Regulation Of Insulin-like Growth factor-I Gene Expression Research Articles

Identification of a Distal STAT5-binding DNA Region That May Mediate Growth Hormone Regulation of Insulin-like Growth Factor-I Gene Expression

Growth hormone (GH) regulates insulin-like growth factor-I (IGF-I) gene expression through signal transducer and activator of transcription 5b (STAT5b) and STAT5a. The objective of this study was to identify the cis-regulatory DNA region involved in this process. By cotransfection analyses of shotgun DNA fragments of a bacterial artificial chromosome sequence containing the entire human IGF-I gene and a large 5'-flanking region, a approximately 700-bp DNA region approximately 75 kb 5' to the IGF-I gene was found to have the ability to enhance gene expression from both heterologous and homologous promoters in the presence of constitutively active STAT5a or STAT5b. This 700-bp DNA region contains two closely located consensus STAT5-binding sites, and its sequence appears to be evolutionarily conserved. Electrophoretic mobility shift assays verified the ability of the two putative STAT5-binding sites to bind to STAT5a and STAT5b. Cotransfection analyses confirmed that both STAT5-binding sites were necessary for the 700-bp DNA region to mediate STAT5a or STAT5b activation of gene transcription. Chromatin immunoprecipitation assays demonstrated that the chromosomal region containing these two STAT5-binding sites was bound by constitutively active STAT5b protein in HepG2 cells and that the binding was accompanied by increased expression of IGF-I mRNA. In reconstituted GH-responsive cells, this 700-bp DNA region was able to mediate GH-induced STAT5a or STAT5b activation of gene expression. These results together suggest that this STAT5-binding site-containing distal 5'-flanking region of IGF-I gene may be an enhancer mediating GH-induced STAT5 activation of IGF-I gene transcription.

Tissue-Specific Increase Of Insulin-Like Growth Factor I Gene Expression In Human Adipose Tissue After Abdominal Surgery

The growth hormone (GH)/insulin-like growth factor I (IGF-I) axis in catabolic conditions is believed to be severely altered, based on measurements of biochemical markers in plasma and clinical observations. To test the hypothesis that GH sensitivity may be differentially regulated in different tissues, we analysed GH receptor (GHR) and IGF-I gene expression in adipose tissue and skeletal muscle, before and after major surgery, and compared it with the corresponding gene products in plasma, IGF-I and GH-binding protein (GHBP). A significant increase (p<0.05) in IGF-I mRNA levels was found in adipose tissue after surgery whereas there were no changes in IGF-I mRNA levels in skeletal muscle or IGF-I concentrations in plasma. Furthermore, GHR mRNA levels did not change after surgery whereas GHBP in plasma decreased postoperatively (p<0.01). This study shows that catabolism induced by surgical trauma is associated with differential regulation of IGF-I gene expression in adipose tissue and skeletal muscle and that plasma measurements of IGF-I and GHBP may not accurately reflect changes in gene expression in different tissues. These observations may have implications in the planning and conduction of clinical studies in catabolic states. Ruth Wickelgren and Gunnel Hellgren contributed equally to this manuscript and should both be considered first

A quantitative real-time RT-PCR assay for salmon IGF-I mRNA, and its application in the study of GH regulation of IGF-I gene expression in primary culture of salmon hepatocytes

