TRANSFORMING GROWTH-FACTOR-BETA (TGF-BETA)-INDUCED DOWN-REGULATION OFCYCLIN-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 com ponents, cyclin A, is inhibited by TGF-beta treatment, We have identif ied a 760-base pair fragment of the human cyclin A gene promoter that is sufficient to confer TGF-beta responsiveness, Using this promoter f ragment, 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 o f the type I (RI) and type II (RII) receptors were required for the an tiproliferative 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 o n the plasminogen activator inhibitor type 1 (PAI-1) promoter, We foun d that TGF-beta-induced inhibition of cyclin A expression was absent i n 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, ex pression of two reciprocal chimeras (RI-RII and RII-RI) resulted in gr owth inhibition, similarly to wildtype receptors, In addition, chimeri c receptors as well as mutant receptors with a deleted cytoplasmic dom ain and kinase-negative receptors inhibited TGF-beta responsiveness. i n 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 w as dispensable, Our results suggest that a functional TGF-beta recepto r 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.