripheral venous catheter 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, and 24 h after the injection. Subjects were then treated with recombinant human GH (2.85 i.u./m2.day); fasting samples were obtained at 3 months (n = 22), and timed sampling was repeated at 6 months (n = 21). Fasting levels of IGF-I, free IGF-I, IGF-II, IGFBP-3, and insulin increased significantly within 3 months of GH treatment, whereas IGFBP-1, IGFBP-2, and IGFBP-6 showed no change. In the timed sampling studies at 0 and 6 months, GH levels peaked 3 h after treatment; the degree of rise and the rate of decline were both greater at 6 months. IGF-I levels increased beginning at 4 h, continuing throughout the 24-h period at month 0, whereas a plateau was observed after 6-8 h during the 6-month study. Free IGF-I paralleled total IGF-I except during fasting, when it varied inversely with IGFBP-1. IGFBP-3 and IGF-II both showed late (> 20 h) responses to a dose of GH, whereas IGFBP-2 and IGFBP-6 showed minimal changes. IGFBP-1 varied inversely with insulin, which, in turn, varied with meal intake. Comparative studies in 2 subjects with GH receptor deficiency showed no response to exogenous GH. However, both IGFBP-1 and IGFBP-2 were several-fold elevated, and IGFBP-1 varied inversely with the low insulin levels. Our data are the first to examine multiple elements of the serum IGF system in response to GH in both GH-deficient and replete states. The relationships of the different response patterns provide insight into the physiology of this system and may guide future studies.

Adolescent↗
PubMed1997

Insulin-like growth factor-binding protein-6 levels are elevated in serum of children with chronic renal failure: a report of the Southwest Pediatric Nephrology Study Group.

Previous studies suggest that growth retardation in children with chronic renal failure (CRF) results in part from inhibition of insulin-like growth factor (IGF) action by excess serum IGF-binding proteins (IGFBPs). Excess IGFBPs in CRF serum include IGFBP-1, -2, and -3 and a diffuse approximately 24- to 28-kDa IGFBP band identified by [125I]IGF ligand blot. The present studies characterized this diffuse approximately 24- to 28-kDa band. Initial studies identified this band as IGFBP-6, because it was immunoprecipitated by antiserum raised against a synthetic peptide of human IGFBP-6 (hIGFBP-6). Additional [125I]IGF ligand blots found that the immunoprecipitated band was 1) recognized by [125I]IGF-II but not [125I]IGF-1, 2) more abundant in CRF than in normal serum, and 3) more abundant in serum from dialyzed than nondialyzed prepubertal CRF children. Using the hIGFBP-6 antiserum in a specific and sensitive RIA, we found that serum IGFBP-6 levels were 4.7 +/- 1.7 nmol/L in 10 normal prepubertal children, 21.4 +/- 6.1 nmol/L in 44 nondialyzed prepubertal CRF children, 73.5 +/- 14.4 nmol/L in 7 dialyzed prepubertal CRF children, and 94.6 +/- 26.2 nmol/L in 14 dialyzed pubertal CRF children. IGFBP-6 levels were also elevated in 71 nondialyzed European children with CRF. In nondialyzed CRF children, serum IGFBP-6 levels 1) correlated inversely with the glomerular filtration rate, 2) did not correlate with height SD score, and 3) were not altered by 12 months of daily recombinant hGH treatment. In summary, a specific antiserum and RIA were used to demonstrate elevated levels of intact IGF-II-binding IGFBP-6 in serum of CRF children. We postulate that the excess IGFBP-6 may modulate the action of IGF-II on target tissues.

Adolescent↗
PubMed1997

Analysis of candidate genes for susceptibility to type I diabetes: a case-control and family-association study of genes on chromosome 2q31-35.

Recent genome searches suggest a putative linkage of many loci to susceptibility to type I diabetes. The chromosome 2q31-35 region is reported to be linked to susceptibility to type I diabetes and is thought to contain several diabetes susceptibility loci. These candidate genes include the HOXD gene cluster, BETA2, CTLA4, CD28, IGFBP2, and IGFBP5. Association studies in populations and families are required to confirm and/or identify the actual susceptibility loci. We hereby report several previously unknown DNA polymorphisms for HOXD8, BETA2, and IGFBP5, which we have used along with previously known polymorphisms of HOXD8 and CTLA4 to test whether these candidate loci are the susceptibility genes on chromosome 2q31-35. Using a case-control design with a subsequent family-association approach to confirm associations, we find no evidence that these candidate genes are associated with susceptibility to type I diabetes.

Alleles↗
PubMed1997

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)

Amino Acid Sequence↗
PubMed1997

Implementing a self care program. Effect on employee health care utilization.