The hormone insulin-like growth factor-I (IGF-I) regulates vertebrate growth. The liver produces most circulating IGF-I, under the control of pituitary growth hormone (GH) and nutritional status. To study the regulation of liver IGF-I production in salmon, we established a primary hepatocyte culture system and developed a TaqMan quantitative real-time RT-PCR assay for salmon IGF-I gene expression. A portion of the coho salmon acidic ribosomal phosphoprotein P0 (ARP) cDNA was sequenced for use as a reference gene. A systematic bias across the 96 well PCR plate was discovered in an initial IGF-I assay, which was corrected when the assay was redesigned. IGF-I mRNA levels measured with the validated assay correlated well with levels measured with an RNase protection assay, and were highest in liver compared with other tissues. We examined the time course of hepatocyte IGF-I gene expression over 48 h in culture, the response to a range of GH concentrations in hepatocytes from fed and fasted fish, and potential effects of variation in IGF-I in the medium. IGF-I gene expression decreased over time in culture in hepatocytes in plain medium, and in cells treated with 5 nM GH with or without a combination of metabolic hormones (1 μM insulin, 100 nM triiodothyronine, and 0.1 nM dexamethasone). GH stimulated IGF-I gene expression at all time points. In cells treated with GH plus metabolic hormones, IGF-I gene expression was intermediate between the controls and GH alone. Increasing concentrations of GH resulted in biphasic IGF-I gene expression response curves in cells from fed and fasted fish, with the threshold for stimulation from 0.5 to 2.5 nM GH, maximal response from 5 to 50 nM, and a reduced response at 500 nM. Medium IGF-I (5 nM) did not affect basal or GH stimulated IGF-I gene expression. This study shows that primary hepatocyte culture and the TaqMan IGF-I assay can be used to study the regulation of hepatic IGF-I gene expression in salmon, and provides the first evidence of a biphasic response to GH concentration in fish hepatocyte culture.

Common carp insulin-like growth factor-I gene: complete nucleotide sequence and functional characterization of the 5′-flanking region

Insulin-like growth factor-I (IGF-I) plays an important role in the growth and development of fish. To understand the molecular mechanism which controls the transcription of the IGF-I gene in common carp, we have cloned and completely sequenced the IGF-I gene and the 5′-flanking region from a local tropical fish, the common carp ( Cyprinus carpio), and characterized its promoter activity by transfection into human embryonic kidney (293GHR) cells which express the human growth hormone (GH) receptor. The common carp gene is the smallest IGF-I gene known so far, spanning approximately 13 kb, and is consisted of five exons and four introns. The sequence of the gene is consistent with the single type of IGF-I cDNA that we have isolated previously from a common carp liver cDNA library. The expression pattern of IGF-I is similar between juvenile carp and adult carp. While liver was found to be the major site of IGF-I gene expression in common carp, the expression levels in other tissues are relatively low. Like many other IGF-I gene promoters, there are no apparent TATA box and CCAAT box upstream of the transcription initiation site. However, sequence analysis of the common carp IGF-I promoter region identified several consensus liver-enriched transcription factor binding sites, including HNF-1α, HNF-3β, C/EBP, and one STAT5. We have analyzed the promoter activity of the 5′-flanking region of the common carp IGF-I gene by performing luciferase reporter assays in transfected 293GHR cells. Addition of human GH to the transfected cells led to an increased expression of the reporter gene, indicating that the cloned genomic fragment possessed promoter activity. This was confirmed by the lack of promoter activity of a construct in which the putative promoter was cloned in a reverse orientation upstream of the reporter gene. The liver-specific transcription factor, hepatic nuclear factor (HNF)-1α, was also found to be involved in the regulation of the common carp IGF-I transcription. Transfection results from a set of deletion mutants helped to map the locations of the responsive elements on the promoter responsible for the GH effect and for the interaction with HNF-1α. These observations provide information for delineating the transcriptional regulation of IGF-I gene expression in common carp.

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.

Insulin-like growth factor-I, but not growth hormone, is dependent on a high protein intake to increase nitrogen balance in the rat

The aim of the present study was to investigate the influence of dietary protein level on the protein anabolic effects of growth hormone (GH) and insulin-like growth factor-I (IGF-I). Female growing rats were fed on either a high- or a low-protein diet with crude protein contents of 222 and 83 g/kg respectively. The diets contained the same amount of metabolizable energy (15.1 MJ/kg) and were given during a 14 d period. During the same time, three groups of rats (n 8) on each diet received subcutaneous infusions of either saline, recombinant human GH (rhGH) or recombinant human IGF-I (rhIGF-I). rhGH and rhIGF-I were given in doses of 360 and 500 micrograms/d respectively. The low-protein diet alone reduced significantly (P < 0.05) IGF-I concentrations in serum and in tissue taken from the gastrocnemius muscle as well as IGF-I mRNA from the same muscle. The responses to rhGH and rhIGF-I in terms of muscle IGF-I and its mRNA were variable. However, when rhIGF-I was infused into rats on the high-protein diet, significantly elevated levels of IGF-I in muscle tissues could be observed. This was associated with a significantly (P < 0.05) increased N balance, whereas rhGH significantly (P < 0.05) enhanced the N balance in rats on the low-protein diet. Thus, it can be concluded that the level of dietary protein ingested regulates not only the effect of IGF-I on whole-body N economy but also the regulation of IGF-I gene expression in muscles. The exact mechanism by which GH exerts its protein anabolic effect, however, remains to be elucidated.

