Insulin-like Growth factor-II Gene Research Articles

Rearrangements at the 11p15 locus and overexpression of insulin-like growth factor-II gene in sporadic adrenocortical tumors

Rearrangements at the 11p15 locus and overexpression of insulin-like growth factor-II gene in sporadic adrenocortical tumors

Parallel regulation of parentally imprinted H19 and insulin-like growth factor-II genes in cultured human fetal adrenal cells.

Adjacent, parentally imprinted, insulin-like growth factor-II (IGF-II) and H19 genes are highly expressed during embryogenesis and are important for fetal growth. Human fetal adrenals express abundantly both IGF-II and H19 genes. To clarify the significance and regulation of the H19 gene, we studied its expression in fetal adrenals. In situ hybridization experiments showed H19 RNA expression throughout the fetal adrenal cortex, with slightly higher expression in the outer definitive (adult) than in the inner fetal zone. In primary cultures of fetal adrenal cells, ACTH and other activators of the protein kinase-A signal transduction pathway increased both H19 and IGF-II RNA accumulation 1.7- to 10-fold. Staurosporine, a protein kinase-C inhibitor, increased H19 and IGF-II RNA to the same extent as did ACTH. The protein kinase-C activator 12-O-tetradecanoyl phorbol-13-acetate and cytokines, tumor necrosis factor-alpha and interferon-gamma, inhibited H19 and IGF-II RNA accumulation. Transforming growth factor-beta 1 caused a decrease in levels of H19 and IGF-II RNA, whereas the IGFs caused a slight increase. Our data show parallel multifactorial regulation of H19 and IGF-II RNAs in human fetal adrenal cells. This suggests common regulatory mechanisms for these adjacent genes.

Parallel regulation of parentally imprinted H19 and insulin-like growth factor-II genes in cultured human fetal adrenal cells

Adjacent, parentally imprinted, insulin-like growth factor-II (IGF-II) and H19 genes are highly expressed during embryogenesis and are important for fetal growth. Human fetal adrenals express abundantly both IGF-II and H19 genes. To clarify the significance and regulation of the H19 gene, we studied its expression in fetal adrenals. In situ hybridization experiments showed H19 RNA expression throughout the fetal adrenal cortex, with slightly higher expression in the outer definitive (adult) than in the inner fetal zone. In primary cultures of fetal adrenal cells, ACTH and other activators of the protein kinase-A signal transduction pathway increased both H19 and IGF-II RNA accumulation 1.7- to 10-fold. Staurosporine, a protein kinase-C inhibitor, increased H19 and IGF-II RNA to the same extent as did ACTH. The protein kinase-C activator 12-O-tetradecanoyl phorbol-13-acetate and cytokines, tumor necrosis factor-alpha and interferon-gamma, inhibited H19 and IGF-II RNA accumulation. Transforming growth factor-beta 1 caused a decrease in levels of H19 and IGF-II RNA, whereas the IGFs caused a slight increase. Our data show parallel multifactorial regulation of H19 and IGF-II RNAs in human fetal adrenal cells. This suggests common regulatory mechanisms for these adjacent genes.

Characterization of the Linked Ovine Insulin and Insulin-Like Growth Factor-II Genes

The ovine insulin-like growth factor-II (oIGF-II) gene is comprised of 9 exons that span approximately 25 kb. Approximately 750 nucleotides upstream of oIGF-II exon 1 are the three exons of the ovine insulin gene that are transcribed in the same direction as oIGF-II. The genomic organization and expression of the oIGF-II gene is similar to that of the human IGF-II gene. Four putative promoters direct the transcription of six 5' noncoding exons (1, 3, 4, 5, 6, and 7), which are alternatively spliced to the shared exons 8, 9, and 10. An ovine exon comparable to human exon 2 has not been identified. Multiple transcription initiation sites were identified for exons 1 and 6 by primer extension analysis. Using a reverse transcriptase polymerase chain reaction (RT-PCR) assay, exon 1 and 3 transcripts were shown to be expressed in adult but not fetal liver. In addition, a novel transcript, which contained exon 1 spliced directly to exon 8, was detected in adult liver. Exon 4 transcripts were not detected in either fetal or adult liver, whereas exon 6 and 7 transcripts were detected in both fetal and adult liver. Exon 5 transcripts were also expressed in both fetal and adult liver, which is in contrast to the tumor cell-specific expression of human exon 5. Like the human and rodent genes, the regulation of expression of the oIGF-II gene is under complex control.

