Rat Insulin-like Growth factor-I Gene Research Articles

Dispersed Chromosomal Stat5b-binding Elements Mediate Growth Hormone-activated Insulin-like Growth Factor-I Gene Transcription

The growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis regulates somatic growth during childhood and orchestrates tissue repair throughout the life span. Recently described inactivating mutations in Stat5b in humans with impaired growth have focused attention on this transcription factor as a key agent linking GH-stimulated signals to IGF-I gene expression, and several putative Stat5b sites have been identified in the IGF-I gene. Here, we define and characterize potential GH- and Stat5b-activated chromosomal enhancers that can regulate IGF-I gene transcription. Of 89 recognizable Stat5 sequences in 200 kb centering on the rat IGF-I gene, 22 resided within conserved regions and/or were identical among different species. Only 15 of these sites, organized into 7 distinct domains, were found to bind Stat5b by quantitative chromatin immunoprecipitation assays in liver chromatin of rats, but only after acute GH treatment. These sites could bind Stat5b in vitro, and individual domains could mediate GH- and Stat5b-stimulated IGF-I promoter activity in cultured cells. Further analyses revealed that four Stat5b domains possessed chromatin signatures of enhancers, including binding of co-activators p300 and Med1, and RNA polymerase II. These modifications preceded GH-stimulated recruitment of Stat5b, as did lysine 4 monomethylation of histone H3, which was enriched in 6/7 Stat5b-binding elements. In contrast, histone acetylation was induced by GH but was limited to Stat5b binding domains found within the IGF-I transcription unit. We conclude that GH stimulates recruitment of Stat5b to multiple dispersed regions within the igf1 locus, including several with properties consistent with long range transcriptional enhancers that collectively regulate GH-activated IGF-I gene transcription.

Mechanisms of Growth Hormone (GH) Action: IDENTIFICATION OF CONSERVED Stat5 BINDING SITES THAT MEDIATE GH-INDUCED INSULIN-LIKE GROWTH FACTOR-I GENE ACTIVATION

Many of the actions of growth hormone (GH) on somatic growth and tissue maintenance are mediated by insulin-like growth factor-I (IGF-I), a secreted protein whose gene expression is rapidly and potently induced by GH by unknown mechanisms. Recent studies implicating Stat5a and Stat5b in the growth response to GH in mice and observations linking Stat5b to control of IGF-I gene transcription in rats have prompted the current investigations into the molecular determinants of a putative regulatory network extending from GH through Stat5b to IGF-I. Here we characterize as critical components of this hormone-activated transcriptional pathway two adjacent Stat5 binding sites in the second intron of the rat IGF-I gene located within a conserved region previously found to undergo acute and reversible changes in chromatin structure after in vivo GH treatment. As assessed by chromatin immunoprecipitation assays, GH rapidly induced binding of Stat5 to this DNA segment in the liver of GH-deficient rats, just prior to the onset of transcription from both major and minor IGF-I gene promoters. Biochemical reconstitution experiments showed that the two intronic Stat5 DNA elements were able to bind Stat5b in vitro after GH treatment could transmit GH- and Stat5b-dependent transcriptional responsiveness to the major IGF-I promoter and to a minimal neutral gene promoter and were required for full stimulation of reporter gene activity by GH. Taken together, these results identify an intronic enhancer as a key mediator of GH-induced IGF-I gene transcription working through Stat5b and provide new insight into the molecular architecture of this transcriptional pathway.