Metabolic Regulation of IGF-I Gene Expression

Insulin-like growth factor-I (IGF-I) is structurally similar to proinsulin and has wide-ranging actions in the promotion of cellular growth, proliferation, and differentiation. Circulating IGF-I is produced mainly in the liver and acts in an endocrine manner at many sites throughout the body, but IGF-I is also produced in a wide variety of organs and tissues, where it has local paracrine and autocrine effects. Consistent with its role as an important regulator of cell growth, IGF-I is tightly modulated by hormonal and nutritional status. In intact animals, reduction in levels of growth hormone or insulin, reduced food intake, and reduction in dietary protein all lead to a fall in circulating IGF-I levels and a parallel fall in hepatic IGF-I expression. Much of this regulation has been confirmed in cultured cell models showing responses to growth hormone, insulin, and availability of amino acids. Regulation takes place at the level of gene transcription since growth hormone, insulin, and dietary protein all increase both hepatic IGF-I expression and rates of transcript elongation in nuclear runon assays. Although the IGF-I gene is complex, extending over 80 kb and including six exons, most transcripts are initiated at several sites in exon 1. We have used the model of streptozotocin-induced diabetes in rats to study mechanisms of metabolic regulation. Diabetic rats exhibit dose-dependent decreases in serum IGF-I levels and hepatic IGF-I expression, preceded by a reduction in IGF-I gene transcription. A similar impact of diabetes is seen with in vitro transcription systems driven by hepatic nuclear extracts from normal and diabetic rats. In such systems, the study of deletion mutants reveals that a region downstream from the major initiation sites in exon 1 is necessary for the diabetes-associated fall in IGF-I gene transcription. We have localized a metabolism-responsive element to a footprinted region ∼ 140 bp downstream and found that mutation of this element blunts the decrease in IGF-I transcription that occurs with diabetic nuclear extracts. Gel mobility shift analysis with oligonucleotides containing this element reveals reduced DNA-protein interactions with diabetic nuclear extracts. Fasting or protein restriction also lead to diminished hepatic IGF-I expression and a fall in IGF-I gene transcription. Although the downstream region is also involved in nutrition-regulated transcription, the sequence elements that confer this control have not yet been identified. Work now in progress is directed toward identification of the transcription factors that mediate IGF-I regulation by alterations in insulin levels and nutritional status. Such understanding should provide further insight into the role and regulation of IGF-I.

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.

CCAAT/Enhancer-binding Protein δ Activates Insulin-like Growth Factor-I Gene Transcription in Osteoblasts: IDENTIFICATION OF A NOVEL CYCLIC AMP SIGNALING PATHWAY IN BONE

Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast nuclear proteins. These results identify C/EBPdelta as a hormonally activated inducer of IGF-I gene transcription in osteoblasts and show that the HS3D element within IGF-I promoter 1 is a high affinity binding site for this protein.

Prostaglandin A2 Specifically Represses Insulin-Like Growth Factor-I Gene Expression in C6 Rat Glioma Cells