Negative and positive regulation of IGF-II mRNA expression in cultured rat cells by chicken serum

We have investigated the possibility that some serum factors might negatively regulate the expression of the insulin-like growth factor-II (IGF-II) gene in 18–54,SF cells. Northern blot analyses indicated that there were three major transcripts (3.8 kb, 1.8 kb, and 1.2 kb) of the IGF-II gene in these cells. We found that incubation of 18–54,SF cells in medium containing very high concentrations (50–100%) of chicken serum greatly inhibited the steady-state level of all three IGF-II mRNA species. In addition, we also found that incubation of 18–54,SF cells in medium containing lower concentrations (10–50%) of chicken serum induced a 3.5 kb IGF-II mRNA. The inhibitory effect of high concentrations of chicken serum on IGF-II mRNA expression was not due to a cytotoxic effect of the serum, because these cells were maintained in 100% chicken serum for up to two weeks without loss of cell viability. The inhibitory effect of chicken serum on IGF-II mRNA was reversible after withdrawl of the serum. Nuclear run-on assays suggested that this negative regulation of IGF-II mRNA in 18–54,SF cells by chicken serum was not the result of transcriptional inhibition. Treatment of 18–54,SF cells that had been previously incubated in 100% chicken serum for 24 h with actinomycin D resulted in a partial restoration of the expression of the 3.8 kb and 1.2 kb IGF-II mRNA in these cells. Finally, we also obtained a crude fraction of chicken serum and showed that treatment of 18–54.SF cells with this fraction not only inhibited the steady-state level of the 3.8 kb, 1.8 kb and 1.2 kb IGF-II mRNA, but also induced the appearance of new species of IGF-II mRNAs in these cells. These observations suggest that chicken serum may serve as a potential source for the isolation of putative factors that regulate IGF-II mRNA expression in cultured cells.

In situ localization of muscle insulin-like growth factor-II mRNA in developing bovine fetuses

Insulin-like growth factor-II (IGF-II) modulates myogenesis in muscle cell cultures, in utero. IGF-II gene expression is developmentally regulated in several tissues including muscle. Determining whether IGF-II is expressed by developing muscle cells or by neighbouring cells in developing muscle tissue is crucial for determining whether IGF-II exerts a paracrine or an autocrine affect on myogenesis. Semitendinosus muscle samples from 12 bovine fetuses ranging from 60 to 274 days post conception (pc) were analysed for the amount and localization of muscle IGF-II mRNA using Northern, dot blot and in situ hybridization analyses. Northern blot analysis revealed multiple IGF-II transcripts of 5.1, 3.7, 2.6, 2.0, 1.7 and 1.1 kb in developing bovine muscle tissue. The relative amount of muscle IGF-II mRNA increased (P < 0.05) until 162 days pc, then decreased (P < 0.01) to near undetectable levels by the end of gestation (approximately 284 days pc). Between 60 and 162 days pc, in situ hybridization revealed that the majority of the IGF-II transcripts were localized to developing muscle cells rather than connective tissue. After 162 days pc the IGF-II hybridization signal shifted away from muscle cells and greater accumulation was observed in the connective tissue at 274 days pc. These data confirm that the expression of IGF-II in developing bovine muscle tissue is primarily localized in muscle cells and support the claim that IGF-II acts as an autocrine-acting growth factor during myogenesis in vivo.

Inhibition of protein phosphatases activates P4 promoter of the human insulin-like growth factor II gene through the specific promoter element.

