IGFBP-rP_1在原发性肝细胞肝癌中的表达及意义

目的探讨胰岛素样生长因子结合蛋白相关蛋白1(insulin-like growth factor binding proteins-related protein 1,IGFBP-rP1)在原发性肝细胞肝癌(human hepatocellar carcinomas,HCC)中的表达及意义。方法应用免疫组织化学法检测43例HCC、32例癌旁肝组织及9例正常肝组织IGFBP-rP1的表达。结果IGFBP-rP1在HCC的表达显著低于癌旁肝组织(P0.01)及正常肝组织(P0.01),但正常肝组织和癌旁肝组织IGFBP-rP1的表达无明显差异(P0.05)。IGFBP-rP1的表达强度与HCC的恶性生物学行为关系密切。结论IGFBP-rP1抑制HCC的进展,IGFBP-rP1表达下调在HCC的进展中起重要作用。     

Role of a new member of IGFBP superfamily, IGFBP-rP10, in proliferation and differentiation of osteoblastic cells

Bone regeneration is critically regulated by various molecules. To identify the new genes involved in bone regeneration, we performed microarray-based gene expression analysis using a mouse bone regeneration model. We identified a new member of the IGFBP superfamily, designated IGFBP-rP10, whose expression is up-regulated at the early phase of bone regeneration. IGFBP-rP10 consists of an IGFBP homologous domain followed by a Kazal-type protein inhibitor domain and an immunoglobulin G-like domain. A real-time-based RT-PCR analysis demonstrated that various tissues including bone expressed IGFBP-rP10 mRNA in various degrees, and confirmed an up-regulation at the early phase of bone regeneration. In situ hybridization revealed that osteoblastic cells expressed IGFPB-rP10 mRNA during bone regeneration. Bone morphogenetic protein-2 increased the expression level of IGFBP-rP10 mRNA in various cells including C3H10T1/2, MC3T3-E1, C2C12, and primary murine osteoblastic cells. The addition of recombinant mouse IGFBP-rP10 promoted the proliferation of these cells but failed to stimulate alkaline phosphatase activity. These results suggest that IGFBP-rP10 is involved in the proliferation of osteoblasts during bone formation and bone regeneration.     

TGF-beta1 通过PI3K及ERK信号通路调节肺动脉高压过程中IGFBPs蛋白表达

目的：探讨大鼠肺动脉高压(PAH)过程中TGF-beta1对胰岛素样生长因子结合蛋白（IGFBP）表达调节是否依赖于PI3K及ERK 信号通路。方法：取健康成年SD 大鼠26 只,随机分成2 组：PAH组,腹腔注射1%的野百合碱,剂量为60 mg/kg;对照(C)组腹腔 注射生理盐水。于4 周后超声检测肺动脉平均压力,取肺组织做HE 染色,应用NIS-Element 系统测量中膜厚度。原代培养肺动脉 平滑肌（PASMC）细胞,分别加入TGF-beta1 及TGF-beta1 中和抗体后,Western-blot 检测IGFBP3,IGFBP5,Smad2/Smad3 表达。加入 ERK特异性抑制剂PD98059 或PI3K 抑制剂LY294002,检测IGFBP3,IGFBP5 表达。结果：野百合碱处理4 周后,肺动脉高压组 的平均肺动脉压力及右室/(左室+室间隔)比值显著高于对照组。TGF-茁1 可显著升高IGFBP3,IGFBP5 及p-Smad3 的表达（P<0. 05）,而抑制TGF-beta1 则可显著降低三种蛋白的表达（P<0.05）。加入LY294002 抑制PI3K ERK 后,IGFBP3 和p-Smad2 两种蛋白的 表达量显著下调（P<0.05）。加入PD98059 抑制ERK 后可显著降低IGFBP3 及IGFBP5 的表达水平（P<0.05）。结论：PAH 中 TGF-茁1 升高可通过活化Smad2/Smad3 上调IGFBP3和IGFBP5 的表达。TGF-beta1 促进IGFBP3,IGFBP5表达的作用依赖于PI3K 及ERK 信号通路。     

Insulin-like growth factors.

The insulin-like growth factors I and II are single chain polypeptides homologous to proinsulin. IGF I and IGF II contribute to cell regulation and stimulate protein synthesis via signaling through type I receptors which are homologous to insulin receptors and activate phosphorylation cascades. IGFs enhance the proliferation of chondocytes and the proliferation of their collagen and proteoglycan matrix; IGFs stimulate longitudinal (endochondral) bone growth. Throughout life, IGFs are constitutvely expressed ubiquitous factors which help to maintain the survival of differentiated cells, Increased expression is found during growth and tissue repair, Six specific binding proteins, IGFBP 1-6, allow additional tissue compartment specific control of IGF activity; IGFBP production favours storage and IGFBP cleavage leads to activation.     

Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5′–3′- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.     

IGFBP3, a Transcriptional Target of Homeobox D10, Is Correlated with the Prognosis of Gastric Cancer

Homeobox D10 (HoxD10) plays important roles in the differentiation of embryonic cells and progression of breast cancer. Our previous report revealed that insulin-like growth factor binding protein-3 (IGFBP3) was regulated by HoxD10 in gastric cancer cells; however, the functional roles and underlying mechanisms of IGFBP3 in gastric cancer remain unclear. Here, we found that the expression of IGFBP3 were upregulated after ectopic expression of HoxD10 in gastric cancer cells. Chromatin immunoprecipitation assay showed that HoxD10 bound to three potential regions of IGFBP3 promoter. Exogenous HoxD10 significantly enhanced the activity of luciferase reporter containing these binding regions in gastric cancer cells. Further data showed that all of these binding sites had Hox binding element “TTAT”. Immunohistochemical staining results revealed that IGFBP3 expression was significantly downregulated in 86 gastric adenocarcinomas tissues relative to their adjacent non-cancerous tissues (p<0.001). Moreover, IGFBP3 expression was significantly lower in gastric tumor with lymph node metastasis compared with that without lymph node metastasis (p=0.045). Patients with high expression level of IGFBP3 showed favorable 5 year overall survival (p=0.011). Knockdown of IGFBP3 accelerated gastric cancer cell migration and invasion and induced the expression of invasive factors including MMP14, uPA and uPAR. Thus, our data suggest that HoxD10-targeted gene IGFBP3 may suppress gastric cancer cell invasion and favors the survival of gastric cancer patients.     

IGFBP6 regulates vascular smooth muscle cell proliferation and morphology via cyclin E–CDK2

Despite the high prevalence of varicose veins, the underlying pathogenesis of this disease remains unclear. The present study aims to explore the role of insulin-like growth factor binding protein 6 (IGFBP6) in vascular smooth muscle cells (VSMCs). Using a protein array approach, we identified several differentially expressed proteins between varicose great saphenous veins and normal great saphenous veins. Bioinformatic analysis showed that IGFBP6 was closely related to cell proliferation. Further validation confirmed that IGFBP6 was one of the most highly expressed proteins in varicose vein tissue. Knocking down IGFBP6 in VSMCs significantly attenuated cell proliferation and induced the S phase arrest during the cell cycle. Further experiments demonstrated that IGFBP6 knockdown increased cyclin E ubiquitination, which reduced expression of cyclin E and phosphorylation of CDK2. Furthermore, IGFBP6 knockdown arrested centrosome replication, which subsequently influenced VSMC morphology. Ultimately, IGFBP6 was validated to be involved in VSMC proliferation in varicose vein tissues. The present study reveals that IGFBP6 is closely correlated with VSMC biological function and provides unprecedented insights into the underlying pathogenesis of varicose veins.     

P21 Activated Kinase-1 (Pak1) Promotes Prostate Tumor Growth and Microinvasion via Inhibition of Transforming Growth Factor β Expression and Enhanced Matrix Metalloproteinase 9 Secretion

P21-activated kinases (Paks) are major effectors downstream of the small Rho family of GTPases. Among the six isoforms, Pak1 is the most ubiquitous and the best characterized member. Previous studies have shown that inhibition of Pak6, which is predominantly present in the prostate compared with other tissues, inhibits prostate tumor growth in vivo. Even though Pak1 has been identified in normal prostatic epithelial cells and cancer cells, its specific role in the development of prostate cancer remains unclear. We report here that highly invasive prostate cancer cells express significantly higher levels of Pak1 protein compared with non-invasive prostate cancer cells. Furthermore, prostate tumor tissues and prostate cancer metastasized to lungs showed a higher expression of Pak1 compared with normal tissues. Interestingly, Pak6 protein expression levels did not change with the invasive/metastatic potential of the cancer cells or tumors. Although inhibition of Pak1, and not Pak6, resulted in impaired PC3 cell migration, the effects of Pak1 knockdown on transendothelial migration (microinvasion), tumor growth, and tumor angiogenesis was higher compared with Pak6 knockdown. Finally, gene array data revealed reduced expression of matrix metalloproteinase 9 with the ablation of either Pak1 or Pak6 gene expression in PC3 cells, whereas protein levels of TGFβ was elevated significantly with specific modulation of Pak1 activity or ablation of the Pak1 gene. Our observations suggest that although some level of functional redundancy exists between Pak1 and Pak6 in prostate cancer cells, targeting Pak1 is a potential option for the management of prostate tumor growth, microinvasion, and metastasis.     

