胰岛素样生长因子结合蛋白-3

胰岛素样生长因子结合蛋白(IGFBPs)是能与胰岛素样生长因子(IGFs)结合的调节蛋白,调节IGFs与其受体(ICFR)的结合能力,影响IGFR下游信号转导通路中信号强度,调控靶细胞的生长和增殖。IGFBP-3的作用方式有IGF依赖性和非IgF依赖性两种。IGFs、成纤维细胞生长因子(FgF)、胰岛素、细胞表面受体,甚至转录调节区都有可能成为IGFBP-3的结合对象并引起增殖抑制;IGFBP—3的水解片段化、糖基化和磷酸化修饰都可能影响它对靶细胞的增殖抑制能力。     

ENDOCRINE RESPONSE TO AN ULTRA-MARATHON IN PRE- AND POST-MENOPAUSAL WOMEN

Ultra-endurance competitions are becoming increasingly popular but there is limited research on female participants. The purpose of this study was to examine changes in estrogen and the IGF-I system in women after an ultra-marathon. Six pairs of pre- and post- menopausal women were matched for race finish times;mean finish time was 20 hours. Blood samples were drawn 24 hours before the race, at the finish, and 24 hours into recovery. Samples were analysed for estradiol, total IGF-I, IGFBP-1, and intact IGFBP-3. There was a significant increase in estradiol following the race in both groups (P < 0.05). Total IGF-I decreased after the race (P < 0.01) and remained lower in recovery. IGFBP-1 increased after the race (P < 0.001) but returned to pre-race levels after 24 hours, while intact IGFBP-3 was significantly lower post-race and in recovery (P < 0.001). Postmenopausal women had significantly lower estradiol at baseline, but there were no other group differences. These results demonstrate that among recreational female runners, an ultra-marathon is associated with IGF system changes that are consistent with an energy-deficient, catabolic state. Further research is needed to confirm the effect of these endocrine changes on health and performance.     

Epigenetic regulation of insulin-like growth factor binding protein-3 (IGFBP-3) in cancer

Epigenetics refers to heritable changes in gene expression that are independent of alterations in DNA sequence. It is now accepted that disruption of epigenetic mechanisms plays a key role in the pathogenesis of cancer: culminating in altered gene function and malignant cellular transformation. DNA methylation and histone modifications are the most widely studied changes but non-coding RNAs such as miRNAs are also considered part of the epigenetic machinery. The insulin-like growth factor (IGF) axis is composed of two ligands, IGF-I and –II, their receptors and six high affinity IGF binding proteins (IGFBPs). The IGF axis plays a key role in cancer development and progression. As IGFBP genes have consistently been identified among the most common to be aberrantly altered in tumours, this review will focus on epigenetic regulation of IGFBP-3 in cancer for which the majority of evidence has been obtained.     

Matrix metalloproteinase-7 degrades all insulin-like growth factor binding proteins and facilitates insulin-like growth factor bioavailability

Proteolytic modification of insulin-like growth factor binding proteins (IGFBPs) plays an important physiological role in regulating insulin-like growth factor (IGF) bioavailability. Recently, we demonstrated that matrix metalloproteinase-7 (MMP-7)/Matrilysin produced by various cancer cells catalyzes the proteolysis of IGFBP-3 in vitro and regulates IGF bioavailability, resulting in an anti-apoptotic effect against anchorage-independent culture. In the present study, we investigated whether MMP-7 contributes to proteolysis of the other five IGFBPs, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6, and whether this results in phosphorylation of the IGF type 1 receptor (IGF-1R). MMP-7 cleaved all six IGFBPs, resulting in IGF-mediated IGF-1R phosphorylation, which was inhibited by EDTA treatment. These results suggest that MMP-7 derived from cancer cells can regulate IGF bioavailability in the microenvironment surrounding the tumor, where various kinds of IGF/IGFBP complexes are found, thereby favoring cancer cell growth and survival during the processes of invasion and metastasis.     

Insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mediate TGF-beta- and myostatin-induced suppression of proliferation in porcine embryonic myogenic cell cultures

