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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
Project #
Application #
Study Section
Physiological Chemistry Study Section (PC)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Columbia University (N.Y.)
Schools of Medicine
New York
United States
Zip Code
Wager, R E; Scotto, L; Assoian, R K (1994) Analysis of transforming growth factor beta 1 messenger RNA degradation by the transcript-selective, 12-O-tetradecanoylphorbol-13-acetate-regulated ribonuclease system from U937 promonocytes. Cell Growth Differ 5:117-24
Asiedu, C K; Scotto, L; Assoian, R K et al. (1994) Binding of AP-1/CREB proteins and of MDBP to contiguous sites downstream of the human TGF-beta 1 gene. Biochim Biophys Acta 1219:55-63
Scotto, L; Assoian, R K (1993) A GC-rich domain with bifunctional effects on mRNA and protein levels: implications for control of transforming growth factor beta 1 expression. Mol Cell Biol 13:3588-97
Bottalico, L A; Wager, R E; Agellon, L B et al. (1991) Transforming growth factor-beta 1 inhibits scavenger receptor activity in THP-1 human macrophages. J Biol Chem 266:22866-71
Watrin, F; Scotto, L; Assoian, R K et al. (1991) Cell lineage specificity of expression of the murine transforming growth factor beta 3 and transforming growth factor beta 1 genes. Cell Growth Differ 2:77-83
Guadagno, T M; Assoian, R K (1991) G1/S control of anchorage-independent growth in the fibroblast cell cycle. J Cell Biol 115:1419-25
Wager, R E; Assoian, R K (1990) A phorbol ester-regulated ribonuclease system controlling transforming growth factor beta 1 gene expression in hematopoietic cells. Mol Cell Biol 10:5983-90
Scotto, L; Vaduva, P I; Wager, R E et al. (1990) Type beta 1 transforming growth factor gene expression. A corrected mRNA structure reveals a downstream phorbol ester responsive element in human cells. J Biol Chem 265:2203-8
Assoian, R K; Boardman, L A; Drosinos, S (1989) A preparative suspension culture system permitting quantitation of anchorage-independent growth by direct radiolabeling of cellular DNA. Anal Biochem 177:95-9