Regulation of replicative senescence by insulin-like growth factor-binding protein 3 in human umbilical vein endothelial cells

Insulin/insulin-like growth factor (IGF) signaling pathways are among the most conserved processes in aging in organisms ranging from yeast to mammals. Previously, using cDNA microarray technology, we reported that expression of IGF-binding protein 3 (IGFBP3), one of the IGF-binding proteins, was increased with age in human dermal fibroblasts. In this study, the role of IGFBP3 on cellular senescence was studied in human umbilical vein endothelial cells (HUVEC). The expression levels of IGFBP3 mRNA and protein were increased in HUVECs with age. Knockdown of IGFBP3 in old cells with IGFBP3 short hairpin RNA (shRNA) retrovirus resulted in the partial reduction of a variety of senescent phenotypes, such as changes in cell morphology, and decreases in population doubling times and senescence-associated beta-galactosidase (SA-beta-gal) staining. Down-regulation of IGFBP3 rescued the growth arrest induced by p53 overexpression in young HUVECs. In contrast, up-regulation of IGFBP3 in young cells and prolonged IGFBP3 treatment accelerated cellular senescence, confirmed by cell proliferation and SA-beta-gal staining. The FOXO3a (forkhead box O3a) protein level was increased in old IGFBP3 shRNA cells. The treatment of young HUVECs with IGFBP3 repressed the levels of FOXO3a protein. Furthermore, calorie restriction reduced IGFBP3 protein levels, which were found to be increased with age in the rat liver and serum. These results suggest that IGFBP3 might play an important role in the cellular senescence of HUVECs as well as in vivo aging.     

A secreted tumor-suppressor, mac25, with activin-binding activity

BACKGROUND: mac25 is a follistatin (FS)-like protein that has a growth-suppressing effect on a p53-deficient osteosarcoma cell line (Saos-2). The protein exhibits a strong homology to FS, an activin-binding protein, and part of its sequence includes the consensus sequence of the member of the Kazal serine protease inhibitor family. MATERIALS AND METHODS: Localization of mac25 protein was analyzed using mac25 protein fused with green fluorescent protein (GFP). Recombinant mac25 protein was expressed in E. coli and purified. The recombinant mac25 protein was added in culture medium for analysis of growth suppression and cell cycle analysis. Binding of mac25 protein to activin A was studied by immunoprecipitation and Western blots analysis. RESULTS: mac25 protein was localized in the cytoplasm and secreted into culture medium. Addition of recombinant mac25 protein (10-7 M) into the culture medium induced significant suppression of the growth of human cervical carcinoma cells (HeLa) and murine embryonic carcinoma cells (P19), as well as osteosarcoma cells (Saos-2). mac25 protein was co-immunoprecipitated with activin A, a result that suggests that mac25 may be a secreted tumor-suppressor that binds activin A. CONCLUSION: mac25 exhibits homology to insulin-like growth factor-binding proteins (IGF-BPs) and to fibroblast growth factor receptor. The multi-functional nature of mac25 protein may be important for growth-suppression and/or cellular senescence.     

IGFBP7在甲状腺乳头状癌中的表达及意义

目的观察胰岛素样生长结合蛋白-7(IGFBP7)在甲状腺乳头状癌中的表达及意义。方法对87例甲状腺乳头癌(PTC)组织及癌旁组织(PeT)进行IGFBP7、Ki67、p53免疫组织化学分析,其中10例提取蛋白用半定量western blot方法进行IGFBP7表达水平分析。结果 (1)IGFBP7免疫组化显示,PTC癌组织IGFBP7阳性率达78.16%(68/87),明显高于癌旁组织的31.03%(27/87)(P0.05);(2)Western blot灰度值比较,癌组织IGFBP7表达水平显著高于癌旁组织(t=2.875,P0.05);(3)Ki67、p53与IGFBP7相关性分析显示,p53表达水平与IGFBP7表达水平呈显著正相关性(r=0.261,P0.05),而Ki67表达水平与IGFBP7表达水平无相关性(r=0.148,P=0.170);(4)高水平表达的IGFBP7与PTC淋巴结转移高度相关(r=0.238,P0.05)。结论 IGFBP7表达异常增高可能与甲状腺乳头状癌发生及发展有关,有望成为诊断PTC的一项新型的生物标记物。     