The cyclopentenone PGs (PGA and PGJ series) inhibit tumor cell proliferation in vitro and tumorigenesis in vivo via mechanisms that are at present poorly understood. The C6 rat glioma cell line synthesizes and secretes insulin-like growth factor-I (IGF-I), which is believed to act as an autocrine factor for these cells. PGA2 inhibits the proliferation of the C6 cells and causes an increase in the fraction of cells in the G1 phase of the cell cycle. The inhibition of cell proliferation by PGA2 is accompanied by a decrease in the abundance of IGF-I messenger RNA (mRNA). This regulation of IGF-I gene expression is specific, as the abundance of hypoxanthine-guanine phosphoribosyl transferase (HPRT) and ubiquitin mRNA is not significantly affected by PGA2. The repression of IGF-I gene expression is observed at PGA2 concentrations as low as 10 μm and is evident within 4 h after treatment of the C6 cells with PGA2. In addition to specifically regulating the expression of the IGF-I gene, PGA2 also decreases the abundance of cyclin D1 mRNA and increases the abundance of Waf1 mRNA. The inhibition of cell proliferation by PGA2 is partially reversed by coaddition of IGF-I, indicating partial dominance of IGF-I action over PGA2 action. To investigate the molecular basis for the regulation of IGF-I gene expression by PGA2, we developed a sensitive RT-PCR assay for IGF-I nuclear transcripts. A similar assay was developed for quantifying HPRT transcripts, which were used as a control. Treatment of the C6 cells with 20 μm PGA2 resulted in approximately a 6-fold decrease in IGF-I mRNA and IGF-I nuclear transcripts. In contrast, HPRT mRNA and nuclear transcript levels were not significantly affected by PGA2. These results indicate that the decrease in IGF-I mRNA abundance that occurs in response to PGA2 is caused largely by a decrease in IGF-I nuclear transcript levels. To identify the cis-acting element that mediates the effect of PGA2 on IGF-I transcription, C6 cells were transiently transfected with IGF-I/luciferase expression constructs in which luciferase transcription is driven by IGF-I P1 promoter fragments extending from −1711 to +328 or from −1114 to +328 relative to the beginning of exon 1. Treatment of cells with PGA2 in these transient transfection assays did not decrease luciferase activity. These results suggest that the cis-acting regulatory element required for the response to PGA2 is located outside the −1711 to +328 promoter interval.

Prostaglandin A2 specifically represses insulin-like growth factor-I gene expression in C6 rat glioma cells.

The cyclopentenone PGs (PGA and PGJ series) inhibit tumor cell proliferation in vitro and tumorigenesis in vivo via mechanisms that are at present poorly understood. The C6 rat glioma cell line synthesizes and secretes insulin-like growth factor-I (IGF-I), which is believed to act as an autocrine factor for these cells. PGA2 inhibits the proliferation of the C6 cells and causes an increase in the fraction of cells in the G1 phase of the cell cycle. The inhibition of cell proliferation by PGA2 is accompanied by a decrease in the abundance of IGF-I messenger RNA (mRNA). This regulation of IGF-I gene expression is specific, as the abundance of hypoxanthine-guanine phosphoribosyl transferase (HPRT) and ubiquitin mRNA is not significantly affected by PGA2. The repression of IGF-I gene expression is observed at PGA2 concentrations as low as 10 microM and is evident within 4 h after treatment of the C6 cells with PGA2. In addition to specifically regulating the expression of the IGF-I gene, PGA2 also decreases the abundance of cyclin D1 mRNA and increases the abundance of Waf1 mRNA. The inhibition of cell proliferation by PGA2 is partially reversed by coaddition of IGF-I, indicating partial dominance of IGF-I action over PGA2 action. To investigate the molecular basis for the regulation of IGF-I gene expression by PGA2, we developed a sensitive RT-PCR assay for IGF-I nuclear transcripts. A similar assay was developed for quantifying HPRT transcripts, which were used as a control. Treatment of the C6 cells with 20 microM PGA2 resulted in approximately a 6-fold decrease in IGF-I mRNA and IGF-I nuclear transcripts. In contrast, HPRT mRNA and nuclear transcript levels were not significantly affected by PGA2. These results indicate that the decrease in IGF-I mRNA abundance that occurs in response to PGA2 is caused largely by a decrease in IGF-I nuclear transcript levels. To identify the cis-acting element that mediates the effect of PGA2 on IGF-I transcription, C6 cells were transiently transfected with IGF-I/luciferase expression constructs in which luciferase transcription is driven by IGF-I P1 promoter fragments extending from -1711 to -328 or from -1114 to +328 relative to the beginning of exon 1. Treatment of cells with PGA2 in these transient transfection assays did not decrease luciferase activity. These results suggest that the cis-acting regulatory element required for the response to PGA2 is located outside the -1711 to +328 promoter interval.

