Transforming growth factor-beta (TGF-beta)-induced down-regulation of cyclin A expression requires a functional TGF-beta receptor complex. Characterization of chimeric and truncated type I and type II receptors.
Transforming growth factor-beta (TGF-beta) 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-beta treatment. We have identified a 760-base pair fragment of the human cyclin A gene promoter that is sufficient to confer TGF-beta responsiveness. Using this promoter fragment, we have developed a cyclin A-based luciferase reporter assay that quantitates the growth inhibitory effect of TGF-beta 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-beta. In parallel, the functionality of chimeric receptors, between RI and RII (RI-RII or RII-RI), was tested for TGF-beta effect on gene expression using a reporter assay based on the plasminogen activator inhibitor type 1 (PAI-1) promoter. We found that TGF-beta-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-beta. 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-beta 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-beta receptor complex is required for TGF-beta-dependent down-regulation of cyclin A gene expression and illustrate the identical receptor requirements for TGF-beta-induced growth inhibition and gene expression.
Feng
X H
XH
Department of Growth and Development, University of California, San Francisco 94143, USA.
Filvaroff
E H
EH
Derynck
R
R
D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002522:Chlorocebus aethiops; D016213:Cyclins / Q000096:biosynthesis* / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers; D005786:Gene Expression Regulation / Q000187:drug effects*; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis; D008907:Mink; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D018125:Receptors, Transforming Growth Factor beta / Q000096:biosynthesis / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D016213:Cyclins; D017931:DNA Primers; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D008156:Luciferases