ANCHORAGE INDEPENDENT GROWTH--ROLE OF TGF BETA

β型转化生长因子（TGF-β）是一种广泛的 在正常组织中分布的蛋白质， 富含人类血小板 这些结果与数据 显示出生长因子对细胞增殖的有效作用， 成纤维细胞和主动脉平滑肌细胞，表明TGF-β 可能是伤口修复和 动脉粥样硬化 与这一假设一致，初步研究 已经表明TGF-β在LPS诱导的细胞凋亡过程中释放， 在体外单核细胞向巨噬细胞的分化。 选择性 转录后机制负责控制TGF-β 1的表达。 在这个系统中的beta表达。 这里提出的研究是 1）确定该控制机制是否运行 在翻译、翻译后处理的层面，或 亚细胞运输和2）定义负责机制， 生物化学和分子水平。 为了实现这一目标， 将产生针对合成肽的多克隆抗体 其对应于TGF-β的胰蛋白酶片段。 这些 抗体将与生物合成标记的蛋白质一起孵育 从新鲜分离的人的提取物和条件培养基 单核细胞和人单核细胞样细胞系，以确定 TGF-β mRNA被翻译;单核细胞的胰蛋白酶消化 蛋白质可用于从TGF-β样细胞释放抗原， 蛋白质，并确保随后的三级结构独立 免疫反应性。 标记单核细胞的分离 通过凝集素亲和色谱和HPLC分析蛋白质， 免疫沉淀将确定翻译产物是否 加工成真正的TGF-β 亚细胞分级 生物合成标记的单核细胞，然后是 标记的TGF-β胰蛋白酶片段的免疫沉淀 这些组分的提取物将确定TGF-β表达 受到亚细胞运输控制的限制。 北方分析 从单核细胞核、细胞质和 多聚核糖体将用于检查大小和亚细胞 TGF-β信息的位置。 这些RNA片段还可以 在无细胞系统中翻译; 产生的蛋白质产物将区分翻译的 阻断TGF-β mRNA本身的内在作用， 在完整的细胞。 除了提供关于 调节TGF-β在单核细胞和巨噬细胞中的表达， 这个系统提供了一个机会，检查一个完整的系列 亚细胞事件，在细胞内具有潜在的调节作用， 细胞分化过程中分泌蛋白的表达。

Type beta transforming growth factor (TGF-beta) is a widely distributed protein in normal tissue and one which is particularly abundant in human platelets. These results, together with data showing potent effects of the growth factor on proliferation of fibroblasts and aortic smooth muscle cells, suggest that TGF-beta may be an important mediator in both wound repair and athergenesis. Consistent with this hypothesis, preliminary studies have shown that TGF-beta is released during LPS-induced differentiation of monocytes to macrophages in vitro. A selective post-transcriptional mechanism is responsible for control of TGF- beta expression in this system. The studies proposed here are designed to 1) determine whether this control mechanism operates at the level of translation, post-translational processing, or subcellular transport and 2) define the responsible mechanism at a biochemical and molecular level. To accomplish this goal, polyclonal antibodies will be raised against synthetic peptides which correspond to tryptic fragments of TGF-beta. These antibodies will be incubated with biosynthetically labeled protein from extracts and conditioned medium of freshly isolated human monocytes and human monocyte-like cell lines to determine if TGF-beta mRNA is translated; trypsin digestion of monocyte proteins can be used to release antigen from TGF-beta-like proteins and assure subsequent tertiary structure-independent immunoreactivity. Preparative fractionation of labeled monocyte proteins by lectin-affinity chromatography and HPLC prior to immunoprecipitation will determine if a translation product is processed to authentic TGF-beta. Subcellular fractionation of biosynthetically labeled monocytes followed by immunoprecipitation of labeled TGF-beta tryptic fragments from extracts of these fractions will determine if TGF-beta expression is limited by controls on subcellular transport. Northern analysis of mRNA isolated from monocyte nuclei, cytoplasm and polysomes will be used to examine the size and subcellular location of the TGF-beta message. These RNA fractions can also be translated in cell-free systems; immunoprecipitation of the resulting protein products would distinguish between translational blocks intrinsic to the TGF-beta mRNA itself and those imposed in the intact cell. In addition to providing information on the regulated expression of TGF-beta in monocytes and macrophages, this system provides an opportunity to examine an entire series of subcellular events with potential regulatory roles in the expression of secretory proteins during cellular differentiation.