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(Received for publication, March 30, 1995; and in revised form, July
13, 1995) Transforming growth factor-
Volume 270,
Number 41,
Issue of October 13, 1995 pp. 24237-24245
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
(TGF-
)-induced Down-regulation of Cyclin A
Expression Requires a Functional TGF-
Receptor Complex
CHARACTERIZATION OF CHIMERIC AND TRUNCATED TYPE I AND TYPE II
RECEPTORS
(TGF-) inhibits the
proliferation of epithelial cells by altering the expression or
function of various components of the cell cycle machinery. Expression
of one of these components, cyclin A, is inhibited by TGF-
treatment. We have identified a 760-base pair fragment of the human
cyclin A gene promoter that is sufficient to confer TGF-
responsiveness. Using this promoter fragment, we have developed a
cyclin A-based luciferase reporter assay that quantitates the growth
inhibitory effect of TGF- in transient transfection assays. This
assay was used to determine which domains of the type I (RI) and type
II (RII) receptors were required for the antiproliferative effect of
TGF-. In parallel, the functionality of chimeric receptors,
between RI and RII (RI-RII or RII-RI), was tested for TGF- effect
on gene expression using a reporter assay based on the plasminogen
activator inhibitor type 1 (PAI-1) promoter. We found that
TGF--induced inhibition of cyclin A expression was absent in RI or
RII-deficient Mv1Lu cells and that this response was restored by
expression of wild-type type I or type II receptors in these cells.
Furthermore, expression of a single chimeric receptor, either RI-RII or
RII-RI, did not confer cyclin A regulation by TGF-. However,
expression of two reciprocal chimeras (RI-RII and RII-RI) resulted in
growth inhibition, similarly to wild-type receptors. In addition,
chimeric receptors as well as mutant receptors with a deleted
cytoplasmic domain and kinase-negative receptors inhibited TGF-
responsiveness in the cyclin A reporter assay in a dominant negative
fashion. Finally, in both receptor types, the juxtamembrane domain
preceding the kinase domain was essential for receptor function but the
cytoplasmic tail was dispensable. Our results suggest that a functional
TGF- receptor complex is required for TGF--dependent
down-regulation of cyclin A gene expression and illustrate the
identical receptor requirements for TGF--induced growth inhibition
and gene expression.
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