Inhibition by Interleukin-1β and Tumor Necrosis Factor-α of the Insulin-Like Growth Factor I Messenger Ribonucleic Acid Response to Growth Hormone in Rat Hepatocyte Primary Culture*

The cytokines are the putative mediators of the catabolic reaction that accompanies infection and trauma. Evidence suggests that their catabolic actions are indirect and potentially mediated through changes in hormonal axis such as the hypothalamo-pituitary-adrenal axis. Insulin-like growth factor I (IGF-I) is a GH-dependent growth factor that regulates the protein metabolism. To determine whether cytokines can directly inhibit the production of IGF-I by the liver, we investigated the regulation of IGF-I gene expression by interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha (10 ng/ml) in a model of rat primary cultured hepatocytes. Hepatocytes were isolated by liver collagenase perfusion and cultured on Matrigel 48 h before experiments. Each experiment was performed in at least three different animals. In the absence of GH, IL-1 beta and TNF-alpha did not affect the IGF-I messenger RNA (mRNA) basal levels, whereas IL-6 increased it by a factor of 2.5 after 24 h (P < 0.05). GH (500 ng/ml) alone stimulated the IGF-I gene expression markedly (5-to 10-fold increase) after 24 h (P < 0.001). IL-1 beta, and TNF-alpha to a lesser extent, dramatically inhibited the IGF-I mRNA response to GH (IL-1 beta: -82%, P < 0.001 and TNF-alpha: -47%, P < 0.01). The half-maximal inhibition of the IGF-I mRNA response to GH was observed for a concentration of IL-1 beta between 0.1 and 1 ng/ml. Moreover, IL-1 beta abolished the IL-6-induced IGF-I mRNA response. In contrast, IL-6 did not impair the IGF-I mRNA response to GH. To determine the potential role of the GH receptor (GHR) and the GH-binding protein (GHBP) in this GH resistance, we assessed the GHR and GHBP mRNAs response to these cytokines. GH alone did not affect the GHR/GHBP mRNA levels. IL-1 beta markedly decreased the GHR and GHBP mRNA levels (respectively, -68% and -60%, P < 0.05). Neither TNF-alpha nor IL-6 affected the GHR/GHBP gene expression. In conclusion, our results show that IL-1 beta, and TNF-alpha to a lesser extent, blunt the IGF-I mRNA response to GH. The resistance to GH induced by IL-1 beta might be mediated by a decrease of GH receptors, as suggested by the marked reduction of GHR mRNA. These findings suggest that decreased circulating IGF-I, in response to infection and trauma, may be caused by a direct effect of cytokines at the hepatocyte level.

In Vitro Transcription of the Rat Insulin-like Growth Factor-I Gene

Although the liver is the major source of circulating insulin-like growth factor-I (IGF-I), relatively little is known about the regulation of IGF-I gene transcription in this tissue. Since transcripts are initiated largely in exon 1, we established an in vitro transcription system to evaluate activation of transcription via the major exon 1 initiation site. Transcription of a G-free cassette reporter was directed by rat IGF-I genomic fragments, and the adenovirus major late promoter was used as an internal control. Tissue specificity was demonstrated by a 60-90% decrease in transcripts with spleen extracts as compared with liver. 54 base pairs (bp) of upstream sequence were sufficient to direct IGF-I gene transcription, and activity increased 5-fold with 300 bp of upstream sequence. DNase I footprinting revealed four protected regions between -300 and -60 bp; binding was confirmed by gel shift analysis, and tissue specificity was demonstrated by reduced shifts with spleen extracts. The necessity of transcription factor binding to such sites was established by competition analysis, which revealed a specific decrease in IGF-I transcription in the presence of a competing fragment. Use of this in vitro transcription system should permit analysis of the function of individual transcription factors involved in regulation of IGF-I gene expression.

Granulosa cell DNA synthesis is strictly correlated with the presence of insulin-like growth factor I and absence of c-fos/c-jun expression.

