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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048224-08
Application #
2185714
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1993-07-01
Project End
1996-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
8
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Miami School of Medicine
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33146
Davis, Joseph H; Baker, Tania A; Sauer, Robert T (2009) Engineering synthetic adaptors and substrates for controlled ClpXP degradation. J Biol Chem 284:21848-55
McGinness, Kathleen E; Bolon, Daniel N; Kaganovich, Mark et al. (2007) Altered tethering of the SspB adaptor to the ClpXP protease causes changes in substrate delivery. J Biol Chem 282:11465-73
Bottazzi, M E; Buzzai, M; Zhu, X et al. (2001) Distinct effects of mitogens and the actin cytoskeleton on CREB and pocket protein phosphorylation control the extent and timing of cyclin A promoter activity. Mol Cell Biol 21:7607-16
Zhu, X; Scharf, E; Assoian, R K (2000) Induction of anchorage-independent growth by transforming growth factor-beta linked to anchorage-independent expression of cyclin D1. J Biol Chem 275:6703-6
Davey, G; Buzzai, M; Assoian, R K (1999) Reduced expression of (alpha)5(beta)1 integrin prevents spreading-dependent cell proliferation. J Cell Sci 112 ( Pt 24):4663-72
Dalton, S L; Scharf, E; Davey, G et al. (1999) Transforming growth factor-beta overrides the adhesion requirement for surface expression of alpha(5)beta(1) integrin in normal rat kidney fibroblasts. A necessary effect for induction of anchorage-independent growth. J Biol Chem 274:30139-45
Bohmer, R M; Scharf, E; Assoian, R K (1996) Cytoskeletal integrity is required throughout the mitogen stimulation phase of the cell cycle and mediates the anchorage-dependent expression of cyclin D1. Mol Biol Cell 7:101-111
Zhu, X; Assoian, R K (1995) Integrin-dependent activation of MAP kinase: a link to shape-dependent cell proliferation. Mol Biol Cell 6:273-82
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
Han, E K; Guadagno, T M; Dalton, S L et al. (1993) A cell cycle and mutational analysis of anchorage-independent growth: cell adhesion and TGF-beta 1 control G1/S transit specifically. J Cell Biol 122:461-71

Showing the most recent 10 out of 11 publications