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.

Introduction

The transforming growth factor-beta (TGFβs) superfamily consists of a large number of structurally related polypeptides, each capable of regulating a fascinating array of cellular processes such as cell proliferation, differentiation, migration, extracellular matrix formation, and immunosuppression (Massagué, 1990, Roberts and Sporn, 1990). Three TGFβ isoforms have been identified in mammals: β1, β2, and β3 (Burt and Paton, 1992). These polypeptides exert their biological actions by both autocrine and paracrine mechanisms through binding to specific cell surface receptors. Three types of TGFβ receptors designated type I (TβRI), type II (TβRII) and type III (TβRIII) have been cloned (Lopez-Casillas et al., 1991, Wang et al., 1991, Lin et al., 1992, Ebner et al., 1993) and the role of each type of receptor has been determined. Type III receptor (280–330 kDa), also known as betaglycan, is the most abundant TβR class present in many types of cells. It has a short cytoplasmic domain which appears to lack signaling function. It has been described to have no direct role in TGFβ signaling, but is involved in the presentation of the ligand to the signaling receptors (Lopez-Casillas et al., 1993). However, recent data (Brown et al., 1999) suggest that this receptor could play an essential and nonredundant role in TGFβ signaling. TβRI and TβRII are transmembrane serine-threonine kinases which mediate the multiple effects of the TGFβs. TβRII, a 75–85 kDa glycoprotein, consists of a signal peptide, a small hydrophilic extracellular domain, a single transmembrane domain and a large cytoplasmic domain. The intracellular domain of TβRII contains a constitutively active serine /threonine kinase activity which is essential for signal transduction in concert with TβRI. TβRI, a 50–60 kDa transmembrane glycoprotein, is ubiquitously expressed in various types of cells and has been shown to be structurally similar to TβRII. The kinase activity contained in the cytoplasmic domain of TβRI requires the presence of functional TβRII for the TGFβ signal to be transduced (Wrana et al., 1994, Yamashita et al., 1994).

The requirement of both TβRI and TβRII for TGFβ signaling indicates that removal of either receptor could result in loss of TGFβ signal transduction. Mutations of the TβRII can interrupt the signal transduction pathway by dominant negative mechanisms (Wrana et al., 1992, Brand et al., 1993, Carcamo et al., 1995). Mutated TβRII inhibits endogenous receptor function in a dominant way, most likely by interfering with endogenous receptor complex formation and function. For instance, overexpression of a kinase-deficient mutant in TGFβ sensitive cells has been reported to result in the loss of TGFβ sensitivity (Brand et al., 1993, Wieser et al., 1993, Zhao and Young, 1996).

Green fluorescent protein (GFP) from jellyfish Aequorea victoria (Prasher et al., 1992) is becoming widely used as a reporter molecule in the localization and function of several proteins (Chalfie et al., 1994, Tsien, 1998). EGFP (GFPmut1; Cormack et al., 1996) a red-shifted variant of wild type GFP has been optimized for brighter fluorescence and higher expression in mammalian cells. EGFP emits bright green light when excited with blue light (Heim et al., 1995) and this fluorescence can be detected in living cells using standard fluorescence filters for fluorescein (Chalfie et al., 1994) without the requirement of any substrate or cofactor.

The aim of the present work was to investigate whether a kinase-deficient mutant of TβRII (TβRII-KR, Wrana et al., 1992) was able to block the inhibitory effects of TGFβ1. For that, we stably transfected a TGFß responsive mouse adrenocortical cell line Y-1 (Schimmer, 1981) which respond to TGF with a vector expressing TβRII-KR fused to EGFP. We found, that this chimeric protein was overexpressed when compared to the endogenous type II TGFβ receptor, properly targeted to the cell membrane, blocked the inhibitory effects of TGFβ1 on cell growth and expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) and increased the tumorigenicity of the cells injected in nude mice.