Overexpression of a dominant-negative type II TGFβ receptor tagged with green fluorescent protein inhibits the effects of TGFβ on cell growth and gene expression of mouse adrenal tumor cell line Y-1 and enhances cell tumorigenicity

Abstract

Transforming growth factor β (TGFβ) has been reported to be a potent growth inhibitor of epithelial cells. The purpose of the present work was to study in vitro and in vivo the effects of overexpression of a dominant-negative type II TGFβ receptor on the proliferation and differentiation of Y-1 cells. Stable transfections were performed with a mutant TβRII (TβRII-KR) fused with the Enhanced Fluorescent Green Protein (EGFP). The expression of this fusion protein and its overexpression were demonstrated by northern blot and immunoblot with EGFP and TβRII probes and antibodies respectively. The membrane localization of this fusion protein was confirmed by confocal microscopy. The functionality of this fusion protein was demonstrated by its blocking effects on TGFβ action on DNA synthesis and on Y-1 expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD). Moreover, in nude mice the tumorigenicity of cells stably transfected with the fusion protein was higher than that of cells stably transfected with EGFP alone. Taken together, the present results show that TβRII-KR/EGFP blocks the effects of TGFβ1 on Y-1 cells and acts as a potent dominant-negative receptor preventing TGFβ signaling.