胰岛素样生长因子2研究进展

胰岛素样生长因子2在胎儿生长发育、肿瘤细胞增殖、肌肉生长等方面具有重要的调控作用。本文综述了胰岛素样生长因子2基因结构、基因组印迹和作为影响肌肉重量数量性状基因座的研究进展。Abstract: Insulin-like growth factors play an important role in fetal growth and development, tumour cell proliferation and muscle growth. This review is focused on the insulin-like growth factor 2 gene structures, and their imprintings in mammalian genomes. In addition, we also discussed that IGF2 is the major paternally expressed candidate gene affecting muscle mass.     

Chromatin modification of imprinted H19 gene in mammalian spermatozoa

The allele-specific epigenetic markings of endogenously imprinted genes in placental mammals occur during gametogenesis. The identification of the molecular nature of gametic imprints is the first step towards understanding the mechanistic basis of epigenesis in embryonic and adult somatic tissues. The specific question addressed in this work is whether the closely positioned but oppositely imprinted insulin-like growth factor 2 (IGF 2) and H19 genes, which have similar temporal regulation during development, differ in chromatin structure in mammalian spermatozoa. During terminal differentiation of mammalian spermatozoa, about 3–15% of the haploid genome retains a quasisomatic-type chromatin structure, whereas the remaining genomes interact with protamines that are further cross-linked by -S-S- bridges. Micrococcal nuclease (MNase) and DNase I digestions of human (HSN) and porcine sperm nuclei (PSN) showed that the IGF 2 gene in both types of nuclei retained somatic-type nucleosomes that were close-packed with a periodicity of 150 bp. However, the H19 gene in both species was predominantly organised by unique structural repeats, which were 650–674 bp in PSN and 438–522 bp in HSN, condensing at least 20 kb of chromatin. These results, together with previous studies, suggest that epigenetic chromatin modification leading to preferential condensation of the paternal H19 allele in embryonic tissues is already present in the germ cells. Mol. Reprod. Dev. 50:474–484, 1998. © 1998 Wiley-Liss, Inc.     

Erasure of methylation imprinting of Igf2r during mouse primordial germ-cell development

During germ cell differentiation in mice, the genome undergoes specific epigenetic modifications. These include demethylation of imprinted genes and subsequent establishment of parental allele-specific methylation. The mouse Igf2r gene is an imprinted gene that shows maternal-specific expression. Maternal-specific methylation of differentially methylated region 2 (DMR2) of this gene may be necessary for its maternal-specific expression. Before the allele-specific methylation is established, DMR2 is demethylated in both male and female primordial germ cells (PGCs) by 13.5 days post coitum (dpc), indicating that the demethylation of this region occurs earlier in PGC development. The timing of the demethylation has been, however, unknown. In this study, we attempted to determine the timing of methylation erasure of Igf2r DMR2 in developing PGCs, using transgenic mice expressing green fluorescent protein specifically in the germ line. We purified migrating PGCs from the transgenic mice and examined the methylation status of DMR2. The results show that some CpG sites within DMR2 start demethylation at 9.5 dpc in some migrating PGCs, before the cells colonize genital ridges, and the progression of demethylation is rapid after colonization of the genital ridges. To examine whether the gonadal environment is involved in demethylation, we analyzed the methylation of DMR2 after culturing migrating PGCs in the absence of a gonadal environment. These culture experiments support the idea that a gonadal environment is not required for demethylation of the region in at least a fraction of PGCs.     

生长激素/胰岛素样生长因子1与阿尔茨海默病

生长激素／胰岛素样生长因子-1（GH／IGF-1）轴的合成、分泌、调节及生物学活性与阿尔茨海默病（AD）有密切关系。生长激素（GH）的合成和分泌受生长激素释放激素（GHRH）正向调节。GH／IGF-1轴活性下降导致一系列生理功能变化。GH／IGF-1缺乏可引起衰老及神经退行性变（AD）而导致认知功能的下降,相应激素的补给可以抑制或逆转这种认知障碍。越来越多的证据表明：GH／IGF-1参与AD型痴呆病理过程,对AD有很好的治疗应用前景。本文就生长激素／胰岛素样生长因子1在AD发病中的机理和药理学研究做一综述。     

