Abstract
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.
Highlights
Transforming growth factor- (TGF-) inhibits the proliferation of epithelial cells by altering the expression or function of various components of the cell cycle machinery
TGF- is often primarily studied for its growth inhibitory effect, and considerable evidence suggests that the effects on growth inhibition and gene expression result from different signaling cascades
The TGF--induced inhibition of luciferase expression was most evident when TGF- treatment was initiated within 6 h after release from contact inhibition, and the cyclin A promoter response was maximal when the cells were exposed to TGF- for 48 h (Fig. 1A). These results indicate that the 760-bp cyclin A promoter fragment in pCAL2 contains the TGF--responsive element(s) and that this cyclin A-luciferase assay can be used as transcriptional reporter system to measure the antiproliferative effect of TGF- in transient transfection assays
Summary
Transforming growth factor- (TGF-) inhibits the proliferation of epithelial cells by altering the expression or function of various components of the cell cycle machinery. 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. Induction of PAI-1 expression can be measured using a reporter plasmid in which luciferase expression is driven from the PAI-1 promoter in a TGF--dependent manner [24, 25] This assay is often used in combination with transient transfection experiments to measure cellular responsiveness to TGF- and the corresponding receptor requirements. No convenient reporter assay has been developed to measure the growth inhibitory response of TGF-, even though considerable evidence suggests that TGF-’s effects on growth inhibition and gene expression result from divergent signaling cascades (for review, see Ref. 26)
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