We have previously shown that cultured porcine embryonic myogenic cells (PEMC) produce both insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 and secrete these proteins into their media. Exogenously added recombinant porcine (rp) IGFBP-3 and rpIGFBP-5 act via IGF-dependent and IGF-independent mechanisms to suppress proliferation of PEMC cultures. Furthermore, immunoneutralization of endogenous IGFBP-3 and IGFBP-5 in the PEMC culture medium results in increased DNA synthesis rate suggesting that endogenous IGFBP-3 and IGFBP-5 suppress PEMC proliferation. TGF-beta superfamily members myostatin and TGF-beta1 have also been shown to suppress proliferation of myogenic cells, and treatment of cultured PEMC with either TGF-beta1 or myostatin significantly (P < 0.01) increases levels of IGFBP-3 and -5 mRNA. We have previously shown that immunoneutralization of IGFBP-3 decreases the proliferation-suppressing activity of TGF-beta1 and myostatin. Here, we show that immunoneutralization of IGFBP-5 also significantly (P < 0.05) decreases the DNA synthesis-suppressing activity of these molecules. Simultaneous immunoneutralization of both IGFBP-3 and IGFBP-5 in TGF-beta1 or myostatin-treated PEMC cultures restores Long-R3-IGF-I-stimulated DNA synthesis rates to 90% of the levels observed in control cultures receiving no TGF-beta1 or myostatin treatment (P < 0.05). Even though immunoneutralization of IGFBP-3 and -5 increased DNA synthesis rates in TGF-beta1 or myostatin-treated PEMC cultures, phosphosmad2 levels in these cultures were not affected. These findings strongly suggest that IGFBP-3 and IGFBP-5 affect processes downstream from receptor-mediated Smad phosphorylation that facilitate the ability of TGF-beta and myostatin to suppress proliferation of PEMC.     

Potential of proteomics towards the investigation of the IGF-independent actions of IGFBP-3

Early investigations into the insulin-like growth factor (IGF)-independent actions of insulin-like growth factor-binding protein (IGFBP)-3 have implicated a large array of signaling proteins with links to cell cycle control and apoptosis. However, the actual mechanism of IGFBP-3 action is still unclear. In an effort to clearly understand the mechanism of IGF-independent IGFBP-3 actions, a proteomic approach to identify the actual proteins involved in interaction with IGFBP-3 from different cell compartments, the phosphorylation status of IGFBP-3 under different physiologic conditions and the proteins upregulated by IGFBP-3 are briefly reviewed. The IGF system is a well-recognized key player in diseases such as cancer, diabetes and malnutrition. It is only after the signaling pathways of the IGF-independent actions of IGFBP-3 are clearly understood that the system can be manipulated to affect these disorders.     

IGFBP-3 and apoptosis—a licence to kill?

Insulin-like growth factor binding protein (IGFBP)-3, the major carrier of insulin-like growth factors (IGFs) in the circulation, was first isolated and characterised over a decade ago. More recently, IGFBP-3 has been assigned a role as a putative death-promoting factor, a function that appears, under certain circumstances, to be independent of its IGF-binding ability. This review examines the current evidence for a pro-apoptotic function for IGFBP-3 and speculates on its physiological significance.     

IGFBP-3高效可溶表达、纯化及生物学活性初步研究

克隆胰岛素样生长因子结合蛋白3(IGFBP-3)的cDNA片段,构建原核表达载体pET-DsBA-IGFBP3。将该重组质粒转化大肠杆菌BL21(DE3)plysS中,诱导表达IGFBP-3融合蛋白(简称D-IGFBP3)。经检测融合蛋白主要以可溶形式表达。表达产物用His亲和层析柱纯化,获得了纯度超过95%的重组IGFBP-3融合蛋白。Western-blot结果表明在相应分子量处有一条特异性条带。细胞活性研究显示它对MCF-7细胞生长具有一定的抑制作用,且在体外具有与IGF-I结合的活性。     

Characterisation of N-glycans bound to IGFBP-3 in sera from healthy adults

Human IGFBP-3 contains three potential N-linked glycosylation sites. Published data concerning the type and saccharide composition of the N-glycans is scarce. The aim of this study was to characterise N-glycans covalently attached to IGFBP-3 from sera of healthy adults (men and women). In order to do that a panel of eight lectins covering broad saccharide specificity was used: agarose-immobilised SNA (Sambucus nigra agglutinin), Con A (lectin from Canavalia ensiformis), RCA I (Ricinus communis agglutinin I), PHA-E (Phaseolus vulgaris erythroagglutinin), PHA-L (P. vulgaris leukoagglutinin), succinylated WGA (wheat germ agglutinin), ECL (Erythrina cristagalli lectin) and UEA (Ulex europaeus agglutinin). IGFBP-3 interacted with SNA, Con A, RCA I, PHA-E and, to a much lesser extent, with PHA-L. These results indicate that human IGFBP-3 bears mostly biantennary complex type N-glycans with a very high content of α-2,6-linked Sia at their termini. Hybrid type and high-mannose type N-glycans are present, as well as a bisecting GlcNAc residue, which may be core fucosylated. N-glycosylation of IGFBP-3 follows the N-glycosylation pattern of major serum proteins. This study represents a ground for the future research of glycosylation pattern of IGFBP-3 from the circulation of men and women diagnosed with different illnesses.     