IGFBP7基因的结构与功能的研究进展

胰岛素样生长因子结合蛋白7（IGFBP7）是IGFBPs超家族的新成员,结构上除具有与IGFBPs相似的保守N端结构域外,还有特异的Kazal型丝氨酸蛋白酶抑制结构域和免疫球蛋白样C2结构域。除与IGFs结合发挥作用外,还能独立调控细胞凋亡、增殖和迁移等。而至今尚无对水生无脊椎动物IGFBP7的研究报道,结合本实验室的研究综述了目前IGFBP7基因结构和功能上的研究进展,并对今后的研究工作进行了展望。     

Effects of fibroblast growth factor 2 and insulin-like growth factor II on the development of parthenogenetic mouse embryos in vitro

Most parthenogenetic embryos (PEs) in mammals die shortly after implantation, and this failure to develop is associated with genomic imprinting. We have examined the influence of human recombinant basic fibroblast growth factor 2 (FGF-2) and human recombinant insulin-like growth factor II (ICF-II) on the development of (CBA x C57BL/6)F1 parthenogenetic mouse embryos. Embryos were treated in vitro at the morula stage with different doses of FGF-2 and, after their development to blastocysts, transferred to pseudopregnant recipients. The optimal doses of FGF-2 did not affect the number of forming and implanting blastocysts, but increased, from 20 to 42%, the number of embryos developing to somite stages. PEs (18-21 somites) treated with an optimal dose of FGF-2 were explanted for further development in culture by treatment with the second growth factor, IGF-II. Eighty-three percent of those embryos cultured with IGF-II (2.5 microg/ml) developed to 35 or more somites, as compared with 36% of embryos cultured without any growth factors (P < 0.01). Also, a significantly higher proportion of PEs developed to 40-50 somites in this case. These results show that the in vitro treatment of PEs with FGF-2 at the morula stage increases the number of somite embryos, and the second treatment of somite PEs with IGF-II in culture medium prolongs their development significantly.     

Growth hormone,insulin-like growth factor I,and motoneuron size

In this study we asked whether growth hormone (GH) and one of its key mediators, insulin-like growth factor I (IGF-I), influence spinal motoneuron size in conjunction with whole body size. We present evidence that GH has such a role, possibly without the mediation of IGF-I. Both lumbar motoneuron and body size were found to be increased relative to littermate controls in transgenic mice overexpressing GH, while body size, but not motoneuron size, was increased in mice overexpressing IGF-I. GH overexpression coordinately increased nucleolar, nuclear, and cell body size in lumbar spinal motoneurons, so that their normal size relationships were preserved in the transgenic mice. In addition, spinal cord and brain weights were significantly increased in both types of transgenic animal. We conclude that GH can regulate motoneuron, central nervous system, and body size in the same animal, and that IGF-I can mimic the effects of GH on at least two of these three parameters. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 202–212, 1997.     

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.     

Deregulation of DNA methyltransferases and loss of parental methylation at the insulin-like growth factor II (Igf2)/H19 loci in p53 knockout mice prior to tumor development

To ascertain whether p53 deficiency in vivo leads to the deregulation of DNA methylation machinery prior to tumor development, we investigated the expression profile of DNA methyltransferases in the thymus and the liver of p53(+/+), p53(+/-), and p53(-/-) mice at 7 weeks of age before tumor development. The expression of DNA methyltransferases was examined in the thymus at 7 weeks of age, since the malignant T-cell lymphoma develops most frequently in p53(-/-) mice around 20 weeks of age. Both mRNA and protein levels of Dnmt1 and Dnmt3b were increased in the thymus and the liver of p53-deficient mice. The expression of Dnmt3a was also increased in the liver but not in the thymus of p53-deficient mice. Dnmt3L expression was reduced in the thymus of p53(+/-) and p53(-/-) mice. The total 5-methylcytosine (5-MeC) in the genomic DNA of p53(+/+), p53(+/-), and p53(-/-) mice was quantitated by dot-blot using antibody against 5-MeC. Global methylation was increased in the thymus and the liver of p53-deficient mice. To correlate the deregulated expression of DNA methyltransferases with the disturbance of the epigenetic integrity, we examined the DNA methylation of the imprinting control region (ICR) at the insulin-like growth factor II (Igf2)/H19 loci in the thymus and the liver of p53(+/+), p53(+/-), and p53(-/-) mice. The region containing two CCCTC binding factor (CTCF) binding sites in the 5'-ICR tended to be hypomethylated in the thymus of p53(-/-) mice, but not in the liver. The expression profile of Igf2 and H19 indicated that the thymus-specific changes of Igf2 and H19 expression were coherent to the hypomethylation of the ICR in the thymus. Our results suggest that p53 is required for the maintenance of DNA methylation patterns in vivo.     