IGF-I Transcript Levels in Whole-Liver Tissue, in Freshly Isolated Hepatocytes, and in Cultured Hepatocytes from Lean and Obese Zucker Rats

Background: The mechanisms underlying the maintenance of normal to high rates of linear growth and plasma insulin-like growth factor I (IGF-I) levels in spite of a low growth hormone secretion in obese children remain unknown. Among the animal models of early-onset obesity, obese Zucker (fa/fa) rats (which are homozygous for an inactivating missense mutation in the leptin receptor) are particularly appropriate, because their linear growth shows this growth hormone independence. Methods: To study the regulation of IGF-I synthesis in this model, we have established primary cultures of hepatocytes derived from 12-week-old Zucker male obese and lean rats. The rat IGF-I gene contains six exons, and alternative splicing generates different mRNAs, one of which (called IGF-1B) has been shown to be decreased by fasting. We report steady state mRNA levels for IGF-I (all transcripts) and for IGF-IB in hepatocytes after 3 days in culture, in freshly isolated hepatocytes, and in whole-liver tissue. RT-PCRs using primers specific for IGF-I or IGF-IB were performed with two different internal competitors for quantification. Results: In primary cultures of hepatocytes, the IGF-IB mRNA was increased by >50-fold (p = 0.01) in cells derived from obese animals as compared with cells from lean animals. However, these transcript levels were not significantly different when measured in freshly isolated hepatocytes or in whole-liver tissue. Conclusions: Increased IGF-IB transcription could be an intrinsic characteristic of cultured hepatocytes harbouring leptin receptors that bear the fa mutation. However, the modulation of this characteristic by cell-cell interactions and by in vivo hormone and metabolic status remains to be studied.

Developmental regulation of insulin-like growth factor-I and growth hormone receptor gene expression

During development, the insulin-like growth factor I (IGF-I) gene is expressed in a tissue specific manner; however, the molecular mechanisms governing its developmental regulation remain poorly defined. To examine the hypothesis that expression of the growth hormone (GH) receptor accounts, in part, for the tissue specific expression of the IGF-I gene during development, the developmental regulation of IGF-I and GH receptor gene expression in rat tissues was examined. The level of IGF-I and GH receptor mRNA was quantified in RNA prepared from rats between day 17 of gestation (E17) and 17 months of age (17M) using an RNase protection assay. Developmental regulation of IGF-I gene expression was tissue specific with four different patterns of expression seen. In liver, IGF-I mRNA levels increased markedly between E17 and postnatal day 45 (P45) and declined thereafter. In contrast, in brain, skeletal muscle and testis, IGF-I mRNA levels decreased between P5 and 4M but were relatively unchanged thereafter. In heart and kidney, a small increase in IGF-I mRNA levels was observed between the early postnatal period and 4 months, whereas in lung, minimal changes were observed during development. The changes in GH receptor mRNA levels were, in general, coordinate with the changes in IGF-I mRNA levels, except in skeletal muscle. Interestingly, quantification of GH receptor levels by Western blot analysis in skeletal muscle demonstrated changes coordinate with IGF-I mRNA levels. The levels of the proteins which mediate GH receptor signaling (STAT1, -3, and -5, and JAK2) were quantified by Western blot analysis. These proteins also are expressed in a tissue specific manner during development. In some cases, the pattern of expression was coordinate with IGF-I gene expression, whereas in others it was discordant. To further define molecular mechanisms for the developmental regulation of IGF-I gene expression, protein binding to IGFI-FP1, a protein binding site that is in the major promoter of the rat IGF-I gene and is important for basal promoter activity in vitro, was examined. Gel shift analyses using a 34-base pair oligonucleotide that contained IGFI-FP1 did not demonstrate changes in protein binding that paralleled those in IGF-I gene expression, suggesting that protein binding to IGFI-FP1 does not contribute to the developmental regulation of IGF-I gene expression, at least in brain and liver. In summary, the present studies demonstrate coordinate expression of the IGF-I gene and GH receptor during development and suggest that GH receptor expression contributes to the tissue specific expression of the IGF-I gene during development.

Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I.

