MicroRNA-21 Promotes Glioblastoma Tumorigenesis by Down-regulating Insulin-like Growth Factor-binding Protein-3 (IGFBP3)

Despite advances in surgery, imaging, chemotherapy, and radiation, patients with glioblastoma multiforme (GBM), the most common histological subtype of glioma, have an especially dismal prognosis; >70% of GBM patients die within 2 years of diagnosis. In many human cancers, the microRNA miR-21 is overexpressed, and accumulating evidence indicates that it functions as an oncogene. Here, we report that miR-21 is overexpressed in human GBM cell lines and tumor tissue. Moreover, miR-21 expression in GBM patient samples is inversely correlated with patient survival. Knockdown of miR-21 in GBM cells inhibited cell proliferation in vitro and markedly inhibited tumor formation in vivo. A number of known miR-21 targets have been identified previously. By microarray analysis, we identified and validated insulin-like growth factor (IGF)-binding protein-3 (IGFBP3) as a novel miR-21 target gene. Overexpression of IGFBP3 in glioma cells inhibited cell proliferation in vitro and inhibited tumor formation of glioma xenografts in vivo. The critical role that IGFBP3 plays in miR-21-mediated actions was demonstrated by a rescue experiment, in which IGFBP3 knockdown in miR-21KD glioblastoma cells restored tumorigenesis. Examination of tumors from GBM patients showed that there was an inverse relationship between IGFBP3 and miR-21 expression and that increased IGFBP3 expression correlated with better patient survival. Our results identify IGFBP3 as a novel miR-21 target gene in glioblastoma and suggest that the oncogenic miRNA miR-21 down-regulates the expression of IGFBP3, which acts as a tumor suppressor in human glioblastoma.     

Prohibitin-2 Binding Modulates Insulin-like Growth Factor-binding Protein-6 (IGFBP-6)-induced Rhabdomyosarcoma Cell Migration

Insulin-like growth factor (IGF)-binding protein (IGFBP)-6 decreases cancer cell proliferation and survival by inhibiting the effects of IGF-II. More recently, IGFBP-6 was found to promote the migration of rhabdomyosarcoma (RMS) cells in an IGF-independent manner, and MAPK pathways were involved in this process. However, the precise molecular mechanisms of these IGF-independent migratory actions of IGFBP-6 are largely unknown. Here, we report that prohibitin-2 (PHB2), a single-span membrane protein, is a key regulator of IGFBP-6-induced RMS cell migration. PHB2 and IGFBP-6 co-localize on the RMS cell surface, and they specifically interact, as demonstrated by affinity chromatography, co-immunoprecipitation, biosensor analysis, and confocal microscopy. Binding affinities for PHB2 are 9.0 ± 1.0 nm for IGFBP-6 and 10.2 ± 0.5 nm for mIGFBP-6, a non-IGF-binding mutant of IGFBP-6. The C-domain but not the N-domain of IGFBP-6 is involved in PHB2 binding. In addition, IGFBP-6 indirectly increases PHB2 tyrosine phosphorylation on RMS membranes. Importantly, PHB2 knockdown completely abolished IGFBP-6-mediated RMS cell migration. In contrast, IGFBP-6-induced MAPK pathway activation was not affected, suggesting that PHB2 may act as a downstream effector of these pathways. These results indicate that PHB2 plays a key role in this IGF-independent action of IGFBP-6 and suggest a possible therapeutic target for RMS.     

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。     

Expression of Insulin-like Growth Factor Binding Protein-3 and Regulation of the Insulin-like Growth Factor-I Axis in Pig Testis

The stem cell niche is a complex unit comprising key components, such as the extracellular matrix and various paracrine factors, which regulate the differentiation of adult stem cells. In our previous study, we established pig spermatogonial stem cells (pSSCs) in culture and identified the expression of insulin-like growth factor binding protein-3 (IGFBP-3) in pSSCs. The present study investigated not only the expression of IGFBP-3, but also its possible role in pSSCs. In this study, IGFBP-3-expressing cells responded positively to protein gene product 9.5 (PGP9.5), which is a marker for pig spermatogonia. IGFBP-3 expression was significantly increased in 60-dayold pig testes. Additionally, the expression levels of insulinlike growth factor I (IGF-I) and its receptor (IGF-IR) were observed in pSSCs and pig Sertoli cells (pSCs). Furthermore, IGF-I treatment enhanced the proliferation of pSCs and pSSCs when they were co-cultured. Blocking the IGF-I pathway using a specific IGF-IR inhibitor dramatically reduced the proliferation of pSCs. In addition, when heparan sulfate was used to sequester IGFBP-3 from IGF-I binding, a significant increase in the proliferation of pSCs was observed. Exogenous IGF-I treatment also increased the expression level of IGFBP-3 in cultured pSSCs. Furthermore, pSSCs grew well in IGF-I-treated pSC conditioned media. In summary, IGF-I and IGF-IR signaling are important for the proliferation of pSCs, and the germ cell-derived IGFBP-3 had an inhibitory effect on the mitotic activity of IGF-I in pSCs.     

