Potentiation of Growth Factor Signaling by Insulin-like Growth Factor-binding Protein-3 in Breast Epithelial Cells Requires Sphingosine Kinase Activity

We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1, but not SphK2, expression. Similarly, IGFR1 phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK1 silencing. SphK1 expression and activity were stimulated by IGFBP-3 ∼2-fold over 24 h. Silencing of sphingosine 1-phosphate receptor 1 (S1P₁) or S1P₃, but not S1P₂, abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S1P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S1P₁ and S1P₃. These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-10A cells requires SphK1 activity and S1P₁/S1P₃, suggesting that S1P, the product of SphK activity and ligand for S1P₁ and S1P₃, is the “missing link” mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3.Insulin-like growth factor-binding protein-3 (IGFBP-3)² is one of the family of six IGFBPs that bind the peptide growth factors IGF-I and IGF-II with high affinity and regulate their bioactivity (1). As the predominant carrier of IGFs in the endocrine system, IGFBP-3 regulates the movement of these growth factors from the circulation to target tissues and inhibits their proliferative and antiapoptotic cellular effects by blocking their activation of the type 1 IGF receptor (IGFR1) at the cell surface. In vitro studies in a variety of cell types have revealed that IGFBP-3 may also impact on cell growth and survival independently of modulating IGF bioactivity, inducing cell cycle arrest and apoptosis by regulation of apoptotic effector proteins (2–4) and interaction with nuclear receptors (5–7).There is, however, also evidence of an association between IGFBP-3 and enhanced cell proliferation. Some clinical studies in breast, prostate, pancreatic, renal cell, and non-small cell lung cancers have shown that a high level of tissue expression of IGFBP-3 correlates with increased tumor growth or malignancy (8–13). Although the mechanism linking IGFBP-3 with growth stimulation in vivo remains unclear, we and others have shown that, in vitro, IGFBP-3 can enhance the effects of stimulatory growth factors. Human and bovine skin fibroblasts exposed to low concentrations of exogenous IGFBP-3 exhibit enhanced IGF-stimulated DNA synthesis (14, 15), and similarly, exogenous and endogenous IGFBP-3 enhanced the growth response to IGF-I in the MCF-7 breast cancer cell line (16). We have also shown previously that IGFBP-3 is inhibitory to DNA synthesis in MCF-10A breast epithelial cells in the absence of exogenous growth factors or serum (17), but is growth stimulatory in the presence of EGF in the same cell line (18). There is no evidence that potentiation of EGF or IGF bioactivity by IGFBP-3 requires direct interaction between IGFBP-3 and the growth factor receptors (15, 18), but the mechanism underlying the effects of IGFBP-3 on growth factor signaling has not been elucidated.Recently it was suggested that, in human umbilical vein endothelial cells, an antiapoptotic effect of IGFBP-3 is associated with increased expression and activity of sphingosine kinase 1 (SphK1), and formation of the bioactive sphingolipid sphingosine 1-phosphate (S1P) (19, 20). SphK1 has been shown to have a role in oncogenesis (21), and S1P, acting both as an intracellular second messenger and extracellularly through activation of specific S1P receptors, stimulates cell proliferation and survival (22). In addition to transducing S1P signaling, the G-protein-coupled S1P receptors have been implicated in signal amplification of a variety of growth factors receptors, including the EGF and platelet-derived growth factor receptors, via receptor transactivation (23, 24). In this study we investigated whether the sphingosine kinase system is involved in modulation of growth factor receptor signaling pathways by IGFBP-3 and demonstrate that SphK1 expression is stimulated by IGFBP-3 in MCF-10A cells, and its activity is required for potentiation of EGF and IGF-I signaling by IGFBP-3 in these cells.     

Control of Adipose Tissue Expandability in Response to High Fat Diet by the Insulin-like Growth Factor-binding Protein-4

Adipose tissue expansion requires growth and proliferation of adipocytes and the concomitant expansion of their stromovascular network. We have used an ex vivo angiogenesis assay to study the mechanisms involved in adipose tissue expansion. In this assay, adipose tissue fragments placed under pro-angiogenic conditions form sprouts composed of endothelial, perivascular, and other proliferative cells. We find that sprouting was directly stimulated by insulin and was enhanced by prior treatment of mice with the insulin sensitizer rosiglitazone. Moreover, basal and insulin-stimulated sprouting increased progressively over 30 weeks of high fat diet feeding, correlating with tissue expansion during this period. cDNA microarrays analyzed to identify genes correlating with insulin-stimulated sprouting surprisingly revealed only four positively correlating (Fads3, Tmsb10, Depdc6, and Rasl12) and four negatively correlating (Asph, IGFbp4, Ppm1b, and Adcyap1r1) genes. Among the proteins encoded by these genes, IGFbp4, which suppresses IGF-1 signaling, has been previously implicated in angiogenesis, suggesting a role for IGF-1 in adipose tissue expandability. Indeed, IGF-1 potently stimulated sprouting, and the presence of activated IGF-1 receptors in the vasculature was revealed by immunostaining. Recombinant IGFbp4 blocked the effects of insulin and IGF-1 on mouse adipose tissue sprouting and also suppressed sprouting from human subcutaneous adipose tissue. These results reveal an important role of IGF-1/IGFbp4 signaling in post-developmental adipose tissue expansion.     