The molecular mechanisms underlying myogenic induction by insulin-like growth factor I (IGF-I) are distinct from its proliferative effects on myoblasts. To determine the postmitotic role of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell lines carrying a stably transfected rat IGF-I gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC-IGF-I exclusively in differentiated L6E9 myotubes, which express the embryonic form of myosin heavy chain (MyHC) and no endogenous IGF-I, resulted in pronounced myotube hypertrophy, accompanied by activation of the neonatal MyHC isoform. The hypertrophic myotubes dramatically increased expression of myogenin, muscle creatine kinase, beta-enolase, and IGF binding protein 5 and activated the myocyte enhancer factor 2C gene which is normally silent in this cell line. MLC-IGF-I induction in differentiated L6E9 cells also increased the expression of a transiently transfected LacZ reporter driven by the myogenin promoter, demonstrating activation of the differentiation program at the transcriptional level. Nuclear reorganization, accumulation of skeletal actin protein, and an increased expression of beta1D integrin were also observed. Inhibition of the phosphatidyl inositol (PI) 3-kinase intermediate in IGF-I-mediated signal transduction confirmed that the PI 3-kinase pathway is required only at early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction in these cells. Expression of IGF-I in postmitotic muscle may therefore play an important role in the maturation of the myogenic program.

Regulation of insulin-like growth factor I (IGF-I) gene expression in brain of transgenic mice expressing an IGF-I-luciferase fusion gene.

Insulin-like growth factor I (IGF-I) plays an important role in the development and function of the central nervous system (CNS). Little is known, however, about the factors and mechanisms involved in regulation of CNS IGF-I gene expression. To facilitate our goal to define mechanisms of IGF-I gene regulation in the CNS, we generated several lines of transgenic (Tg) mice that express firefly luciferase (LUC) under control of a 11.3-kb fragment from the 5' region of the rat IGF-I gene. Consistent with expression of the native IGF-I gene in murine brain, expression of the transgene predominated in neurons and astrocytes and used promoter 1, the major IGF-I promoter in the CNS and in most tissues. Transgene messenger RNA and protein expression rapidly increased after birth and peaked at postnatal (P) day 4 in all brain regions studied. LUC activities in all regions then gradually decreased to 0.5-4% of their peak values at P31, except for the olfactory bulb, which maintained about one third of its maximal activity. Compared with littermate controls, administration of dexamethasone decreased LUC activity and transgenic IGF-I messenger RNA abundance, whereas GH significantly increased the expression of the transgene. Addition of GH to cultured fetal brain cells from Tg mice for 12 h also increased LUC activity in a dose-dependent manner (77-388%). These results show that this IGF-I promoter transgene is expressed in a fashion similar to the endogenous IGF-I gene, and thus indicates that the transgene contains cis-elements essential for developmental, GH, and glucocorticoid regulation of IGF-I gene expression in the CNS. These Tg mice should serve as an useful model to study mechanisms of IGF-I gene regulation in the brain.

17beta-estradiol potently suppresses cAMP-induced insulin-like growth factor-I gene activation in primary rat osteoblast cultures.

Insulin-like growth factor-I (IGF-I) is a key factor in bone remodeling. In osteoblasts, IGF-I synthesis is enhanced by parathyroid hormone and prostaglandin E2 (PGE2) through cAMP-activated protein kinase. In rats, estrogen loss after ovariectomy leads to a rise in serum IGF-I and an increase in bone remodeling, both of which are reversed by estrogen treatment. To examine estrogen-dependent regulation of IGF-I expression at the molecular level, primary fetal rat osteoblasts were co-transfected with the estrogen receptor (hER, to ensure active ER expression), and luciferase reporter plasmids controlled by promoter 1 of the rat IGF-I gene (IGF-I P1), used exclusively in these cells. As reported, 1 microM PGE2 increased IGF-I P1 activity by 5-fold. 17beta-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE2 by up to 90% (ED50 approximately 0.1 nM). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17alpha-estradiol was 100-300-fold less effective. By contrast, 17beta-estradiol stimulated estrogen response element (ERE)-dependent reporter expression by up to 10-fold. 17beta-Estradiol also suppressed an IGF-I P1 construct retaining only minimal promoter sequence required for cAMP-dependent gene activation, but did not affect the 60-fold increase in cAMP induced by PGE2. There is no consensus ERE in rat IGF-I P1, suggesting novel downstream interactions in the cAMP pathway that normally enhances IGF-I expression in skeletal cells. To explore this, nuclear extract from osteoblasts expressing hER were examined by electrophoretic mobility shift assay using the atypical cAMP response element in IGF-I P1. Estrogen alone did not cause DNA-protein binding, while PGE2 induced a characteristic gel shift complex. Co-treatment with both hormones caused a gel shift greatly diminished in intensity, consistent with their combined effects on IGF-I promoter activity. Nonetheless, hER did not bind IGF-I cAMP response element or any adjacent sequences. These results provide new molecular evidence that estrogen may temper the biological effects of hormones acting through cAMP to regulate skeletal IGF-I expression and activity.

