Project Details
Description
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.
Status | Finished |
---|---|
Effective start/end date | 7/1/87 → 6/30/93 |
Funding
- National Heart, Lung, and Blood Institute
ASJC Scopus Subject Areas
- Genetics
- Molecular Biology
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