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。     

胰岛素样生长因子结构蛋白功能及其调节机制

胰岛素样生长因子结合蛋白 (ＩＧＦＢＰ)是ＩＧＦ家族的重要成员 ,ＩＧＦ及其生物活性的调节与ＩＧＦＢＰ密切相关。ＩＧＦＢＰ还具有独立于ＩＧＦ的生物活性 ,并拥有自己的调节机制。1 .ＩＧＦＢＰ简介目前 ,已确定了 6种与ＩＧＦ有高亲和力的ＩＧＦＢＰ(ＩＧＦＢＰ1～ 6 ) ,另有 4种与ＩＧＦ有较低亲和力的ＩＧＦＢＰ(ＩＧＦＢＰ相关肽 )也有报道[1] 。ＩＧＦＢＰ1～ 6有 35%的氨基酸序列同源 ,它们都有相同的Ｎ端和Ｃ端模式 ,只是中间区不同。其中 ,前 5种与ＩＧＦ Ｉ的结合优先于与ＩＧＦ ＩＩ的结合 ,而ＩＧＦＢＰ 6与ＩＧＦ ＩＩ的亲…     

Zinc Deficiency Promotes Testicular Cell Apoptosis in Mice

Biological Trace Element Research - Zinc (Zn) plays an important role in spermatogenesis, and carbon tetrachloride (CCl4) induces testicular oxidative damage and cell death. The objective of the...     

胰岛素样生长因子1（IGF-I）通过PI-3K/Akt途径对结肠癌细胞凋亡的影响

目的：探讨胰岛素样生长因子-1（IGF-I）通过磷酯酰肌醇3-激酶/蛋白激酶B（PI-3K/Akt）信号通路对结肠癌细胞株SW480凋亡率的影响及其凋亡抑制蛋白survivin表达水平的变化。方法：培养结肠癌SW480细胞株,实验分成三组：未加IGF-I空白组、IGF-I刺激组、IGF-I＋LY294002阻断组,检测阻断剂LY294002是否阻断PI-3K/Akt通路（Western Blot检测三组P-Akt表达情况）;Western Blot及免疫荧光观察三组survivin蛋白表达变化;MTT法检测细胞增殖活性,流式细胞术检测细胞凋亡情况。结果：Western blot结果显示LY294002可抑制IGF-I诱导的p-Akt的表达（P〈0.05）;阻断IGF-I诱导的PI-3K/Akt通路后MTT显示结肠癌细胞SW480增殖抑制率升高（P〈0.05）,流式细胞术分析显示凋亡率明显上升（P〈0.05）;Western blot及免疫荧光结果显示LY294002可抑制IGF-I诱导的survivin的表达（P〈0.05）。结论：IGF-I可通过PI-3K/Akt通路诱导survivin表达,从而抑制结肠癌细胞SW480的凋亡。     

Hepatoma-derived Growth Factor-related Protein-3 Interacts with Microtubules and Promotes Neurite Outgrowth in Mouse Cortical Neurons