1. One way to reduce health care costs is to reduce the demand for health care services. This can be accomplished by teaching employees to make better decisions about when they should see the health care provider or go to the emergency department versus treating themselves at home using self care. 2. In an effort to reduce health care costs, a manufacturing company implemented a self care program using a publication called the HealthyLife Self Care Guide. The guide was distributed to employees during a 50 minute workshop. 3. Analysis of claims data 1 year prior to distribution of the Guide and 1 year after distribution showed a savings of $39.65 per employee (a 24.4% decrease in costs) due to reduced health care provider and emergency department visits. This amounted to a return on investment of 2.6:1. 4. It appears that implementing a self care program in a worksite setting can be an effective way to reduce employer health care costs.

Adult↗
PubMed1996

Insulin-like growth factor binding proteins as growth inhibitors in children with chronic renal failure.

Children with chronic renal failure (CRF) often fail to attain an adult height consistent with their genetic potential. The growth hormone (GH)/insulin-like growth factor (IGF)/growth plate chondrocyte axis has been intensively studied in these children to determine the basis for this growth failure. Evidence suggests that hepatic GH resistance results in deficient expression of IGF-I. However, serum IGF-I levels are usually normal and it is IGF-I action on target tissues which is inhibited, possibly by the presence of excess high-affinity IGF binding proteins (IGFBPs) in CRF serum. In this paper we evaluate the roles of IGFBP-1, -2, and -3 as growth inhibitors in CRF children. The data support a role for each of these IGFBPs as growth inhibitors. Currently, IGFBP-1 meets most criteria expected of a growth inhibitor, but IGFBP-2 and -3 will likely also meet these criteria and may well be important contributors to the growth failure of CRF. Ultimately, many or all of the six IGFBPs may be found to contribute to the excess high-affinity IGF binding sites which are a hallmark of CRF serum and are possible contributors to the growth failure of CRF children.

Adult↗
PubMed1996

Characterization of insulin-like growth factors and their binding proteins in peritoneal dialysate.

Serum insulin-like growth factors (IGFs), which circulate bound to specific IGF binding proteins (IGFBPs), must exit the intravascular space before acting on target tissues. Little is known about the nature of IGF/IGFBPs in extravascular fluids of patients with chronic renal failure (CRF). Peritoneal dialysate (PD) was studied since, after a short incubation, PD contains proteins which have entered an extravascular space; thus, IGF/IGFBP forms in PD are more likely than serum forms to interact with target tissues. IGF-I and IGF-II, and IGFBPs 1-4, were readily identified by specific immunoassays and/or 125iodine-IGF ligand blotting of simultaneously obtained PD and serum samples from seven CRF children; IGFBP-3 was a major IGFBP in PD as in serum. Where quantitated, IGF and IGFBP levels in PD were approximately 10% of serum concentrations. After separation of PD and serum by size-exclusion chromatography, serum had more IGFBP-3 in 150-kilodalton (kDa) than 35-kDa fractions, while PD had far less IGFBP-3 in 150-kDa than 35-kDa fractions. Immunoblot studies revealed a major 29-kDa IGFBP-3 fragment, in addition to intact 41- and 38-kDa IGFBP-3 forms, in PD and CRF serum; the 29-kDa form predominated in the 35-kDa PD fractions. These data suggest that the 29-kDa fragment is the IGFBP-3 form most likely to modulate IGF effects on target tissues of CRF individuals.

Adolescent↗
PubMed1996

Steroidal allylic fluorination using diethylaminosulfur trifluoride: a convenient method for the synthesis of 3 beta-acetoxy-7 alpha- and 7 beta-fluoroandrost-5-en-17-one.

This paper discusses our findings regarding fluorination of the diastereomeric 3 beta-acetoxy-7-hydroxyandrost-5-en-17-ones (3 and 4) at the allylic 7-hydroxyl group using diethylaminosulfur trifluoride under various experimental conditions. The reaction led to the formation of allylic 7 alpha- and 7 beta-fluoro derivatives, 6 and 7, contaminated with small amounts of 3 beta-acetoxy-5 alpha-fluoroandrost-6-en-17-one (8), the rearrangement product, and 3 beta-acetoxyandrosta-4,6-dien-17-one (9), the elimination product. However, synthesis of 3 beta-acetoxy-7 alpha-fluoroandrost-5-en-17-one (6) and 3 beta-acetoxy-7 beta-fluoroandrost-5-en-17-one (7) has been achieved in high isomeric purity by careful manipulation of the experimental conditions. Also included herein is a convenient chemical synthesis of pure 3 beta-acetoxy-7 alpha-hydroxyandrost-5-en-17-one (4) and 3 beta-acetoxy-7 beta-hydroxyandrost-5-en-17-one (3), the starting materials for the present fluorination reaction. The structure of a degradation product, 3 beta-acetoxy-5 alpha-hydroxyandrost-6-en-17-one (5), has been established by X-ray diffraction analysis to ascertain unambiguously its absolute configuration.