To understand the transcriptional regulation of the human insulin-like growth factor II (IGF-II) gene, we examined the effects of okadaic acid, a potent in vitro inhibitor of protein phosphatases, on the activation of human IGF-II gene expression. Treatment of A-549 human lung adenocarcinoma cells with okadaic acid increased expression of the IGF-II mRNAs. Since the 4.8-kb mRNA is transcribed under the control of human IGF-II P4 promoter, we examined the P4 promoter element responsible for the okadaic acid-mediated transcriptional activation. Transfection of IGF-II P4 promoter-chloramphenicol acetyltransferase constructs demonstrated that the effects of okadaic acid on the induction of IGF-II gene expression are mediated through multiple promoter elements, including an Egr-1 consensus element. We have also shown that okadaic acid induced the expression of the transcription factor Egr-1. Moreover, by using a GAL4-Egr-1 fusion protein, we have directly demonstrated that okadaic acid positively regulates Egr-1 transcriptional activity in vivo. These results indicate that protein phosphatases play an important role in the transcriptional regulation of the IGF-II.

Insulin-Like Growth Factor II (IGF-II) and the IGF-II/ Mannose-6-Phosphate Receptor: The Myth Continues

Insulin-like growth factor II (IGF-II) is a polypeptide hormone with structural and functional homology with IGF-I and pro-insulin. It is now thought that IGF-II acts as a growth factor during fetal life and development. In rats, IGF-II levels in the circulation are high in the fetus and decline rapidly after birth. IGF-II mRNA expression in many tissues including the liver and the choroid plexus is also high during fetal life and low thereafter. Targeted disruption of the IGF-II gene in mice leads to a deficiency in their growth. Alternatively, it has been proposed that IGF-II could act as a growth and differentiation factor in the central nervous system. Administration of IGF-II into the central nervous system in rats leads to increased food intake and altered feeding behaviour. In muscle cells and in colon epithelial cells, IGF-II might also serve as an important regulator of differentiation. A key role for IGF-II as a paracrine or autocrine growth factor in certain tumours has been proposed. The IGFs exert their effects by binding to high-affinity membrane receptors that are expressed in many cells and tissues. The IGF-I receptor, which binds IGF-I with the highest affinity and which is very similar to the insulin receptor, is thought to mediate most, if not all, of the IGF-induced biological functions. The IGF-II/mannose-6-phosphate (M6P) receptor is a bifunctional glycoprotein with no homology to the insulin receptor. This receptor binds IGF-II and lysosomal enzymes bearing the M6P recognition marker at distinct binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Infrequency of ras, p53, WT1, or RB gene alterations in Wilms tumors.

Alteration of the ras family of oncogenes and of the tumor suppressor genes p53 and RB are the most common genetic events in human tumors. Although there have been no reports of the prevalence of these alterations in Wilms tumors, overexpression of the N-myc and insulin-like growth factor-II (IGF-II) genes have been observed, and alteration of another tumor suppressor gene (WT1) has been demonstrated. Forty-four Wilms tumor specimens were tested for the presence of N-, K-, and H-ras mutations in codons 12, 13, and 61 by single-strand conformation polymorphism (SSCP) analysis and direct DNA sequence analysis. Sixteen tumors were tested for abnormalities of WT1 by Southern and northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). N-myc, c-myc, WT1, and IGF-II mRNA expression was measured in 16 tumors by Northern blot analysis. Thirty-eight tumors were screened for p53 mutations by SSCP analysis and direct DNA sequence analysis. Nine tumors were analyzed for loss of heterozygosity (LOH) of RB. Although the authors confirmed that N-myc and IGF-II are overexpressed in Wilms tumors, no mutations of ras family, p53, or RB genes were identified, and no gross alterations of WT1 were detected by Southern or Northern blot analysis. These findings suggest that H-ras, K-ras, N-ras, p53, and RB are not involved in the pathogenesis of Wilms tumor.

Developmental regulation of bovine insulin-like growth factor-II (IGF-II) gene expression: homology between bovine transcripts and human IGF-II exons

