ANCHORAGE INDEPENDENT GROWTH--ROLE OF TGF BETA

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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.