缺氧复氧对滑膜细胞IGF-IGFBP-3 以及线粒体的影响

目的:探讨氧波动环境对原代成纤维样滑膜细胞(fibroblast-like synoviocyte, FLS)胰岛素样生长因子-1(insulin growth factor-1,IGF-1)、胰岛素样生长因子结合蛋白-3(insulin-like growth factor binding protein-3, IGFBP-3)及线粒体的影响。方法:分离并鉴定正常人滑膜细胞,再对滑膜细胞进行分组:对照组、缺氧/再充氧(hypoxia/reoxygenation,H/R)干预组。采用实时定量PCR检测滑膜细胞中IGF-1、IGFBP-3的m RNA水平;Western blot检测滑膜细胞中IGF-1、IGFBP-3的蛋白水平;流式细胞仪检测线粒体膜电位(Mitochondrial membrane potential, MMP)以及线粒体通透性转换孔(Mitochondrial Permeability Transition Pore, MPTP)的变化。结果:与对照组比较,H/R干预组的相对IGF-1和IGFBP-3的m RNA水平和蛋白表达水平显著升高(P0.05),膜电位水平降低(P0.05),线粒体通透性转换孔开放。结论:氧波动环境可促进IGF-1和IGFBP-3的表达及细胞线粒体损伤,其可能是骨关节炎(OA)发病的重要机制之一。     

The amino-terminal region of insulin-like growth factor binding protein-3, (1-95)IGFBP-3, induces apoptosis of MCF-7 breast carcinoma cells

In an earlier study, we reported that an N-terminal proteolytic fragment ((1-95)IGFBP-3) corresponding to the first 95 residues of human insulin-like growth factor binding protein-3 (IGFBP-3) inhibits proliferation in a variety of fibroblasts. With a view to investigating its cytostatic capacity in carcinoma cells, we transiently transfected MCF-7 breast adenocarcinoma cells with an expression vector containing (1-95)IGFBP-3 cDNA. The transfected cells secreted a hyper-glycosylated form of (1-95)IGFBP-3. Twenty-four hours after transfection, cell morphology and viability were similar in control and (1-95)IGFBP-3-secreting cells. However, after 48 h, (1-95)IGFBP-3-secreting cells were apoptotic, with marked cytoplasmic vacuolation and increased free histones in the cytoplasm. Culture media conditioned by (1-95)IGFBP-3-secreting cells also induced morphological changes and apoptosis in wild-type MCF-7 cells, indicating that (1-95)IGFBP-3 was responsible for the effects observed. These results provide further evidence that the N-terminal proteolytic fragment of IGFBP-3 has a functional role.     

大白菜BrMYB34-3基因的3′-RACE克隆及表达

以大白菜叶片为材料,根据NCBI数据库中Br MYB34-3基因序列的已知信息,利用3-RACE获得了Br MYB34-3基因全长,用RT-PCR方法对Br MYB34-3在不同组织中的表达进行分析.结果显示,该基因全长1 135 bp,编码280个氨基酸.Br MYB34-3具有R2R3-MYB(含有2个MYB结构域的转录因子)典型的R2、R3结构域及基序.其氨基酸序列与大白菜的Br MYB34-1和Br MYB34-2、拟南芥的At MYB34具有较高的一致性.Br MYB34-3在莲座叶、当天开的花中表达水平较高,在花序顶端表达水平较低,在根和花序轴中未检测到表达,这与拟南芥At MYB34的表达模式不同.研究表明,Br MYB34-3是大白菜中的MYB34同源基因,在功能上可能与拟南芥At MYB34基因存在差异.     

Single nucleotide polymorphism in Egyptian cattle insulin-like growth factor binding protein-3 gene

Insulin-like growth factor binding protein-3 (IGFBP-3) gene is a structural gene responsible for the multiple influences of insulin-like growth factors (IGFs) system. It is considered as a candidate gene for growth and production traits. In the present study, we aimed to determine the genetic polymorphism of Egyptian cattle IGFBP-3 gene.The amplified fragment of cattle IGFBP-3 gene at 651-bp was digested with three different endonucleases; HaeIII, MspI and TaqI. The digestion of the PCR products with MspI and TaqI endonucleases revealed similar restriction patterns in all tested animals.Digestion of the PCR product with HaeIII restriction enzyme revealed three different genotypes in Egyptian cattle due to the presence of two alleles; allele A with 7 digested fragments resulting from the presence of 6 restriction sites and allele C with 8 digested fragments resulting from the presence of 7 restriction sites; six sites as allele A in the addition of another restriction site at position 298^299 as a result of SNP (A → C) in C allele at position 299. The restriction patterns of IGFBP-3/HaeIII showed that forty-six examined animals are genotyped as AA, CC and AC with frequencies of 21.74%, 21.74% and 56.52%, respectively.It is concluded that the IGFBP-3/HaeIII polymorphism may be utilized as a good marker for genetic differentiation between cattle animals for different body functions such as growth, metabolism, reproduction, immunity and energy balance. The nucleotide sequences of Egyptian cattle IGFBP-3 A and C alleles were submitted to GenBank with the accession numbers KF899893 and KF899894, respectively.