Initial observations have indicated similarities between bovine and human IGF-II production during development. The aim of the present study was to investigate whether cattle could provide an experimental model that would mimic the complex pattern of human IGF-II gene expression. Expression of bovine IGF-II gene during development was studied by RNA hybridization using various human IGF-II probes. In fetal tissues and in adult muscle, the bovine IGF-II gene was expressed as a family of eight transcripts ranging in size from 5.2 to 1.1 kb. In adult bovine liver, a major IGF-II transcript of 4.4 kb was expressed that could not be detected in any fetal or adult extra-hepatic tissue. During fetal life, quantitative IGF-II mRNA expression differed in liver and muscle, and the relative amounts of the different transcripts varied with the tissue of origin. These observations suggest that the regulation of bovine IGF-II gene expression is specific to the stage of development and the tissue concerned. Moreover its pattern is very similar to that in its human counterpart. In order to identify a putative homology between human and bovine gene structures, bovine mRNAs were examined for cross-hybridization with various non-coding exons of the human gene. Cross-hybridization was detected with human untranslated exons 5 and 6, suggesting the presence of two distinct promoters similar to the human promoters P3 and P4. The 4.4 kb mRNA species expressed in adult bovine liver failed to hybridize to a probe for human exons 1 and 2, suggesting that the leader sequences of this transcript were different from those present in the human gene. Finally, results obtained with a probe containing the 3' untranslated end of exon 9 suggested the presence of at least two polyadenylation sites in the bovine gene. Although differences in IGF-II gene structures were found between cattle and man, the similarities in the pattern of gene expression between the two species suggest that cattle may be a useful model to investigate some developmental aspects of the expression of the human IGF-II gene.

Changes in lung expansion alter pulmonary DNA synthesis and IGF-II gene expression in fetal sheep

Our aim was to determine the effect of short-term (7 days) alterations in fetal lung liquid volume on pulmonary DNA synthesis rates and insulin-like growth factor-II (IGF-II) mRNA levels. Fifteen chronically catheterized fetal sheep were divided into three groups. In one, the trachea was obstructed, in another lung liquid was drained by gravity, and the third group served as controls. After 7 days, [3H]thymidine was injected into each fetus and 8 h later fetal tissues were collected. Fetal lung-to-body weight ratios and total lung DNA contents were greatly increased in fetuses with tracheal obstruction compared with control fetuses, whereas the drainage of lung liquid did not affect these measurements. DNA synthesis rates in pulmonary tissue were significantly reduced from a mean control value of 153.3 +/- 25.1 disintegrations per minute (dpm)/microgram DNA to 57.2 +/- 8.6 dpm/microgram DNA by lung liquid drainage (P < 0.05) and were significantly increased to 236.0 +/- 24.0 dpm/microgram DNA by tracheal obstruction (P < 0.05). Following tracheal obstruction, lung IGF-II mRNA levels were increased to 177.0 +/- 18.2% (P < 0.05) of the mean value for control fetuses, whereas they were reduced to 56.1 +/- 7.1% of control in lung liquid-drained fetuses. We conclude that altering fetal lung expansion has a potent and rapid effect on pulmonary DNA synthesis and that this effect may, in part, be mediated by an alteration in IGF-II gene expression.

Wilms’ tumor (WT1) gene expression in rat decidual differentiation

Abstract The Wilm's tumor suppressor gene (WT1) encodes a zinc-finger containing transcription factor that is selectively expressed in the developing urogenital tract, where it is thought to play a role in the differentiation of these tissues. We have used immunocytochemistry and in situ hybridization to study WT1 expression in the rat uterus during normal development and pregnancy from 0 to 20 days post coitum (P.c.). WT1 mRNA was abundant in uterine stroma from juvenile rats, but was much less abundant in uterine tissue from sexually mature rats; WT1 expression is not affected by ovariectomy or by treatment with estradiol or estradiol plus progesterone. WT1 gene was highly expressed, however, in the endometrial cells of early pregnancy. On day 6 p.c. WT1 mRNA was detected in anti-mesometrial decidual cells, and WT1 immunoreactivity was concentrated in the nuclei of these cells. All cells of fully-developed deciduoma at 7-8 days p.c. demonstrated WT1 expression. WT1 was not detected in trophoblast/placental tissues but remained abundant in the decidua basalis until parturition. The expression of WT1 was compared with insulin-like growth factor-II (IGF-II) and its receptor in the decidual since it has been shown that IGF-II gene transcription is repressed by WT1 in vitro. However, no spatiotemporal correlation in the expression of these three genes was found in differentiation of the rat decidua. In summary, these data suggest a role for WT1 in decidualization, since its expression is activated during the differentiation of uterine stromal cells into decidual cells.

