Growth inhibitory signalling by TGFβ is blocked in Ras-transformed intestinal epithelial cells at a post-receptor locus

Cellular Signalling 15 (2003) 699 – 708 www.elsevier.com/locate/cellsig

Growth inhibitory signalling by TGFh is blocked in Ras-transformed intestinal epithelial cells at a post-receptor locus Bo Jiang a, Jin-San Zhang b, Jianguo Du a, Raul Urrutia b, John Barnard a,* a

Department of Pediatrics, Divisions of Molecular Medicine and Gastroenterology, Children’s Hospital and The Ohio State University, Columbus, OH 43205, USA b Gastroenterology Research Unit and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55901, USA Received 8 November 2002; accepted 9 December 2002

Abstract The transforming growth factor h (TGFh) family of growth regulatory peptides plays an important role in the regulation of gastrointestinal epithelial cell homeostasis. Loss of growth inhibitory signalling by TGFh is common in the context of Ras-transformation and it has been hypothesized that loss of TGFh receptor II (TGFhRII) expression accounts for the emergence of TGFh resistance. Here we examine the functional significance of reduced TGFhRII expression in intestinal epithelial cells transformed by oncogenic Ras. TGFhinduced signalling events downstream of TGFhRII were examined in Ras-transformed RIE-1 cells (RIE-Ras) and compared to the parental RIE-1 line. RIE-Ras cells were resistant to growth inhibition by TGFh. Neither overexpression of TGFhRII in RIE-Ras cells nor expression of constitutively active TGFhRI restored sensitivity to TGFh. TGFh-mediated phosphorylation of Smad2 occurred in TGFh-resistant RIERas cells, as well as other TGFh-resistant cells lines (HT-29, SW620) expressing low levels of TGFhRII. Nuclear translocation of Smad2 and Smad4 occurred equally in RIE-Ras and parental RIE cells. The activity of TIEG2, a TGFh-inducible SP1-like transcription factor, was reduced in RIE-Ras cells, implying that resistance in Ras-transformed RIE cells occurs by a transcriptional mechanism. D 2003 Elsevier Science Inc. All rights reserved. Keywords: TGFh; Ras; Smad; TGFh receptor; Intracellular signalling

1. Introduction The transforming growth factor h (TGFh) family of growth regulatory peptides plays an important role in the regulation of gastrointestinal epithelial cell homeostasis. Growth inhibitory TGFh signalling is initiated by ligand binding to the extracellular domain of the type II TGFh receptor (TGFhRII). Ligand-bound TGFhRII activates the type I TGFh receptor (TGFhRI) by formation of a heterotetrameric signalling complex and phosphorylation of TGFhRI in the GS region of the cytoplasmic tail. TGFhRI phosphorylates the cytoplasmic mediators Smad2 and Smad3, which recruit Smad4 into a transcriptional complex and translocate into the nucleus where they associate with coactivators and corepressors to effect TGFh-mediated tran-

* Corresponding author. Children’s Research Institute, 700 Children’s Drive, W502 Columbus, OH 43205, USA. Tel.: +1-614-722-2725; fax: +1614-722-3273. E-mail address: [email protected] (J. Barnard).

scriptional events [1]. The entire TGFh signalling pathway is subject to extensive negative regulation by additional signalling pathways and extracellular and intracellular processes. Although TGFh is a potent growth inhibitor for normal, nontransformed intestinal epithelial cells [2,3], more than 75% of transformed intestinal epithelial cells are resistant to TGFh-mediated growth inhibition [4,5]. Precise genetic defects leading to loss of TGFh tumour suppressive function have been described in human colorectal cancers. For example, inactivating frameshift mutations in the type II TGFh receptor (TGFhRII) occur in transformed colon cell lines and cancers with microsatellite instability (MSI) due to a germline defect in DNA base –base mismatch repair [6]. Reconstitution of a normal TGFh signalling pathway by transfection of a normal TGFhRII into MSI cell lines restores TGFh sensitivity [7]. It is estimated that approximately 13% of all colorectal cancers demonstrate MSI and loss of TGFhRII [6], while an additional 15% of microsatellite stable (MSS) colorectal cancer cell lines also harbour inactivating mutations in TGFhRII [4]. Additional genetic defects