Hepatoma-derived growth factor-related proteins (HRP) comprise a family of 6 members, which the biological functions are still largely unclear. Here we show that during embryogenesis HRP-3 is strongly expressed in the developing nervous system. At early stages of development HRP-3 is located in the cytoplasm and neurites of cortical neurons. Upon maturation HRP-3 relocalizes continuously to the nuclei and in the majority of neurons of adult mice it is located exclusively in the nucleus. This redistribution from neurites to nuclei is also found in embryonic cortical neurons maturing in cell culture. We show that HRP-3 is necessary for proper neurite outgrowth in primary cortical neurons. To identify possible mechanisms of how HRP-3 modulate neuritogenesis we isolated HRP-3 interaction partners and demonstrate that it binds tubulin through the N-terminal so called HATH region, which is strongly conserved among members of the HRP family. It promotes tubulin polymerization, stabilizes and bundles microtubules. This activity depends on the extranuclear localization of HRP-3. HRP-3 thus could play an important role during neuronal development by its modulation of the neuronal cytoskeleton.Neuritogenesis is a key step in nervous system development in which neurons extend dendrites and axons and connect to different targets in and outside the nervous system. The proper regulation of this process is controlled by a number of extra- and intracellular molecules expressed by neurons themselves or non-neuronal cells in their surroundings. Multiple studies indicate that rearrangement of the neuronal cytoskeleton in response to extracellular signals is an important mechanism during neurite extension and pathfinding (1-3). Manipulation of the polymerization and depolymerization of microtubules has shown that regulation of microtubule assembly and maintenance is important for neuritogenesis (4). Microtubule dynamics are regulated by a huge number of regulatory proteins like tau or other microtubule-associated proteins (MAPs)⁴ (5). In addition, proteins like CRMP-2 that interact with tubulin dimers and accelerate the assembly of tubulin into microtubles have been shown to be involved in the regulation of neuronal polarity and neuritogenesis (6-10). Despite all advances, however, made in the understanding of the role of the cytoskeleton and its regulatory proteins during neuritic growth there are still many open questions regarding the regulation of these processes. Therefore identifying new molecules binding to and modulating the turnover of microtubules is of high interest for the understanding of how neurite outgrowth is regulated.Hepatoma-derived growth factor (HDGF) is a protein that was purified from secretions of hepatoma cells by virtue of its growth factor activity. Subsequently 5 additional proteins were identified in which the 97 N-terminal amino acid residues show strong similarity to HDGF. Accordingly this family of proteins has been termed HDGF-related proteins (HRP) (11-13). HDGF has neurotrophic activity for hippocampal, spinal, and facial motor neurons (14, 15). So far, however, no receptor or signal transduction pathway involved has been identified for any of the HRPs.Most HRPs are expressed in a variety of tissues. HRP-3, however, is the only family member in whose expression is restricted. It is only expressed in neurons and to a low extent in glial cells (16, 17). Like HDGF after transfection into human embryonic kidney cells HRP-3 exhibits proliferative activity (12). The strong and almost exclusive expression of HRP-3 in postmitotic neurons, however, suggests biological functions other than its growth factor activity (16).In the present study we examine the expression and function of HRP-3 protein during mouse embryonic neuronal development. We demonstrate that the protein locates to the cytoplasm and neurites during early nervous system development, whereas most of HRP-3 can be found in the nucleus in adult neurons. We show that HRP-3 promotes neurite growth and suggest that this is due to the interaction of HRP-3 with the tubulin component of the neuronal cytoskeleton.     

A Mechanism of Male Germ Cell Apoptosis Induced by Bisphenol-A and Nonylphenol Involving ADAM17 and p38 MAPK Activation

Germ cell apoptosis regulation is pivotal in order to maintain proper daily sperm production. Several reports have shown that endocrine disruptors such as Bisphenol-A (BPA) and Nonylphenol (NP) induce germ cell apoptosis along with a decrease in sperm production. Given their ubiquitous distribution in plastic products used by humans it is important to clarify their mechanism of action. TACE/ADAM17 is a widely distributed extracellular metalloprotease and participates in the physiological apoptosis of germ cells during spermatogenesis. The aims of this work were: 1) to determine whether BPA and NP induce ADAM17 activation; and 2) to study whether ADAM17 and/or ADAM10 are involved in germ cell apoptosis induced by BPA and NP in the pubertal rat testis. A single dose of BPA or NP (50 mg/kg) induces germ cell apoptosis in 21-day-old male rats, which was prevented by a pharmacological inhibitor of ADAM17, but not by an inhibitor of ADAM10. In vitro, we showed that BPA and NP, at similar concentrations to those found in human samples, induce the shedding of exogenous and endogenous (TNF-α) ADAM17 substrates in primary rat Sertoli cell cultures and TM4 cell line. In addition, pharmacological inhibitors of metalloproteases and genetic silencing of ADAM17 prevent the shedding induced in vitro by BPA and NP. Finally, we showed that in vivo BPA and NP induced early activation (phosphorylation) of p38 MAPK and translocation of ADAM17 to the cell surface. Interestingly, the inhibition of p38 MAPK prevents germ cell apoptosis and translocation of ADAM17 to the cell surface. These results show for the first time that xenoestrogens can induce activation of ADAM17 at concentrations similar to those found in human samples, suggesting a mechanism by which they could imbalance para/juxtacrine cell-to-cell-communication and induce germ cell apoptosis.     