Wilms' tumor (WT1) gene expression in rat decidual differentiation

Abstract The Wilm's tumor suppressor gene (WT1) encodes a zinc-finger containing transcription factor that is selectively expressed in the developing urogenital tract, where it is thought to play a role in the differentiation of these tissues. We have used immunocytochemistry and in situ hybridization to study WT1 expression in the rat uterus during normal development and pregnancy from 0 to 20 days post coitum (p.c.). WT1 mRNA was abundant in uterine stroma from juvenile rats, but was much less abundant in uterine tissue from sexually mature rats; WT1 expression is not affected by ovariectomy or by treatment with estradiol or estradiol plus progesterone. WT1 gene was highly expressed, however, in the endometrial cells of early pregnancy. On day 6 p.c. WT1 mRNA was detected in anti-mesometrial decidual cells, and WT1 immunorcactivity was concentrated in the nuclei of these cells. All cells of fully-developed deciduoma at 7–8 days p.c. demonstrated WT1 expression. WT1 was not detected in trophoblast/placental tissues but remained abundant in the decidua basalis until parturition. The expression of WT1 was compared with insulin-like growth factor-II (IGF-II) and its receptor in the decidual since it has been shown that IGF-II gene transcription is repressed by WT1 in vitro. However, no spatiotemporal correlation in the expression of these three genes was found in differentiation of the rat decidua. In summary, these data suggest a role for WT1 in decidualization, since its expression is activated during the differentiation of uterine stromal cells into decidual cells.

Transcriptional regulation of the major promoters of the human IGF‐II gene

The human insulin-like growth factor-II (IGF-II) gene constitutes a complex transcriptional unit that is controlled by four different promoters preceding multiple 5'-untranslated exons. Expression of the IGF-II gene is development and tissue specific and yields a family of mRNAs that all encode preproIGF-II. We have analyzed the sequences and factors involved in the transcriptional activation of promoters P1 and P3. Detailed analysis revealed several characteristic elements and protein binding sequences. P1, which is expressed only in adult liver tissue, contains a region that can be specifically activated by the CCAAT/enhancer binding protein (C/EBP). P3 consists of a proximal promoter region that supports general transcription, whereas cell-specific elements are located upstream.

Insulin-like growth factor-II messenger ribonucleic acid expression in fetal tissues of the sheep during late gestation: effects of cortisol.

Using RNase protection analysis, insulin-like growth factor-II (IGF-II) mRNA levels were measured in various tissues from fetal sheep during late gestation (term, 146 +/- 2 days) and after experimental manipulation of fetal plasma cortisol levels. No gestational trend in IGF-II mRNA levels was observed in the fetal lung, kidney, or skeletal muscle. However, in the fetal liver, there was a marked decline in IGF-II mRNA abundance immediately before term, which closely paralleled the normal prepartum surge in fetal plasma cortisol. This decrease in hepatic IGF-II mRNA levels toward term was prevented when the cortisol surge was abolished by fetal adrenalectomy and was stimulated prematurely in fetuses younger than 130 days by exogenous infusion of cortisol. Hepatic and renal IGF-II mRNA abundances were also reduced when fetal cortisol levels were raised endogenously by maternal fasting in late gestation. Muscle IGF-II mRNA levels were reduced by fetal cortisol infusion, but not by maternal fasting, and were higher in adrenalectomized than in intact fetuses in late gestation. No change in IGF-II mRNA levels were observed in the fetal lung in response to altering the fetal cortisol level either exogenously or endogenously. When the data from all fetuses were combined regardless of treatment or gestational age, there was a significant inverse correlation between the plasma cortisol level in utero and IGF-II mRNA abundance in the fetal liver (P < 0.001), but not in any of the other fetal tissues studied. These findings show that cortisol suppresses IGF-II gene expression in the liver of the sheep fetus and indicate that the developmental change in hepatic IGF status toward term may be due to the prepartum cortisol surge.

Differential promoter activation in two human insulin-like growth factor-II-producing tumor cell lines.