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…     

p53蛋白在周期调节蛋白A1变异引起的雄性小鼠生殖细胞凋亡中的作用

为研究p5 3蛋白在周期调节蛋白A1(cyclinA1)变异引起的雄性小鼠生殖细胞凋亡中的作用 ,以p5 3基因敲除的小鼠和周期调节蛋白A1基因敲除的小鼠杂交 ,获取同胎生单基因变异和双基因同时变异的雄性后代共 4组 12只 .比较它们的性腺和生殖细胞发育 ,并用TUNEL染色法观察和比较生殖细胞的凋亡情况 .在睾丸最大横切面上观察到 :周期调节蛋白A1变异组凋亡细胞最多 (348± 10 4个 ) ,明显高于p5 3 周期调节蛋白A1双基因变异组 (12 1± 38个 ) ,t=3 2 5 79,P =0 0 4 72 .p5 3变异组凋亡细胞最少 (45± 2 4个 ) ,配对t检验显示有非常显著性差异 ,t=8 4 0 13,P =0 0 0 35 .这一研究结果提示 ,p5 3基因可能在雄性生殖细胞的发育中起监视作用 ,并在周期调节蛋白A1变异引起发育异常时启动p5 3途径造成异常细胞的凋亡 .     

Dexpanthenol Promotes Cell Growth by Preventing Cell Senescence and Apoptosis in Cultured Human Hair Follicle Cells

Dexpanthenol (D-panthenol) is a precursor of vitamin B5 (pantothenic acid) and is widely used for dietary supplements and topical applications. D-panthenol has long been used in hair care products for the purpose of anti-hair loss, its effects and the underlying mechanisms, however, were barely reported. In this study, the effects of D-panthenol on human hair follicle cells, including dermal papilla cells (hDPCs) and outer root sheath cells (hORSCs), were investigated. D-panthenol enhanced the cell viability, increasing the cellular proliferation marker Ki67 in cultured hDPCs. The markers for apoptosis (Caspase3/9) and cell senescence (p21/p16), reported to be expressed in aged or resting phase follicles, were significantly reduced by D-panthenol. Anagen-inducing factors (ALP; β-catenin; versican), which trigger or elongate the anagen phase, were stimulated by D-panthenol. On the other hand, D-panthenol reduced TGF-β1 expressions in both mRNA and protein levels. The expression of VEGF, which is important for peripheral blood vessel activation; was up-regulated by D-panthenol treatment. In cultured hORSCs, cell proliferation and viability were enhanced, while the mRNA expression of cell senescence markers (p21/p16) was significantly down-regulated. The expressions of both VEGF and its receptor (VEGFR) were up-regulated by D-panthenol. In conclusion, our data suggest that the hair growth stimulating activity of D-panthenol was exerted by increasing the cell viability, suppressing the apoptotic markers, and elongating the anagen phase in hair follicles.     

Germ Plasm and Germ-line Cell Determination: The Role of Mitochondria

Literature data on the structure and origin of material of the germ cell line determinants and on the presence of products of nuclear and mitochondrial genomes in the structured germ determinants (nuage) are reviewed. The personal data, obtained on spermatogenic cells of sea urchin and of other marine invertebrates, evidence the transformation of the mitochondrial matrix into the nuage material. The presence of matrix structures of mitochondrial origin in the germ plasm, which ensure reproduction and the function of the structured macromolecular complex of germ determinants relatively independent from the nuclear genome, is suggested.     

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...     

Adaptor Protein-2 Interaction with Arrestin Regulates GPCR Recycling and Apoptosis

G protein-coupled receptors (GPCRs) are integral to cellular function in nearly all physiologic and many pathologic processes. GPCR signaling represents an intricate balance between receptor activation, inactivation (desensitization, internalization and degradation) and resensitization (recycling and de novo synthesis). Complex formation between phosphorylated GPCRs, arrestins and an ever-increasing number of effector molecules is known to regulate cellular function. Previous studies have demonstrated that, although N -formyl peptide receptor (FPR) internalization occurs in the absence of arrestins, FPR recycling is arrestin-dependent. Furthermore, FPR stimulation in the absence of arrestins leads to receptor accumulation in perinuclear endosomes and apoptosis. In this study, we show that the interaction of GPCR-bound arrestin with adaptor protein-2 (AP-2) is a critical anti-apoptotic event. In addition, AP-2 associates with the receptor-arrestin complex in perinuclear endosomes and is required for proper post-endocytic GPCR trafficking. Finally, we observed that depletion of endogenous AP-2 results in the initiation of apoptosis upon stimulation of multiple GPCRs, including P2Y purinergic receptors and CXCR2, but not CXCR4. We propose a model in which the abnormal accumulation of internalized GPCR-arrestin complexes in recycling endosomes, resulting from defective arrestin-AP-2 interactions, leads to the specific initiation of aberrant signaling pathways and apoptosis.     

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。     

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。     

胰岛素样生长因子结合蛋白-1启动子的克隆与序列分析

用酚氯仿抽提法提取SD大鼠的肝脏基因组DNA,PCR扩增胰岛素样生长因子结合蛋白-1（IGFBP-1）启动子并克隆至pUC118载体。从含IGFBP-1启动子的质粒中酶切分离出IGFBP-1启动子并测序。PCR扩增出420bp的目的DNA片段,与文献报道的IGFBP-1启动子DNA大小一致。经正、反两方向序列分析显示,克隆的基因序列和GenBank数据库中的IGFBP-1启动子基因序列一致。以上结果表明克隆成功了IGFBP-1启动子基因,为构建具双向调节的胰岛素分泌调控基因质粒奠定了基础。