Transcription initiation of the rat insulin-like growth factor-I gene in hepatocyte primary culture.

Transcription initiation in the insulin-like growth factor-I (IGF-I) gene is complex, involving multiple sites in two exons. While most transcripts are initiated in exon 1 in vivo, critical regulatory mechanisms are difficult to assess in intact animals. To examine the impact of insulin and growth hormone (GH) under more controlled conditions, we have studied the utilization of different exon 1 and exon 2 transcription-initiation sites in normal rat hepatocytes in primary culture. Normal rat hepatocytes were cultured for 48 h in serum-free medium, with insulin at 10(-6) or 10(-11) M, and with or without human GH 200 ng/ml. Relative abundance of IGF-I transcripts was evaluated by the RNase-protection assay, using a probe which permitted identification of initiation in exon 1 (site 1 (-380 bp from the 3' end of exon 1), site 2 (-343 bp), site 3 (-242 bp), sites 1 and 2 spliced, and site 4 (-32 bp)), as well as in exon 2. After normalization of signal intensity to adjust for differences in length of protected probe, the utilization of initiation sites in vitro was remarkably similar to that in vivo: 1, 14, 6, 23, 19 and 37% for sites 1, 2, 3, 1 and 2 spliced, 4 and exon 2 respectively in the cultured hepatocytes, compared with 1, 12, 18, 21, 18 and 40% for these sites in normal liver. Insulin alone increased transcripts initiated from exon 1, site 2 by over 3 times, and both sites 1 and 2 spliced and exon 2 transcripts by about 5 times. GH alone had similar effects, producing a 4-5 times increase in transcripts from these initiation sites. Addition of both insulin and GH had additive effects, increasing transcripts from exon 1, sites 2, 3 and 4 by 4-6 times, and from exon 1, sites 1 and 2 spliced, and exon 2 by over 8 times. Of the total IGF-I mRNA transcripts, 37% were initiated from sites 2 and/or sites 1 and 2 spliced, and 37% from exon 2. Analysis of the relative contribution of individual initiation sites revealed hormone-induced increases which were statistically significant only for exon 2, in the presence of insulin alone and in combination with GH. In conclusion, in cultured hepatocytes, insulin or GH alone produced a coordinated increase in all exon 1 transcripts, and the effect of the combination of insulin and GH was additive for these transcripts. Exon 2 appeared to be more sensitive to insulin alone, and to GH in the presence of insulin, than exon 1. Since utilization of initiation sites in hepatocytes mimics that found in liver, this in vitro system should be useful for examining underlying transcriptional regulatory mechanisms.

The major promoter of the rat insulin-like growth factor-I gene binds a protein complex that is required for basal expression