Effects of cadmium and zinc on plasma levels of growth hormone, insulin-like growth factor I, and insulin-like growth factor-binding protein 3

Humans are constantly exposed to cadmium (Cd) as a result of the increase in air pollution and cigaret use. Zinc (Zn), which is an essential element for the metabolism of and the constituent of many enzymes, causes growth retardation in the deficiency status so at present it is often added to the diet without measuring blood levels of this element. We also aimed to observe the effects of both Cd and Zn on the plasma levels of growth hormone (GH), insulin-like growth factor I(IGF-I), and insulin-like growth factor-binding protein 3 (IGFBP-3) in this study. For this purpose, 27 young Wistar albino male rats were divided into three groups. The first group was given 50 mg/L of CdCl₂, the second group received 500 mg/L of ZnSO₄, and the third group, as a control, received only drinking water for 1 mo. At the end of this period, plasma GH, IGF-I, and IGFBP-3 of the animals were analyzed in the blood obtained. The significance between groups was evaluated with the Mann-Whitney U-test. According to our results, levels of IGF-I and IGFBP-3 in the Cd-administered group were significantly lower than those of controls (p<0.05 and p<0.01 respectively). No statistically significant difference was observed between Zn administered and control groups in terms of all three parameters. These results show that although the addition of Zn to the diet of healthy rats had no effect on the levels of GH, IGF-I, and IGFBP-3, Cd addition lowered the levels of IGF-I and IGFBP-3 but did not change the levels of GH compared to controls.     

Nucleosome positioning signals in the DNA sequence of the human and mouse H19 imprinting control regions

We have investigated the sequences of the mouse and human H19 imprinting control regions (ICRs) to see whether they contain nucleosome positioning information pertinent to their function as a methylation-regulated chromatin boundary. Positioning signals were identified by an in vitro approach that employs reconstituted chromatin to comprehensively describe the contribution of the DNA to the most basic, underlying level of chromatin structure. Signals in the DNA sequence of both ICRs directed nucleosomes to flank and encompass the short conserved sequences that constitute the binding sites for the zinc finger protein CTCF, an essential mediator of insulator activity. The repeat structure of the human ICR presented a conserved array of strong positioning signals that would preferentially flank these CTCF binding sites with positioned nucleosomes, a chromatin structure that would tend to maintain their accessibility. Conversely, all four CTCF binding sites in the mouse sequence were located close to the centre of positioning signals that were stronger than those in their flanks; these binding sites might therefore be expected to be more readily incorporated into positioned nucleosomes. We found that CpG methylation did not effect widespread repositioning of nucleosomes on either ICR, indicating that allelic methylation patterns were unlikely to establish allele-specific chromatin structures for H19 by operating directly upon the underlying DNA-histone interactions; instead, epigenetic modulation of ICR chromatin structure is likely to be mediated principally at higher levels of control. DNA methylation did, however, both promote and inhibit nucleosome positioning at several sites in both ICRs and substantially negated one of the strongest nucleosome positioning signals in the human sequence, observations that underline the fact that this epigenetic modification can, nevertheless, directly and decisively modulate core histone-DNA interactions within the nucleosome.     