Insulin-like growth factor I (IGF-I) mRNA is selectively expressed by a subset of rat ovarian follicles. To elucidate the biological role and regulation of follicular IGF-I, we have analyzed individual follicles with respect to IGF-I production, DNA synthesis, and c-fos and c-jun expression, since AP-1 complex components have been implicated both in regulation of IGF-I gene expression and cell proliferation. IGF-I mRNA localization, bromodeoxyuridine (BRDU) incorporation, and c-fos and c-jun immunoreactivity were compared in serial sections from prepubertal and mature rat ovaries. BRDU incorporation is exclusively concentrated in IGF-I-expressing follicles in both prepubertal and mature rats, with further intrafollicular correlation between local concentrations of BRDU-positive granulosa cells and IGF-I mRNA. Furthermore, there is a striking negative correlation between IGF-I mRNA localization and follicular fos/jun immunoreactivity. Both c-fos and c-jun are readily detected in granulosa cells of luteinized follicles where IGF-I mRNA is never found. Immunoreactive c-jun but not c-fos is detected in granulosa cells of atretic follicles, while neither c-fos nor c-jun is detected in IGF-I mRNA-positive granulosa cells. These data implicate IGF-I in granulosa cell proliferation and the fos/jun heterodimer in granulosa cell luteinization, while expression of c-jun in the absence of c-fos may be implicated in programmed granulosa cell death.

Tissue-specific transcription start site usage in the leader exons of the rat insulin-like growth factor-I gene: evidence for differential regulation in the developing kidney

The production of insulin-like growth factor-I (IGF-I) in extrahepatic tissues supports both autocrine and paracrine functions and is regulated differently from that in liver, which supports endocrine function. In rat liver, transcription initiation primarily occurs at four distinct, widely separated sites in exon 1 of the IGF-I gene, whereas in exon 2, transcription initiation occurs at a cluster of sites. To understand the molecular basis for tissue-specific regulation of IGF-I gene expression, we have mapped transcription start site usage in the following extrahepatic tissues: testes, lung, kidney, heart, brain, muscle, and stomach, with liver serving as a control. In adult rats, kidney and brain exhibited a pattern of exon 1 transcription similar to that seen in liver, i.e. roughly equivalent use of start sites 2 and 3. In contrast, testes and lung preferentially used start site 3, while stomach, heart, and muscle predominantly used start site 3. Start sites 1 and 4 were used in all tissues at extremely low levels. In those tissues studied in which exon 2 transcripts are expressed (testes, lung, stomach, and kidney), the pattern of exon 2 transcription initiation was identical to that in adult rat liver. During postnatal development, the use of all transcription start sites in exons 1 and 2 was coordinate in lung and stomach. Selection of transcription start sites in the kidney, on the other hand, was subject to regulation during postnatal development. Specifically, within exon 1, start site 3 was expressed constitutively throughout peri- and postnatal development. In contrast, the usage of start site 2 was not detected at late fetal or early postnatal stages, but appeared and rapidly increased only at the stage of weaning. Exon 2 transcripts in kidney also did not appear until the postnatal period. These data suggest tissue-specific and developmentally regulated transcription factors regulating IGF-I promoter activity or, alternatively, tissue-specific and developmental stage-dependent differences in the stability of IGF-I mRNAs resulting from the use of different transcription start sites. These different mRNAs may be of significance in the differential regulation of IGF-I production for autocrine or paracrine function.

Insulin-like growth factor-I gene expression during development and estrous cycle in the rat uterus

The ontogeny and estrous cycle-dependent variation of insulin-like growth factor-I (IGF-I) gene expression was analyzed in the rat uterus. RNA extracted from rat uteri contained transcripts with estimated sizes of 7.0, 1.7, and 1.2-0.8 kb that were recognized by a 32P-labelled mouse IGF-I RNA probe. A solution hybridization RNase protection assay was used to measure the abundance of IGF-I mRNAs in uteri from rats of different ages. The highest levels were found in adult rats ( p < 0.01). The levels of IGF-I transcripts changed markedly during the estrous cycle with the highest levels at proestrus (< 0.01). There was an 8-fold increase in the abundance of IGF-I mRNA between diestrus-2 and proestrus. The corresponding livers had no significant variation of IGF-I gene expression during the estrous cycle, demonstrating a tissue-specific regulation of the IGF-I gene. The time and dose dependency of estrogen regulation of IGF-I gene expression was studied in hypophysectomized rats. The levels of IGF-I mRNA in the uterus decreased after hypophysectomy. A single s.c. injection of estradiol significantly increased the levels of IGF-I transcripts after 3 h ( p < 0.01). A low dose of estradiol (0.1 /gmg/100 g) increased the levels of IGF-I transcripts but progesterone in higher doses (5 μg/100 g) was without effect, indicating that the effect was specific for estradiol. However, the present study provides no information regarding whether this regulation is at the level of transcription or mRNA stability. The present study provides further support for estrogen as a major regulator of IGF-I gene expression in the uterus in vivo.