IGF-I gene transcription is regulated by two promoters — the major promoter which is active in all tissues and regulates transcription of IGF-I mRNAs that contain exon 1 and a second promoter which regulates transcription of IGF-I mRNAs that contain exon 2 and from which significant transcription is restricted to the liver. The major promoter is a TATAA-less promoter that lacks both a CAAT box and SP1 binding sites and that utilizes multiple transcription initiation sites. The current studies were designed to delineate the functional elements of the major promoter. Transient transfection assays using rat C6 glioma cells and rat dermal fibroblasts in primary culture demonstrated that basal activity of the major promoter was located between −18 (with +1 defined as the most 5′ transcription initiation site in exon 1) and +78 of exon 1. DNase I footprinting, which was performed using nuclear extracts from rat C6 glioma cells, demonstrated protein binding to a sequence that extended from −10 to +9 (termed IGFI-FP1). In gel shift assays, binding of C6 cell nuclear proteins to a 34-basepair (bp) oligonucleotide that contained IGFI-FP1 resulted in the formation of three specific protein-DNa complexes. The functional role of protein binding to IGFI-FP1 was examined by mutating the sequences between either −4 and −2 or −9 and −7 in IGF-I-luciferase fusion genes that contained either 412 or 18 bp of 5′-flanking region and 302 bp of exon 1. Both of these mutations altered protein binding to IGFI-FP1 as demonstrated by gel shift analysis. Transfection of the wild-type and mutant fusion genes into C6 glioma cells demonstrated that mutation of the nucleotides between −4 and −2 decreased luciferase activity to ∼50% of wild-type activity, whereas mutation of the nucleotides between −9 and −7 decreased luciferase activity to 11–35% of wild-type activity. These data demonstrate that: (i) basal activity of the major promoter of the rat IGF-I gene is localized to the region between −18 and +78 of exon 1; (ii) protein binding sites are present within this region of the major promoter; and (iii) protein binding to this region contributes to basal expression of the IGF-I gene.

Transcriptional Regulation of the Rat Insulin-like Growth Factor-I Gene Involves Metabolism-dependent Binding of Nuclear Proteins to a Downstream Region

Insulin-like growth factor-I (IGF-I) gene transcription is mediated largely via exon 1. In an initial search for regulatory regions, rat hepatocytes were transfected with IGF-I constructs. Since omission of downstream sequences led to reduced expression, we then used in vitro transcription to evaluate potential metabolic regulation via downstream regions. With templates including 219 base pairs of downstream sequence, transcriptional activity was reduced 70-90% with hepatic nuclear extracts from diabetic versus normal rats. However, activity was comparable with templates lacking downstream sequences. The downstream region contained six DNase I footprints, and templates with deletion of either region III or V no longer provided reduced transcriptional activity with nuclear extracts from diabetic rats. Nuclear protein binding to regions III and V appeared to be metabolically regulated, as shown by reduced DNase I protection and activity in gel mobility shift assays with nuclear extracts from diabetic rats. Southwestern blotting probes corresponding to regions III and V recognized a approximately 65-kDa nuclear factor present at reduced levels in diabetic rats. These findings indicate that a downstream region in exon 1 may be important for both IGF-I expression and metabolic regulation. Altered concentration or activity of a transcription factor(s) binding to this region may contribute to reduced IGF-I gene transcription associated with diabetes mellitus.

Rapid activation of rat insulin-like growth factor-I gene transcription by growth hormone reveals no changes in deoxyribonucleic acid-protein interactions within the second promoter

Rapid activation of rat insulin-like growth factor-I gene transcription by growth hormone reveals no changes in deoxyribonucleic acid-protein interactions within the second promoter

Rapid activation of rat insulin-like growth factor-I gene transcription by growth hormone reveals no changes in deoxyribonucleic acid-protein interactions within the second promoter.

Insulin-like growth factor-I (IGF-I) is a highly conserved 70-residue circulating peptide that mediates many of the systemic growth-promoting effects of GH. This laboratory has found previously that GH rapidly stimulates hepatic IGF-I transcription in hypophysectomized (hypox) rats by activating promoter 1, the major rat IGF-I gene promoter. In this study, the hormonal regulation of IGF-I expression through promoter 2, a minor promoter in most tissues but active in the liver, was investigated. Through use of a sensitive RNase protection assay, GH was shown to rapidly induce the accumulation of correctly initiated transcripts directed by this promoter in hepatic nuclei. Using in vitro DNase-I footprinting, six DNA-protein interactions were identified within promoter 2 with hepatic nuclear extracts from juvenile male hypox rats given a single ip injection of GH or saline 60 min before death. These DNA-protein-binding complexes also were investigated for specificity and for regulation by GH by gel mobility shift assays. All DNA-protein interactions were detected in hepatic nuclear protein extracts from hypox rats and did not change within 15-120 min after GH treatment. These results thus identify and characterize a series of constitutive nuclear protein-binding sites within the second rat IGF-I promoter that may be involved in mediating its transcriptional activity.

