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
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.
Section snippets
Materials
[α-32P]dCTP (>3000 Ci/mmol) and [3H] Thymidine (20 Ci/mmol) were obtained from ICN Biomedicals France (Orsay). Recombinant human TGFβ1 and anti-TβRII antibody (directed against the extracellular domain of human TβRII) were supplied by R&D Systems (Minneapolis, MN). A second anti-TβRII antibody (directed against the conserved kinase domain from amino-acids 246–266 of human TβRII) was obtained from Santa Cruz Biotechnology (CA, USA). A specific anti-TGFβ1 rabbit polyclonal antibody was prepared
Y-1 cells express and respond to TGFβ1
Before transfection we investigated whether Y-1 cells express TGFß1 and whether they respond to TGFβ1. Fig. 2A shows that Y-1 cells (lane 1) as well as another mouse cell line M3 (lane 2) express a single 2.5 kb TGFβ1 transcript, which is similar in size to the transcript expressed by three human cell lines H295R, HepG2 and SW13 (lanes 3, 4, 5 respectively). Immunocytochemistry of Y-1 cells (Fig. 2B) using a specific TGFβ1 antibody revealed a strong immunoreactivity in the cells. Moreover, the
Discussion
Molecular cloning (Prasher et al., 1992) and heterologous expression of jellyfish cDNA (Chalfie et al., 1994) and generation of GFP variants with greatly increased brightness and rate of fluorophore production (Cormack et al., 1996) have triggered the widespread and growing use of GFP as a reporter molecule in the localization and function of several proteins (Chalfie et al., 1994, Tsien, 1998). Here we describe the characterization of the first EGFP chimera of a mutated receptor
Acknowledgements
We thank Dr J. Massagué for p3TP-Lux and TβRII-KR vectors, Dr R. Derynck for TGFβ1 cDNA, Dr F. Labrie and Dr V. Luu The for 3β-HSD cDNA, Dr R. Ivel for StAR cDNA, Dr.W. Rainey and Dr.M. Ascoli for H295R and MA10 cells respectively. We thank also, Dr M. Benchaid Menchabi for his help in confocal microscopy, and J. Bois for her secretarial help. This work was supported in part by grants from Association de la Recherche contre le cancer (ARC) and University Claude Bernard-Lyon1.
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