Androstenes↗
PubMed1996

Insulin-like growth factor-binding protein-1 expression in cultured human bone cells: regulation by insulin and glucocorticoid.

Insulin-like growth factors (IGFs) and their specific regulatory binding proteins (IGFBPs) are postulated to play a key role in bone metabolism. To date, IGFBP-2 through -6 have been characterized in bone cell systems. In this study we focused on IGFBP-1. Primary cultures of normal human osteoblasts derived from trabecular bone (hOB cells) expressed low levels of IGFBP-1 messenger RNA (mRNA), as determined by Northern analyses. Treatment of hOB cells with 1 microM cortisol or 100 nM dexamethasone for 20 h stimulated IGFBP-1 mRNA expression 5-fold and increased levels of immunoassayable IGFBP-1 in the conditioned medium 3-fold. Estradiol and progesterone had no effect. IGFBP-1 expression was not observed in U-2, TE-85, or MG-63 human osteosarcoma cell lines or in normal human fibroblasts. Insulin (1-100 nM) potently inhibited both basal and glucocorticoid-stimulated IGFBP-1 expression in hOB cells. Insulin had little or no effect on steady state levels of the other IGFBP mRNA. A monoclonal antibody to the insulin receptor blocked insulin binding to insulin receptors and completely prevented insulin-induced suppression of IGFBP-1. In summary, we have documented IGFBP-1 mRNA and protein expression in normal nontransformed human osteoblastic cells. This expression was stimulated by glucocorticoids and inhibited by insulin in a manner similar to IGFBP-1 regulation in hepatocytes. Insulin acts through insulin receptors on hOB cells. We postulate that IGFBP-1 produced by osteoblasts in vivo can modulate local actions of IGF on bone formation in response to changes in glucocorticoid and insulin concentrations.

Bone and Bones↗
PubMed1995

Multiple proteins bind the insulin response element in the human IGFBP-1 promoter.

An insulin response element (IRE) has been identified approximately 100 base pairs (bp) 5' to the transcription start site of the human insulin-like growth factor binding protein-1 (hIGFBP-1) gene. This cis element appears crucial to the multihormonal regulation of hIGFBP-1 expression in liver, since (i) an intact IRE is required for maximal stimulation of hIGFBP-1 promoter activity by dexamethasone, and (ii) the IRE confers insulin inhibition of both basal and dexamethasone-stimulated hIGFBP-1 promoter activity. Further progress in understanding how the IRE confers insulin and glucocorticoid effects requires identification of transcription factors confering effects of these hormones. D-site binding protein (DBP), and members of the hepatic nuclear factor 3 (HNF 3) and high mobility group I/Y (HMG I/Y) protein families, each known to bind DNA elements similar in sequence to the IRE, were tested for IRE binding. DBP, HMGI and HNF 3 beta each protected the hIGFBP-1 IRE from DNAseI digestion. Additional studies are required to establish whether binding of any of these proteins to the IRE is important to the regulation of hIGFBP-1 expression by insulin and/or glucocorticoids.

DNA↗
PubMed1995

Conservation of IGFBP structure during evolution: cloning of chicken insulin-like growth factor binding protein-5.

The insulin-like growth factor binding proteins (IGFBPs) have conserved characteristics of their genomic organization, including similar locations of exon borders relative to nucleotides encoding conserved cysteine residues. Furthermore, the human IGFBP genes, as well as the human homeobox (HOX) genes, are localized to chromosomes 2, 7, 12, and 17. Although little is known about the evolution of the IGFBP genes, the association of human IGFBP and homeobox (HOX) genes at four chromosomal loci may indicate that their ancestral genes were linked prior to the first duplication of chromosomal DNA containing the ancestral HOX cluster. The hypothesis that IGFBPs are ancient proteins is supported by the reported detection of IGFBP activity in serum from the Agnathan species, Geotria australis, a primitive vertebrate. Further studies of IGFBPs in different species are needed to understand the evolution of this protein/gene family. Chicken provides a good intermediate model, since birds diverged from mammals approximately 300 million years ago. A complementary DNA (cDNA) clone encoding chicken insulin-like growth factor binding protein-5 (cIGFBP-5) was isolated. The deduced amino acid sequence is 83% identical to human IGFBP-5 and encodes a mature polypeptide of 251 amino acids. The conservation of IGFBP-5 primary structure across vertebrate species suggests maintenance of important functions during evolution.