0898-6568/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0898-6568(03)00010-X

700

B. Jiang et al. / Cellular Signalling 15 (2003) 699–708

in colorectal cancers include inactivating mutations in Smad2, which account for 6% or less of colon cancers [8,9] and Smad4 mutations in the range of 16– 22% [10 – 12]. Germline Smad4 mutations have also been detected patients with familial juvenile polyposis coli, a rare disorder predisposing to colorectal carcinoma (JPC) [13]. In aggregate, the aforementioned genetic defects in TGFh signalling occur in approximately 50% of human colorectal cancers. Undiscovered additional mutations in the TGFh pathway or epigenetic events involving TGFh signalling must therefore be operative in the remaining TGFh-resistant colorectal cancers. TGFh resistance in Ras-transformed cells has been invoked as one such epigenetic mechanism. The Ras gene product, a membraneassociated guanine nucleotide-binding signalling protein, is mutationally activated in about 50% of colorectal carcinomas [14,15]. Nontransformed intestinal epithelial cells transfected with an activated H-Ras oncogene become resistant to growth inhibition by TGFh [16,17], but precisely how oncogenic Ras blocks growth inhibitory signalling in these cells is unproven. Multiple reports have described a marked down-regulation of TGFhRII in TGFh-resistant Ras-transformed cells, including intestinal epithelial cells [16 –22]. In intestinal epithelial cells, downregulation of TGFhRII by oncogenic Ras is transcriptional, reversible and involves Raf-independent Ras signalling pathways [19,23]. In K-Ras-transformed thyroid cells, transfection of TGFhRII restores TGFh sensitivity, suggesting the Ras-induced reduction in TGFhRII is functionally important [22]. Oncogenic Ras signalling also intersects with TGFh signalling at multiple post-receptor loci. Kretzschmar et al. [24] provided evidence that increased ERK1/2 activity in Ras-transformed mammary epithelial cells results in phosphorylation of Smad2 and Smad3 in the linker region of the protein, which appears to disturb translocation of Smad2 and Smad3 into the nucleus following TGFh treatment. Conversely, others have found no effect [25] or a stimulatory effect [26,27] of Ras signalling on Smad nuclear translocation and transcriptional activity. Other loci of TGFh signalling interference have also been recently described. For example, in intestinal epithelial cells, overexpression of oncogenic Ras using an inducible vector causes proteasome-mediated degradation of Smad4 and emergence of TGFh resistance, an effect that can be overcome by enforced overexpression of Smad4 [28]. Lo et al. [29] found that phosphorylation by Erk1/2 leads to stabilization of TGIF, a Smad corepressor protein, the result being inhibition of TGFh growth inhibitory signalling. Collectively, these observations suggest that the signalling pathways activated by oncogenic Ras may impact TGFh signalling at a multiple loci, depending in part at least on the cell type and experimental context. In the present report, we have addressed the functional significance of decreased TGFhRII expression in Ras-transformed intestinal epithelial cells. The marked reduction of

TGFhRII in Ras-transformed intestinal epithelial cells is insufficient to attenuate TGFh-induced Smad2 phosphorylation and nuclear translocation of the Smad transcriptional complex, indicating that the block in growth inhibitory signalling by TGFh occurs distal to Smad translocation into the nucleus. In support of this, we show that mutant activated Ras interferes with the activity of TIEG2, a TGFh-inducible transcriptional repressor, which is impaired by Erk1/2 activation [30].

