NOVEL TGF-BETA INDUCIBLE GENE IN HUMAN OSTEOBLASTS

描述：（改编自研究者摘要）转化生长 β因子（TGF-β）被认为在人类骨细胞中起主要作用， physiology. 它高度集中在人体骨骼中，由两种 正常骨形成成骨细胞（hOB）和骨吸收破骨细胞（OC）， 并对这些细胞的活性有重大影响。 其生产和 hOB细胞中的活化受类固醇，甲状旁腺激素， TGF-β本身和其他药物。 TGF-β有可能在 在hOB和OC之间的偶联以及在衰老和骨质疏松症中的作用。 申请人实验室已经确定了一种新的TGF-β诱导的早期 差异显示聚合酶链反应检测TIEG基因。 当 将此差异表达的cDNA作为探针插入hOB cDNA文库中， 筛选后，分离到该基因的2.9kb cDNA。 北方分析 使用该cDNA显示TIEG mRNA为3.5kb长。 其水平是 在TGF-β或BMP-2治疗的30分钟内增加，并达到 在人细胞中，2小时时最多为对照值的10倍， 胎儿来源（hF 0 Bs）和成人来源（hOBs）。 十几个其他的增长 因子和细胞因子不调节TIEG表达。 该2.9kb cDNA 计算机序列分析表明，TIEG基因是一个 mRNA编码480个氨基酸的蛋白质。 TIEG的C末端 蛋白质显示出与含锌指的 转录因子家族的基因，而N-末端区域似乎 独一无二 最近，申请人已经：1）制备了多克隆 TIEG蛋白的抗体; 2）显示TIEG在小鼠中表达， hOB和MCF-7乳腺癌细胞;以及 各种其他组织。 后者显示hOB细胞（但不显示其他细胞）。 骨细胞）和乳腺上皮细胞（但不是基质细胞） 含有/表达TIEG。 3)表明其 调节是高度生长因子/细胞因子特异性的，（限于TGF-β 1）。 4）Western blotting鉴定TIEG蛋白; 5)确定TIEG蛋白具有src同源性-3结合结构域， 似乎是酪氨酸磷酸化的;和6）表达TIEG蛋白 在细菌中。 他们建议利用新开发的人类胚胎 成骨细胞系，用温度敏感的SV-40 T 抗原，以进一步确定：1）组织/细胞类型特异性的抗原， TIEG表达和TGF-β调节; 2）生长因子/细胞因子 其调节的特异性; 3）使用TIEG蛋白的调节 PAb和MAb（通过蛋白质印迹法）、细胞内定位、半衰期 分析等; 4)蛋白质是否被磷酸化， Src-酪氨酸激酶通路参与; 5）TIEG蛋白的功能 通过其对TGF-β调节基因的启动子活性的作用，如 及其对锌指和SP-1共有序列的作用，和 TIEG反义寡核苷酸和过表达在人乳腺癌中的作用 稳定的细胞转染子对hFOB细胞增殖和骨形成的影响 最后6）基因中的TIEG蛋白DNA结合元件 启动子，并确定TIEG蛋白DNA结合元件在 基因启动子，并确定TIEG蛋白磷酸化对 这个绑定。 因为TIEG是一种早期诱导的假定转录， 因子基因，其蛋白质产物可能作为一个重要的作用， 成骨细胞中的信号分子。

DESCRIPTION: (Adapted from investigator's abstract) Transforming Growth Factor-beta (TGF-beta) is thought to play a major role in human bone cell physiology. It is highly concentrated in human bone, produced by both normal bone forming osteoblasts (hOB) and bone resorbing osteoclasts (OC), and has major effects on activities of these cells. Its production and activation in hOB cells are regulated by steroids, parathyroid hormone, TGF-beta itself, and other agents. It is possible that TGF-beta plays a role in the coupling between hOB and OC, and in aging and osteoporosis. The applicant laboratories have identified a new TGF-beta inducible early gene (TIEG) using differential display polymerase chain reaction. When this differentially expressed cDNA was used as a probe in hOB cDNA library screening, a 2.9 kb cDNA of this gene was isolated. Northern analyses using this cDNA has shown that TIEG mRNA is 3.5 kb long. Its levels are increased within 30 minutes of TGF-beta or BMP-2 treatment, and reaches a maximum of 10-fold above control values at 2 hours in human cells from both fetal sources (hF0Bs) and adults (hOBs). A dozen other growth factors and cytokines do not regulate TIEG expression. This 2.9 kb cDNA has been sequenced and the computer sequence analyses indicates that TIEG mRNA encodes for a 480 amino acid protein. The C-terminal end of TIEG protein shows significant homology to a zinc finger-containing transcription factor family of genes, whereas the N-terminal region seems to be unique. Recently, the applicants have: 1) prepared polyclonal antibodies to the TIEG protein; 2) shown that the TIEG is expressed in both hOB and MCF-7 breast cancer cells; as well as in select cell types in a variety of other tissues. The latter shows hOB cells (but not other bone cells) and epithelial cells of the breast (but not stromal cells) contain/express TIEG using immunohistochemistry. 3) shown that its regulation is highly growth factor/cytokine specific, (restricted to TGF- beta family and EGF); 4) identified the TIEG protein by Western blotting; 5) determined that the TIEG protein has src homology-3 binding domains and appears to be tyrosine phosphorylated; and 6) expressed the TIEG protein in bacteria. They propose to utilize newly developed human fetal osteoblast cell lines, immortalized with a temperature-sensitive SV-40 T antigen, to further determine: 1) the tissue/cell type specificity of the TIEG expression and the TGF-beta regulation; 2) the growth factor/cytokine specificity of its regulation; 3) the regulation of the TIEG protein using PAb and MAb via Western blotting, intracellular localization, half-life analyses, etc.; 4) whether the protein is phosphorylated and whether the src-tyrosine kinase pathway is involved; 5) the function of TIEG protein via its action on the promoter activities of TGF-beta regulated genes, as well as its action on the zinc finger and SP-1 consensus sequences, and the effects of TIEG antisense oligonucleotides and overexpression in stable cell transfectants on hFOB cell proliferation and bone forming functions; and finally 6) the TIEG protein DNA binding element in gene promoters and determine the effect of TIEG protein DNA binding element in gene promoters and determine the effect of TIEG protein phosphorylation on this binding. Because TIEG is an early induced putative transcription factor gene, its protein products might play an important role as a signaling molecule in osteoblastic cells.