Amino Acid Sequence↗
PubMed1995

Hepatic nuclear factor 3- and hormone-regulated expression of the phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein 1 genes.

The rate of transcription of the hepatic phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein 1 (IGFBP-1) genes is stimulated by glucocorticoids and inhibited by insulin. In both cases, the effect of insulin is dominant, since it suppresses both basal and glucocorticoid-stimulated PEPCK or IGFBP-1 gene transcription. Analyses of both promoters by transfection of PEPCK or IGFBP-1-chloramphenicol acetyltransferase fusion genes into rat hepatoma cells has led to the identification of insulin response sequences (IRSs) in both genes. The core IRS, T(G/A)TTTTG, is the same in both genes, but the PEPCK promoter has a single copy of this element whereas the IGFBP-1 promoter has two copies arranged as an inverted palindrome. The IGFBP-1 IRS and PEPCK IRS both bind the alpha and beta forms of hepatic nuclear factor 3 (HNF-3), although the latter does so with a sixfold-lower relative affinity. Both the PEPCK and the IGFBP-1 IRSs also function as accessory factor binding sites required for the full induction of gene transcription by glucocorticoids. A combination of transient transfection and DNA binding studies suggests that HNF-3 is the accessory factor that supports glucocorticoid-induced gene transcription. In both genes, the HNF-3 binding site overlaps the IRS core motif(s). A model in which insulin is postulated to mediate its negative effect on glucocorticoid-induced PEPCK and IGFBP-1 gene transcription indirectly by inhibiting HNF-3 action is proposed.

Animals↗
PubMed1994

Glucocorticoids and insulin regulate expression of the human gene for insulin-like growth factor-binding protein-1 through proximal promoter elements.

Glucocorticoids stimulate, while insulin inhibits, the hepatic transcription of insulin-like growth factor-binding protein-1 (IGFBP-1). In the present studies, human HEP G2 hepatoma cells were transiently transfected with human (h)IGFBP-1 promoter constructs. Activity of a construct containing the first 1205 base pairs (bp) of the hIGFBP-1 promoter was stimulated 6-9.5-fold by dexamethasone, and this increase was inhibited approximately 76% by insulin. Deletion and site-directed mutations of the hIGFBP-1 promoter (a) identified two glucocorticoid response elements, located within the first 200 bp of the promoter, which are essential for dexamethasone-stimulated promoter activity and which specifically bind human glucocorticoid receptor; (b) showed that a recently characterized insulin-responsive element, located approximately 110 bp 5' to the transcription start site (Suwanichkul, A., Morris, S.L., and Powell, D. R. (1993) J. Biol. Chem. 268, 17063-17068), confers the entire inhibitory effect of insulin not only on basal but also on glucocorticoid-stimulated promoter activity; and (c) showed that this insulin-responsive element is essential for maximal glucocorticoid-stimulated activity. These studies suggest that the interaction of proteins that bind to a cluster of cis elements located in the first 200 bp of the hIGFBP-1 promoter are of major importance in modulating the opposing effects of glucocorticoids and insulin on hepatic hIGFBP-1 expression.

Base Sequence↗
PubMed1994

Characterization of the chromosomal gene and promoter for human insulin-like growth factor binding protein-5.

To better understand the regulation of insulin-like growth factor binding protein-5 (IGFBP-5) expression, we cloned the IGFBP-5 gene from human genomic libraries and identified a region in the 5' flanking sequence which functions as a promoter. The human IGFBP-5 gene is divided into four exons which, primarily due to a first intron of approximately 25 kilobases, span approximately 33 kilobases of DNA. Southern analysis identified a single copy of the IGFBP-5 gene in the haploid human genome, and several independent mapping strategies found this gene tightly linked with, and in opposite transcriptional orientation to, the IGFBP-2 gene at chromosomal region 2q33-34. Primer extension studies identified the IGFBP-5 mRNA cap site 772 base pairs (bp) 5' to the first nucleotide of the translation start codon. Analysis of the 5'-flanking sequence identified a potential TATA element beginning 33 bp 5' to the mRNA cap site. When a DNA fragment containing this cap site and 461 bp of upstream sequence was placed 5' to the chloramphenicol acetyltransferase reporter gene and transfected into MDA-MB-468 human breast cancer cells, it directed chloramphenicol acetyltransferase expression in an orientation-specific manner, suggesting that this region contains elements essential for IGFBP-5 promoter activity.

Amino Acid Sequence↗