丙烯酰胺对雄性小鼠生殖细胞遗传损伤的研究

以雄性小鼠的生殖细胞为研究对象,经腹腔注射丙烯酰胺建立动物模型,采用生殖细胞早期精细胞微核和初级精母细胞染色体制作技术联合评价丙烯酰胺对雄性小鼠的生殖毒性及遗传毒性,观察染毒后精子发育的不同阶段对丙烯酰胺的损伤反应,为丙烯酰胺的生殖损伤提供一定依据。     

Nogo-B与Clusterin结合对细胞凋亡的影响

神经轴突生长抑制因子Nogo—B在体分布广泛,提示其除了具有抑制中枢神经系统轴突再生作用外,可能还扮演其他重要的功能角色。该研究为探讨Nogo-B下游新的结合分子及其功能开展相应研究。通过设计诱饵蛋白筛选人脑cDNA文库、免疫共沉淀方法,寻找Nogo-B下游结合分子：通过流式细胞术,检测结合对于细胞凋亡的影响：通过绿色荧光蛋白标记和免疫组织化学方法,探讨Nogo-B诱导细胞凋亡的机制。结果提示,Clusterin除了与Nogo-66功能域在酵母双杂交系统中存在结合,与Nogo—B在哺乳细胞中也能发生结合。过表达Nogo-B可明显诱导HEK293细胞凋亡,与Clusterin共表达可下调早期细胞凋亡率,但后期Nogo—B可通过调节Clusterin由胞浆到胞核转位,进一步诱导细胞凋亡进程。该研究首次提出Nogo—B与Clusterin之间存在结合,且结合参与了Nogo-B诱导的细胞凋亡进程。     

Mouse Tafazzin Is Required for Male Germ Cell Meiosis and Spermatogenesis

Barth syndrome is an X-linked mitochondrial disease, symptoms of which include neutropenia and cardiac myopathy. These symptoms are the most significant clinical consequences of a disease, which is increasingly recognised to have a variable presentation. Mutation in the Taz gene in Xq28 is thought to be responsible for the condition, by altering mitochondrial lipid content and mitochondrial function. Male chimeras carrying a targeted mutation of Taz on their X-chromosome were infertile. Testes from the Taz knockout chimeras were smaller than their control counterparts and this was associated with a disruption of the progression of spermatocytes through meiosis to spermiogenesis. Taz knockout ES cells also showed a defect when differentiated to germ cells in vitro. Mutant spermatocytes failed to progress past the pachytene stage of meiosis and had higher levels of DNA double strand damage and increased levels of endogenous retrotransposon activity. Altogether these data revealed a novel role for Taz in helping to maintain genome integrity in meiosis and facilitating germ cell differentiation. We have unravelled a novel function for the Taz protein, which should contribute to an understanding of how a disruption of the Taz gene results in the complex symptoms underlying Barth Syndrome.     

Knockdown of miR-214 Promotes Apoptosis and Inhibits Cell Proliferation in Nasopharyngeal Carcinoma

MicroRNA-214 (MiR-214) is aberrantly expressed in several human tumors such as ovarian cancer and breast cancer. However, the role of miR-214 in nasopharyngeal carcinoma (NPC) is still unknown. In this study, we report that miR-214 was overexpressed in NPC cell lines and tissues. Silencing of miR-214 by LNA-antimiR-214 in NPC cells resulted in promoting apoptosis and suppressing cell proliferation in vitro, and suppressed tumor growth in nude mice in vivo. Luciferase reporter assay was performed to identify Bim as a direct target of miR-214. Furthermore, this study showed that low Bim expression in NPC tissues correlated with poor survival of NPC patients. Taken together, our findings suggest that miR-214 plays an important role in NPC carcinogenesis.     

Chromosome Location and Association of Haplotypes of Insulin-like Growth Factor Binding Protein-2 with Production Performance in Swine

Insulin-like growth factor binding protein-2 is a member of the insulin-like growth factor families. Using a porcine RH panel, the gene was mapped on chromosome 15q22-23. Meanwhile, using polymerase chain reaction single strand conformation polymorphism, genotypic and allelic frequencies were analyzed in 17 pig breeds (total animals 570), together with a chi-square test of Hardy-Weinberg equilibrium. Also the association between haplotypes and production performance was analyzed in a Lantang x Landrace population family (n = 133, total 43 traits). At each locus we investigated, all the breeds showed different genotypic and allelic frequency distributions. In general, the Chinese native pig breeds carried a higher allele A frequency (over 50%) than the European pigs. For production performance, pigs with the CAG haplotype had higher fore-body and rear-body weight than those with the TGT and TAG haplotypes (P < 0.05). Also, pigs with the CAG haplotype had higher bone weight of the rear-body than those with the CAT haplotype (P < 0.05); pigs with the TGT and CAG haplotypes had higher forelimb and rearlimb weight than those with the CAT haplotype (P < 0.01 and P < 0.05, respectively); pigs with the TGG haplotype had higher leaf fat weight than those with the TGT and CAG haplotypes (P < 0.05); and pigs with the CAG haplotype had more stomach weight than those with the CAT and CGT haplotypes (P < 0.01); pigs with the TGT and CAG haplotypes had more ribs and longer body than those with the CGT-TGG, and CAT-TAG haplotypes (P < 0.05). These results suggest that IGFBP-2 is associated with production performance, but our population family was small. More studies with large samples are needed before the IGFBP-2 locus will be useful for a selection program.