特异AT序列结合蛋白-1促进胰岛素样生长因子 结合蛋白-2在K562细胞中的表达

探讨过表达特异AT序列结合蛋白-1 ( special AT-rich sequence binding protein ,SATB1)核基质结合区（MAR）结合蛋白对胰岛素样生长因子结合蛋白-2（IGFBP2）基因表 达的影响,并对其影响机制进行初步探索．首先用脂质体将SATB1的真核表达载体pcDNA3.1-SATB1转染至K562细胞,通过6周G418的筛选获得阳性克隆,RT-PCR、实时PCR及Western 印迹验证过表达情况,对阳性克隆细胞中IGFBP2的表达用RT-PCR、实时PCR及Western 印迹方法进行检测;然后用RNAi的方法干扰阳性细胞中SATB1 的表达后,同样用上述3种方法再次检测IGFBP2的表达状况;用生物信息学方法对IGFBP2基因进行MAR序列与SATB1结合位点搜索分析,寻找SATB1影响IGFBP2基因表达的机制．结果显示,在稳定转染的情况下,实验组K562-SATB1细胞与转染空载体pcDNA3.1的K562-3.1细胞和未转染细胞K562相比,IGFBP2 mRNA水平上调了近7倍,而蛋白水平变化不明显．RNA干扰后,IGFBP2的表达在mRNA水平也相应下调,蛋白水平的变化同样不明显．通过生物信息学分析发现,IGFBP2第1个内含子中可能存在2. 5 kb MAR样序列,且MAR样序列上存在多个SATB1的潜在结合位点．综上所述,过表达SATB1可以使K562细胞中IGFBP2 mRNA表达水平提高,而且其调控机制可能与SATB1直接和IGFBP2基因中的MAR样序列结合有关．     

IGFBP5 suppresses oleate-induced intramyocellular lipids deposition and enhances insulin signaling

Excess intramyocellular lipids are often accompanied by muscle insulin resistance (IR) and type 2 diabetes. The mechanism of the formation of intramyocellular lipids is unclear yet. In this study, we optimized the cellular model of intramyocellular lipids from differentiated C2C12 cells and identified that the expression of insulin-like growth factor-binding protein 5 (IGFBP5) is diminished in this process. Then, we added exogenous recombinant IGFBP5 during myocyte triglyceride (TAG) formation and found decreased lipids accumulation. In addition, IGFBP5 could promote lipolysis when added to the cellular model after the formation of intramyocellular lipids. Moreover, IGFBP5 could enhance myocyte insulin sensitivity by inhibiting the expression of the thioredoxin-interacting protein (TXNIP) and arrestin domain-containing 4 (ARRDC4), which are a negative regulator of insulin signaling in both cases. Meanwhile, IGFBP5 also inhibited the expression of glycerol-3-phosphate acyltransferase (GPAM) and diglyceride acyltransferase 2 (DGAT2), which were involved in TAG synthesis from a fatty acid. IGFBP5 also reduced TAG storage by promoting lipolysis. Therefore, IGFBP5 may play a role in the excess accumulation of lipid in muscle cells of diabetic patients and serve as a reference for further research and treatment of muscle IR and diabetes.     

前列腺组织中神经生长因子(NGF)表达的研究

研究前列腺组织中神经生长因子（ＮＧＦ） 的生理学意义。采用原位杂交和免疫组化法, 检测４３ 例前列腺增生组织, ８ 例腺癌组织和８ 例正常组织中β－ＮＧＦｍ ＲＮＡ及其蛋白的表达及分布。结果显示β－ＮＧＦｍ ＲＮＡ 在正常组织及增生组织中定位于间质细胞, 偶见于上皮细胞中; 而在癌组织中, 上皮细胞和间质细胞有同样强度的β－ＮＧＦｍ ＲＮＡ染色。其蛋白在良性组织中表达主要着色在间质细胞中,上皮细胞呈弱表达,而癌组织中上皮细胞见着色明显增强（Ｐ＜ ０．０５）。ＮＧＦ的自分泌异常可见是前列腺组织由良性向恶性转变的原因之一。     

Identification of a novel insulin-like growth factor binding protein gene homologue with tumor suppressor like properties

Here we report the identification of a new insulin-like growth factor binding protein homologue, provisionally designated insulin-like growth factor binding related protein-4 (IGFBP-rP4). IGFBP-rP4 was found to be most closely related to IGFBP-7 with 52% amino acid homology and 43% amino acid identity, and shares a similar domain structure. Semi-quantitative RT-PCR expression analysis demonstrated a pattern of downregulation of this gene in multiple tumor samples including lung and colon cancer, compared to matched adjacent normal tissue. Western blotting revealed a protein of approximately 38kDa expressed in both the cell pellet and secreted into the supernatant of transiently transfected Cos-7 cells. Cos-7 supernatants containing IGFBP-RP4 protein were observed to suppress the growth of HeLa cells in culture compared to vector controls. IGFBP-RP4 directly transiently transfected into HeLa cells also further confirmed the growth suppressive properties of this protein. Together these data suggest that IGFBP-RP4 may be a novel putative tumor suppressor protein.