Differential expression of insulin-like growth factor-I and insulin-like growth factor binding protein-1 in the diabetic rat

Summary Multiple factors contribute to the growth retardation which is a characteristic feature of uncontrolled diabetes. In this report we have examined the effects of streptozotocin-induced (STZ) diabetes on expression of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-1 (IGFBP-1) in various tissues. As early as 7 days after STZ administration there was a modest reduction in IGF-I mRNA abundance. The reduction (10–30%) was of similar magnitude in each of the 7 tissues examined; liver, kidney, lung, diaphragm, quadraceps, heart and adipose tissue. However, the reduction achieved statistical significance only in the lung (p < 0.05) and diaphragm (p < 0.01). A further reduction in IGF-I mRNA abundance was seen in many tissues, 32 and 91 days after STZ administration. In contrast to the decrease in IGF-I mRNA, IGFBP-1 mRNA was significantly increased in the liver and kidney of diabetic rats. IGFBP-1 mRNA was detectable at only very low levels in other tissues but was increased in diabetic rats compared non-diabetic rats. In diabetic rats, a highly significant correlation (R = 0.75, p < 0.001) between hepatic IGFBP-1 mRNA and glucose was observed whereas there was no significant correlation between serum glucose and hepatic IGF-I mRNA abundance (R = 0.24, p = NS). Treatment of diabetic rats with insulin resulted in a small, non significant increase in hepatic and renal IGF-I mRNA and a significant decrease in renal IGFBP-1 mRNA abundance. The observations reported here are consistent with the hypothesis that diminished IGF-I expression and inhibition of available IGF-1 by increased levels of IGFBP-1 may explain the impaired growth seen in diabetic animals.     

Analysis of polymorphisms in the insulin-like growth factor 1 receptor (IGF1R) gene from Japanese quail selected for body weight

Insulin-like growth factor 1 receptor ( IGF1R ) is essential for the signalling of growth. In this study, we performed single nucleotide polymorphism (SNP) detection in the Japanese quail IGF1R coding region and an association study between SNPs and body weight in two lines (SS and LL) selected for large and small body weight. Of 21 SNPs obtained, a SNP at position AB292766:c.2293G>A led to the replacement of a valine with an isoleucine (V765I). The two lines were fixed for alternate alleles, with allele encoding valine fixed in the LL line. A significant effect of the SNP genotype was found on 10-week body weight ( P  < 0.01) and on 4- to 10-week and 6- to 10-week average daily gain ( P  < 0.05) in the F₂ family obtained from lines LL and SS. In six populations maintained in Japan or France, the frequency of allele encoding valine was higher than the allele encoding isoleucine.     

The expression of insulin-like growth factor binding proteins in human hepatocellular carcinoma

Insulin-like growth factors (IGF), IGF receptors and IGF binding proteins (IGFBPs) play an important role in cell growth and differentiation. The liver is the major source of IGF-1 and at least two IGFBPs (IGFBP-1 and IGFBP-3). IGFBPs most often serve to attenuate the effects of IGF at the receptor level and thereby limit IGF-induced cell growth and differentiation. Although changes in IGFBP expression have been described during controlled liver growth such as hepatic regeneration following partial hepatectomy, there is limited knowledge of IGFBPs gene expression in uncontrolled growth or hepatocellular carcinoma. In the present study, we employed Northern blotting techniques to document the expression of IGFBP-1, 3 and 4 in normal human livers, cirrhotic and hepatocellular carcinoma tissues. The results revealed no differences in IGFBP-1, 3 and 4 mRNA levels between normal and cirrhotic tissues. However, the expression of all three IGFBPs mRNA were significantly down regulated in hepatocellular carcinoma tissues. These findings are in keeping with IGFBPs playing an important inhibitory role in the development and/or growth of hepatocellular carcinoma in humans.     

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8‐cell stage (named as a₂PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4‐cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4‐cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post‐implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP‐a₄PgESCs which derived from GFP‐4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5‐day fetus totally derived from GFP‐a₄PgESCs with germline contribution by 8‐cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a₄PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study.     

Paradoxical methylation of the tyrosine hydroxylase gene in mouse preimplantation embryos

The mouse tyrosine hydroxylase (Th) gene is located in an evolutionarily conserved imprinted gene cluster on distal chromosome 7. It is associated with a CpG island that spans the promoter of the gene. Using a bisulfite sequencing method we show that the Th promoter is fully methylated in both male and female mouse germ cells and in human spermatozoa, suggesting that it belongs to the newly identified category of CpG islands, the similarly methylated regions (SMRs). Contrary to other tissue-specific gene sequences, the mouse Th promoter escapes the initial wave of genome demethylation during the first few cell cycles, but becomes demethylated between the morula and the blastocyst stages. This unusual methylation ontogeny may be a characteristic of the SMRs and/or related to the localization of the Th gene in an imprinted gene cluster.     