Characterization of deoxyribonucleic acid-protein interactions at a growth hormone-inducible nuclease hypersensitive site in the rat insulin-like growth factor-I gene.

Many of the growth-promoting effects of GH are mediated by insulin-like growth factor-I (IGF-I), a highly conserved, 70-residue basic peptide. Previous studies have demonstrated that GH rapidly stimulates IGF-I expression in vivo, and our laboratory has identified a GH-regulated alteration in chromatin configuration, manifested as a hormonally induced deoxyribonuclease-I (DNase-I)-hypersensitive site in the second IGF-I intron. In the current study, we have used in vivo DNase-I footprinting to map this hormonally responsive chromatin domain to an approximately 350-nucleotide region and have identified DNA-protein interactions within the hypersensitive site by in vitro gel mobility shift experiments and DNase-I footprinting studies. DNA-protein binding was localized to two adjacent segments of 32 and 48 nucleotides. In 1 of these regions, protein-DNA contacts were also detected in vivo on guanine residues by dimethylsulfate footprinting. DNA-binding activity was present in GH-deficient rats, but was not modified by hormone treatment. Our results define a rapid and reversible genomic alteration in response to GH in a GH-regulated gene and delineate a target within chromatin for GH action.

Characterization of deoxyribonucleic acid-protein interactions at a growth hormone-inducible nuclease hypersensitive site in the rat insulin-like growth factor-I gene

Many of the growth-promoting effects of GH are mediated by insulin-like growth factor-I (IGF-I), a highly conserved, 70-residue basic peptide. Previous studies have demonstrated that GH rapidly stimulates IGF-I expression in vivo, and our laboratory has identified a GH-regulated alteration in chromatin configuration, manifested as a hormonally induced deoxyribonuclease-I (DNase-I)-hypersensitive site in the second IGF-I intron. In the current study, we have used in vivo DNase-I footprinting to map this hormonally responsive chromatin domain to an approximately 350-nucleotide region and have identified DNA-protein interactions within the hypersensitive site by in vitro gel mobility shift experiments and DNase-I footprinting studies. DNA-protein binding was localized to two adjacent segments of 32 and 48 nucleotides. In 1 of these regions, protein-DNA contacts were also detected in vivo on guanine residues by dimethylsulfate footprinting. DNA-binding activity was present in GH-deficient rats, but was not modified by hormone treatment. Our results define a rapid and reversible genomic alteration in response to GH in a GH-regulated gene and delineate a target within chromatin for GH action.

Rapid activation of rat insulin-like growth factor-I gene transcription by growth hormone reveals no alterations in deoxyribonucleic acid-protein interactions within the major promoter.

Insulin-like growth factor-I (IGF-I) is an important mediator of prenatal and postnatal growth, but little is known about the control of IGF-I gene expression. Previously, we demonstrated that GH rapidly stimulates hepatic IGF-I transcription in vivo in hypophysectomized (hypox) rats. In this study, we show that GH induces IGF-I gene transcription through the major promoter, promoter 1, and identify and characterize DNA-protein interactions throughout the promoter. In vitro deoxyribonuclease-I footprinting was used to analyze 1711 nucleotides of promoter 1 and the entire 328-nucleotide 5'-untranslated region of exon 1, using hepatic nuclear protein extracts from male juvenile hypox rats given a single ip injection of GH or saline 60 min before death. Fourteen DNA-protein binding sites were identified, with 6 located in the highly conserved 5'-untranslated region of exon 1. These latter sites were further characterized for specificity and regulation by GH, using gel mobility shift assays. Two of these DNA-protein interactions were also detected by in vivo dimethylsulfate footprinting. All DNA-protein binding was seen using hepatic nuclear protein extracts from hypox rats and did not change within 15, 30, 60, or 120 min after treatment with GH. Our results thus define a series of constitutive DNA-protein interactions within the major rat IGF-I gene promoter that may be involved in mediating GH-activated nuclear signals to initiate IGF-I transcription.