Expression of Insulin-Like Growth Factor-I during Chicken Development*

We have investigated the expression of insulin-like growth factor-I (IGF-I) during ontogeny in the chick. IGF-I mRNA was first detectable in whole embryos on day 6, while serum IGF-I could be measured on day 9, the earliest time point examined. Serum IGF-I values rose 10-fold from about 3 ng/ml on day 10 to a prehatch peak of 30-35 ng/ml during days 15-17, and then declined to about 10 ng/ml at the time of hatching. On days 17 and 20 of incubation, IGF-I mRNA was detected in eye, skeletal muscle, and brain, but could not be found in liver or heart until after hatching. During the posthatch period, serum IGF-I rose from 10 ng/ml in the first week to 35-40 ng/ml during weeks 3-6, and liver IGF-I mRNA increased nearly 5-fold from weeks 1-7. The increases observed during weeks 1-3 correlated with a posthatch rise in serum GH from 21 to 37 ng/ml, although GH levels declined over the subsequent 4 weeks without an appreciable change in serum IGF-I values. By contrast, before embryonic day 12 no GH could be detected in the circulation, while IGF-I was 19 ng/ml on day 11. These observations suggest that there is both GH-dependent and GH-independent regulation of IGF-I gene expression in the chick, as has been found in mammals, and support the idea that IGF-I plays a role in chicken embryonic development.

Pituitary Insulin-Like Growth Factor-I Content and Gene Expression in the Streptozotocin-Diabetic Rat: Evidence for Tissue-Specific Regulation*

Insulinopenic diabetes mellitus in the rat is associated with reduced circulating levels of insulin-like growth factor-I (IGF-I), resulting primarily from decreased IGF-I synthesis in liver and extrahepatic sites. Plasma GH levels in these animals are also suppressed, with loss of episodic secretion and decreased pituitary synthesis. Intrapituitary IGF-I has been postulated to exert local autocrine/paracrine negative feedback regulation on GH synthesis and secretion. The present studies were designed to examine regulation of pituitary IGF-I peptide content and gene expression in insulinopenic streptozotocin (STZ)-diabetic rats compared to that in liver and testis. Serum IGF-I levels were reduced by 86% in STZ-diabetic rats together with reduction of IGF-I content in liver (53%) and testis (74%; all P less than 0.001 vs. control). Concomitantly, liver and testicular IGF-I mRNA levels were reduced by 90% (P less than 0.001 vs. control). Insulin treatment restored IGF-I peptide levels in serum, liver, and testis toward normal, with a partial but significant increase in liver IGF-I mRNA. In contrast, pituitary IFG-I peptide content increased by 69% in STZ-diabetic rats (P less than 0.001 vs. control), with no change in IGF-I gene expression. Insulin treatment completely reversed the rise of pituitary IGF-I peptide content. These results demonstrate a novel discordance in the regulation of IGF-I gene expression and peptide content between pituitary and other tissues in STZ-induced diabetic rats. Elevated IGF-I levels in the pituitaries of these animals may partly explain the suppressed GH synthesis and secretion seen in STZ-diabetic rats and provide further evidence for a potential autocrine or paracrine role of pituitary IGF-I in GH regulation.

Acute regulation of insulin-like growth factor-I gene expression by growth hormone during adipose cell differentiation.

Insulin-like growth factor I (IGF-I) is a mitogenic polypeptide that is thought to play, under the control of growth hormone, a role in fetal development as well as post-natally. The direct effect of growth hormone on the regulation of the expression of IGF-I gene was examined in adipose Ob1771 cells. Growth hormone regulates the abundance of multiple species of IGF-I mRNAs of 15, 7.5, 1.5 and 0.8 kb in a differentiation-dependent manner. The regulation of IGF-I gene expression is strikingly rapid (less than 2 h), reversible and takes place primarily at transcriptional level. Thus growth hormone can increase the cellular content of IGF-I mRNA encoding for a protein which could be involved in a paracrine/autocrine action during adipose tissue development.