Several lines of evidence suggest that the growth factor insulin-like growth factor-II (IGF-II) is involved in the genesis of several types of tumors and may have autocrine effects on tumor progression when overexpressed. The human IGF-II gene is a complex transcription unit. Multiple transcripts are synthesized as a result of alternate promoter usage and the splicing of unique 5'-untranslated regions to common coding exons. We have examined two human tumor cell lines that both produce IGF-II protein which can function as an autocrine growth factor. Interestingly, each cell line uses a different IGF-II promoter; Hep3B cells predominantly activate P3, whereas in SW613 cells P4 is the preferred promoter. Here, we examine the differential transcriptional activation of the human IGF-II gene in Hep3B and SW613 cells by means of transient transfection assays and electrophoretic mobility shift analysis. The results indicate that production of IGF-II protein is mainly regulated at the level of transcription. Additional regulation takes place at the levels of messenger RNA stability and efficiency of translation.

Tumor necrosis factor-α and interferon-γ inhibit insulin-like growth factor II gene expression in human fetal adrenal cell cultures

Insulin-like growth factor-II (IGF-II) gene expression is induced by adrenocorticotropic hormone (ACTH) in human fetal adrenals (HFA), which suggests an important role for IGF-II in HFA growth and differentiation. Many cytokines have different regulatory actions in the endocrine glands. In the present study we have investigated the effects of two cytokines, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), on the regulation of IGF-II gene expression in cultured HFA cells. Both TNF-α and IFN-γ inhibited basal and ACTH-induced accumulation of IGF-II mRNA dose-dependently. Cell viability was not altered by treatment with TNF-α or IFN-γ. In addition, the combination of TNF-α and IFN-γ decreased ACTH-induced IGF-II mRNAs more potently than each cytokine alone. Our results suggest that TNF-α and IFN-γ may be involved in the regulation of HFA growth and differentiation via local IGF-II production.

Transcriptional activation of the insulin-like growth factor-II gene during myoblast differentiation.

Insulin-like growth factor-II (IGF-II) is a secreted 67-amino acid peptide that functions principally as a prenatal growth regulator in mammals. To date, the mechanisms involved in the stimulation of IGF-II expression in the embryo and its attenuation after birth are unknown. Recent studies have shown that IGF-II mRNA and protein are induced during the terminal stages of muscle development in vitro, and that IGF-II may act as an autocrine differentiation agent for skeletal myoblasts. We now have investigated the regulation of IGF-II gene expression in muscle cells. Steady state levels of IGF-II mRNA increased by more than 30-fold during terminal differentiation of the C2 mouse myoblast cell line. Transcription run-on experiments using isolated muscle cell nuclei and direct analysis of nuclear RNA each demonstrated a greater than 10-fold rise in nascent IGF-II mRNA during cellular differentiation, and ribonuclease protection experiments showed that more than 95% of IGF-II mRNAs initiated in noncoding exon 3, implying that transcriptional activation occurs principally through promoter 3, the most 3' of the three mouse IGF-II gene promoters. Analysis of chromatin structure around the IGF-II gene in C2 cells revealed four major and four minor DNase-I-hypersensitive sites, but did not provide insight into the mechanisms of gene activation, since all sites were present in proliferating and differentiating cells. Gene transfer experiments showed that promoter 3 was at least 50-fold more active than promoter 1 or 2 in C2 cells, but the functional assessment of nearly 26 kilobases of additional DNA within the IGF-II locus by an "enhancer trap" approach did not delineate any chromosomal regions capable of mediating differentiation-specific gene activation. Our results demonstrate that muscle cells encode mechanisms for activating IGF-II gene transcription and suggest that these cells may be excellent models for identifying the developmentally regulated factors that control IGF-II gene expression.

Transcriptional activation of the insulin-like growth factor-II gene during myoblast differentiation

Transcriptional activation of the insulin-like growth factor-II gene during myoblast differentiation

Structural analysis of the human insulin-like growth factor-II P3 promoter

The expression of insulin-like growth factor-II (IGF-II) has been observed previously in many human cancers. The human IGF-II P3 promoter has been shown by others to give rise to abundant 6.0 kb and 2.2 kb fetal transcripts which are expressed in a variety of both paediatric and adult tumours. In order to determine the mechanism by which the P3 promoter is controlled, the promoter was analysed in cell lines using chloramphenicol acetyltransferase (CAT) assay and DNAase I footprinting techniques. The data indicated that P3 is a complex promoter involving at least nine transcription factor binding sites. Furthermore, high levels of 5-methylcytosine detected in the P3 promoter of HeLa genomic DNA suggest that IGF-II gene expression may also be influenced by DNA methylation.