Rapid activation of rat insulin-like growth factor-I gene transcription by growth hormone reveals no alterations in deoxyribonucleic acid- protein interactions within the major promoter

Rapid activation of rat insulin-like growth factor-I gene transcription by growth hormone reveals no alterations in deoxyribonucleic acid- protein interactions within the major promoter

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) treatment of the GH-deficient dwarf rat: differential effects on IGF-I transcription start site expression in hepatic and extrahepatic tissues and lack of effect on type I IGF receptor mRNA expression

The rat IGF-I gene consists of six exons, with exons 3 and 4 forming a ‘core’ mature IGF-I coding region to which alternate 5′ and 3′ regions are spliced. Transcription occurs from four dispersed start sites (ss) ≈ 382 (ss 1), ≈ 343 (ss 2), ≈ 245 (ss ≈ 30–40 (ss 4) basepairs (bp) from the 3′ end of exon 1, and from a region 50–70 bp from the 3′ end of exon 2. The expression of ss mRNAs displays tissue-specific and ontogenic regulation. Alternate splicing of exon 5 produces E-peptide coding domain variants (Ea and Eb mRNAs), with the Eb form found predominantly in the liver. The regulation of IGF-I mRNA expression by GH and IGF-I in the GH-deficient dwarf ( dw / dw) rat was investigated using antisense RNA probes in a solution hybridization RNase protection assay to detect leader exon and E domain variant mRNAs. GH treatment of dw / dw and normal Lewis rats increased the expression of all liver leader exon ss and E domain variants coordinately (1.6–1.9-fold increase, p < 0.01), although the increase observed in Eb transcripts was significantly higher in the dw / dw compared to the normal rat ( p < 0.05). In kidney, GH treatment significantly increased exon 1 ss 3 and ss 4 transcripts by approximately 40% ( p < 0.05). The expression of the other start sites was not affected by GH, suggesting that transcription factors may regulate start site usage independently. GH treatment was associated with a significant increase in IGF-I mRNA expression in skeletal muscle ( p < 0.05) but not cardiac muscle or spleen. IGF-I treatment was associated with minor (≈ 20%) but significant ( p < 0.05) reductions in IGF-I mRNA expression in the liver and kidney of dw / dw rats, suggesting that IGF-I can suppress IGF-I mRNA expression. IGF-I treatment did not affect IGF-I mRNA expression in cardiac and skeletal muscle of dw / dw rats. IGF-I receptor mRNA was detected in extrahepatic tissues only, and was not affected by either GH or IGF-I treatment. In summary, start site-specific regulation by GH was observed in kidney. GH increased IGF-I mRNA expression in muscle, kidney and liver, but had no effect in heart or spleen in the dw / dw rat. Our data suggest that systemic IGF-I can feedback on hepatic and renal IGF-I mRNA expression in the GH-deficient state.

Prostaglandin E2 rapidly stimulates insulin-like growth factor-I gene expression in primary rat osteoblast cultures: evidence for transcriptional control.