2. Materials and methods 2.1. Cell lines and reagents RIE-1 rat intestinal epithelial cells were obtained from Ken Brown (Cambridge, UK) and were maintained in DMEM supplemented with 5% foetal calf serum. RIE-Ras cells were kindly supplied by Dr. Robert Coffey (Vanderbilt University) and were stably transfected with pSV2-H-ras (12V) containing human sequences encoding the transforming H-Ras(12V) protein. Multiple G418-resistant RIE-Ras clones (>50) were pooled for use in subsequent studies. SW620, HT-29 and DLD-1 cells were obtained from the American Type Culture Collection. TGFh1 was obtained from R&D Systems (Minneapolis, MN). Anti-phosphoSmad 2 was obtained from Upstate Biotechnology, Waltham, MA. Anti-TGFhRII (sc#400) and anti-Smad 4 antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA). Ras activity was assayed based on binding of GTP-Ras to immobilized Raf-1 and detection of Ras using a pan-Ras antibody (Upstate Biotechnology). 2.2. Transient transfection of constitutively active, mutant TGFbRI and wild-type TGFbRII RIE-1 and RIE-Ras cells in six-well plates were transiently cotransfected with 0.65 Ag/well pCMV5TGFhRI/HA(T204D) (kindly provided by Dr. Jeffrey Wrana), 0.65 Ag/well p3TP-Lux and 0.35 Ag pCMV-h-Gal control plasmid (both kindly provided by Dr. R.D. Beauchamp) using the Lipofectamine 2000 protocol supplied by Invitrogen (Carlsbad, CA). In separate experiments, cells were transfected with 0.65 Ag/well pCMV5-TGFhRII/HA (kindly provided by Dr. Ed Leof). Transfected cells were cultured in DMEM containing 10% foetal calf serum for 24 h to permit recovery. TGFh1 (10 ng/ml) was then added for 24 h. Cell lystes were prepared in reporter lysis buffer and luciferase activities were measured using the Promega Luciferase Assay System (Promega, Madison WI) using a model TD-20/20 luminometer from Turner Designs (Sunnyvale, CA). Values were normalized to h-galactosidase activity to adjust for transfection efficiency. Two TGFh responsive luciferase reporters were used, p3TP-lux (kindly supplied by Dr. R. Daniel Beauchamp) and SBE4-luciferase (kindly supplied by Dr. Bert Vogelstein).

B. Jiang et al. / Cellular Signalling 15 (2003) 699–708

701

2.3. Immunoprecipitation and Western blotting

2.6. Transcriptional reporter assays

After treatments described in the figure legends, cells were lysed in 20 mM Tris –HCl, pH 7.4, 120 mM NaCl, 100 mM NaF, 200 AM Na3VO4, 4 mM PMSF, 10 Ag/ml leupeptin, 10 Ag/ml aprotinin, 0.5% NP40 and 2 mM benzamidine for 30 min at 4 jC. When phospho-Smad2specific antibody was used, 50 mM beta-glycerophosphate was added. The lysate was clarified by centrifugation. The supernatant was incubated with the primary antibody of interest overnight at 4 jC, followed by protein A/G-agarose beads. After centrifugation, the pellet was washed repeatedly with PBS, and boiled in SDS sample buffer and subjected to SDS-PAGE. Proteins were transferred to PVDF membrane using the semidry transblot system from BioRad (Hercules, CA) in 1  transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol), at 20 V for 45 min. Blots were blocked in 5% nonfat milk in 1  TBST (10 mM Tris – HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature, followed by incubation with the primary antibody of interest for 2 h. After four washes with 1  TBST, the blots were incubated with HRP-conjugated secondary antibody at room temperature for 30 min. After the final wash, the ECL Plus detection system (Amersham-Pharmacia, Piscataway, NJ) was used to detect antigen– antibody complexes.

The amino terminus of TIEG2 (a.a. 2 – 371) was subcloned into pM vector (BD Biosciences Clontech, Palo Alto, CA) in frame with the Gal4 DNA binding domain (Gal4DBD) as previously described [30]. RIE and RIE-Ras cells were transiently transfected using Lipofectamine 2000 protocol (Invitrogen) as described above. Briefly, cells were cultured in 24-well tissue culture plates and transfected with Gal4 constructs and the reporter vector, which carries the firefly luciferase gene downstream from five tandem Gal4 DNA binding sites and the thymidine kinase promoter. As a control for transfection efficiency, both Gal4-DBD and Gal4TIEG2 Nterm constructs were cotransfected with the RSVRenilla luciferase control plasmid. Twenty four hours after transfection, luciferase assays were performed with a Turner 20/20 luminometer and the Dual-Luciferase-Reporter Assay System in accordance with the manufacturer’s suggestions (Promega) as described previously [31]. Relative luciferase activity is expressed as 5  GAL4 luciferase values normalized to Renilla luciferase values F S.D.