Histone H1 affects gene imprinting and DNA methylation in Arabidopsis

Imprinting, i.e. parent-of-origin expression of alleles, plays an important role in regulating development in mammals and plants. DNA methylation catalyzed by DNA methyltransferases plays a pivotal role in regulating imprinting by silencing parental alleles. DEMETER (DME), a DNA glycosylase functioning in the base-excision DNA repair pathway, can excise 5-methylcytosine from DNA and regulate genomic imprinting in Arabidopsis. DME demethylates the maternal MEDEA (MEA) promoter in endosperm, resulting in expression of the maternal MEA allele. However, it is not known whether DME interacts with other proteins in regulating gene imprinting. Here we report the identification of histone H1.2 as a DME-interacting protein in a yeast two-hybrid screen, and confirmation of their interaction by the in vitro pull-down assay. Genetic analysis of the loss-of-function histone h1 mutant showed that the maternal histone H1 allele is required for DME regulation of MEA, FWA and FIS2 imprinting in Arabidopsis endosperm but the paternal allele is dispensable. Furthermore, we show that mutations in histone H1 result in an increase of DNA methylation in the maternal MEA and FWA promoter in endosperm. Our results suggest that histone H1 is involved in DME-mediated DNA methylation and gene regulation at imprinted loci.     

Widespread disruption of genomic imprinting in adult interspecies mouse (Mus) hybrids

Mammalian interspecies hybrids exhibit parent-of-origin effects in that offspring of reciprocal matings, even though genetically identical, frequently exhibit opposite phenotypes, especially in growth. This was also observed in hybridization with the genus Mus. These parent-of-origin effects suggested that imbalance in the expression of imprinted genes, which are expressed differentially, depending on their transmission through the maternal or paternal germline, and/or differential loss-of-imprinting (LOI) could underlie these opposite growth phenotypes in reciprocal mammalian hybrids. Here we report that tissue-specific LOI occurs in adult Mus hybrids. Contrary to expectations, LOI patterns were not consistent with a direct influence of altered expression levels of imprinted genes on growth. Bisulfite sequencing revealed that reactivation of maternal alleles of Peg3 and Snrpn in specific tissues was accompanied by partial demethylation at their potential imprinting control regions. We propose that abnormal reprogramming after fertilization and during preimplantation development is in part responsible for hybrid dysgenesis, for which a strong epigenetic basis has been demonstrated.     

Genomic imprinting of the insulin-like growth factor 2 gene in sheep

A number of genes in the human and mouse genomes are subject to genomic imprinting, with selective inactivation of one allele of a gene in a parent-of-origin specific manner. One of the first imprinted genes identified was the Insulin-like Growth Factor 2 gene (IGF2), which promotes growth of the fetus and is expressed from only the paternal allele in most tissues in both the mouse and human. The aim of this study was to establish the imprinting status of IGF2 in sheep (Ovis aries). Sheep provide an interesting model to study imprinting, owing to differences in their placental development and the fact that they have been subject to strong artificial selection for various production traits. We report the identification of a length polymorphism in the transcribed 3′-untranslated region of the ovine IGF2 gene. This polymorphism was used to map IGF2 to sheep Chromosome (Chr) 21 and demonstrate that IGF2 is indeed imprinted in sheep, being expressed from the paternal allele. We also report that the developmental switch from imprinted IGF2 expression in the fetal liver to biallelic IGF2 expression in the adult liver, which occurs in the human but not mouse, also occurs in sheep. Differences in male- and female-specific recombination values reported around the IGF2 locus in the human were also observed around the ovine IGF2 locus. The techniques developed in this study will enable the imprinting status of IGF2 to be assessed in a variety of tissues and stages of development in normal sheep. Received: 3 October 1998 / Accepted 29 January 1999