Osteoblast-enriched (Ob) cultures isolated from fetal rat bone synthesize insulin-like growth factor-I (IGF-I), which functions as a locally acting growth and differentiation factor in the skeleton. Consistent with prior studies demonstrating that IGF-I production is enhanced in bone by agents that induce cAMP, prostaglandin E2 (PGE2) stimulates both cAMP synthesis and IGF-I mRNA in Ob cells. However, little is known about how cAMP regulates IGF-I expression in this or any other cell system. In rat tissues, multiple mechanisms influence levels of IGF-I mRNA, including transcription from two promoters, differential RNA splicing and stability, and alternative RNA polyadenylation. To determine how cAMP influences IGF-I gene expression in Ob cultures, we examined the responses of these cells to treatment with PGE2. PGE2 rapidly enhanced the accumulation of both large and small IGF-I transcripts, with increases in IGF-I mRNA detected within 2 h of treatment and persisting for 24 h. Analysis of precursor RNA by a highly specific and sensitive ribonuclease protection assay demonstrated a rise in nascent IGF-I mRNA within 30 min of exposure to PGE2, with a peak stimulation of 4-fold above control levels seen by 2 h and levels remaining elevated for up to 24 h. IGF-I transcripts in Ob cells were directed only by promoter 1, the more 5' of the two rat IGF-I gene promoters. As additionally assessed using the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole, PGE2 treatment had little effect on IGF-I mRNA stability. In aggregate, these studies show that in fetal rat Ob cultures, PGE2 enhances IGF-I gene expression primarily through transcriptional mechanisms that are limited to a single IGF-I gene promoter. Ob cells, therefore, may be an excellent model for determining how cAMP regulates IGF-I gene transcription.

Multifactorial regulation of IGF‐I gene expression

Insulin-like growth factor-I (IGF-I) is a highly conserved 70-residue circulating peptide with diverse biological effects. In mammals IGF-I is an essential mediator of normal postnatal growth and its expression is influenced by hormonal, nutritional, tissue-specific, and developmental factors. Recent studies have demonstrated that the IGF-I gene is more complicated than might have been predicted from its simple protein sequence. In rats and in humans the single-copy six-exon gene is transcribed by adjacent promoters into nascent RNAs with different 5' leader sequences that undergo both alternative RNA splicing and differential polyadenylation to yield multiple mature transcripts. These observations suggest that trophic agents may modulate expression of IGF-I at any of several nodal points. In this report we review several of the mechanisms responsible for regulating production of IGF-I in the rat. During neonatal development IGF-I gene transcription is progressively activated, leading to a rise in both hepatic IGF-I mRNA and in serum IGF-I. The induction of IGF-I expression is limited to mRNAs directed by promoter 1, the more 5' of two rat IGF-I gene promoters, and precedes the ontogenic appearance of liver growth hormone (GH) receptors, indicating that mechanisms independent of GH activate IGF-I expression during early postnatal life. By contrast, in adult GH-deficient rats, a single intraperitoneal injection of GH causes a prompt rise in IGF-I gene transcription that is mediated equivalently by promoters 1 and 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple transcription start sites in the rat insulin-like growth factor-I gene give rise to IGF-I mRNAs that encode different IGF-I precursors and are processed differently in vitro.

Two distinct class 1 and class 2 rat liver IGF-I mRNAs contain different 5' leader exons, 1 and 2. RNase protection, primer extension, RACE PCR and ribonuclease H mapping established the complete structure of the 5' end of class 1 and class 2 IGF-I mRNAs. Two major transcription start sites in exon 1 yield class 1 IGF-I mRNAs, including 345 or 245 bases of exon 1. Multiple, clustered transcription start sites in exon 2 yield class 2 IGF-I mRNAs with 84-50 bases of exon 2. Cell-free translation of in vitro transcribed IGF-I mRNAs suggests that class 1 and class 2 mRNAs preferentially initiate translation at distinct AUG codons to result in IGF-I precursors with either 48 residue class 1 pre-peptides or 32 residue class 2 pre-peptides. Some translation initiation also occurs at a downstream AUG common to class 1 and 2 mRNAs to yield IGF-I precursors with a 22 residue pre-peptide. Inclusion of microsomal membranes in translations suggests that the three different pre-peptides each function as co-translationally cleaved signal peptides. However, treatment of processed precursors with endoglycosidase H indicates that co-translational processing of precursors with 22 and 32 residue pre-peptides leads to glycosylation of downstream IGF-I precursor sequences whereas co-translational processing of precursors with 48 residue pre-peptide is not associated with glycosylation.