2.4. Immunofluorescence Immunofluorescence staining was accomplished in multichambered slides. Cells were treated with 10 ng/ml TGFh1 for 1 h, fixed in 10 jC methanol, air dried and permeabilized using 0.2% Triton. Cells were blocked in 10% normal goat or normal donkey serum for 20 min to suppress nonspecific binding of IgG followed by an overnight incubation with a 1:75 dilution of an affinity-purified mouse monoclonal anti-human Smad4 antibody (Santa Cruz). The secondary antibody (FITC-conjugated goat anti-mouse antibody at 1:200) in 10% normal goat or donkey serum was added for 45 min. Nuclei were stained with propidium iodide and fluorescence was viewed using a Zeiss LSM 410 confocal microscope. 2.5. Cell proliferation assays 3

H-thymidine incorporation assays were carried out in 24-well tissue culture plates. Cells were seeded at a density of 20,000 cells/well, allowed to attach for at least 24 h, then treated as described in the figure legends. A 3Hthymidine (Amersham) pulse (1 ACi/well) was provided between the 18th and 21st hours of treatment. Radioactivity incorporated into trichloroacetic-acid-insoluble material was determined by scintillation counting and results are presented as the mean F S.E.M. for triplicate or quadruplicate measurements. Each experiment was repeated at least three times.

3. Results Cell lines used in this study were tested for growth inhibition by TGFh1 using 3H-thymidine incorporation assays. Rapidly growing, subconfluent RIE and RIE-Ras cells were treated with varying concentrations of TGFh1 and these data were supplemented with additional experiments from three human colon cancer lines (HT-29, SW-620 and DLD-1) with potentially unique properties with respect to TGFh sensitivity. The transformed lines (RIE-Ras, HT-29, SW-620 and DLD-1) were resistant to inhibition by TGFh1 while the parental RIE-1 line was not (Fig. 1). The results are congruous with either previously published observations [17,32] or the expected growth response to TGFh based on known defects in TGFh signalling [33 – 35], thus confirming the suitability of these lines for studies reported herein. Intestinal epithelial cells transformed by oncogenic Ras become resistant to growth inhibition by TGFh, assume a transformed morphology, and form colonies in soft agar [16,17,36]. TGFhRII mRNA and protein levels are 5- to 10fold reduced in Ras-transformed intestinal epithelial cells, suggesting a possible mechanism by which oncogenic Ras causes TGFh resistance [17,23]. In the present study, the significance of decreased TGFhRII expression in RIE-Ras cells was examined in more detail by several approaches, including transfection of a constitutively active TGFhRI mutant, enforced expression of TGFhRII and finally determination of the level of Smad2 phosphorylation and other downstream signalling events. The fidelity of TGFh signalling downstream of TGFhRII was first examined by transient transfection of both RIE and RIE-Ras cells with constitutively active TGFhRI, generated by mutation of Thr204 in the GS domain to aspartic acid

702

B. Jiang et al. / Cellular Signalling 15 (2003) 699–708

Fig. 1. TGFh sensitivity in intestinal epithelial cell lines. 3H-thymidine incorporation assays were performed as described in Materials and methods. Rapidly growing, subconfluent cells were treated with the indicated concentration of TGFh1 or vehicle for 18 h prior to a 3-h 3Hthymidine pulse. 3H-thymidine incorporation into trichloroacetic-acidinsoluble material was counted in a scintillation counter and expressed as the mean of quadruplicate experiments done in triplicate assays.

[37]. This TGFhRI(204D) mutation mimics ligand-mediated activation of the TGFhRI serine/threonine kinase, under normal conditions accomplished by interaction of TGFh with the extracellular domain of TGFhRII. Cells were

cotransfected with two different TGFh-sensitive reporters (p3TP-lux and SBE4-LUC), a plasmid encoding h-galactosidase to control for transfection efficiency. As shown in Fig. 2, the p3TP-lux reporter gene was activated approximately 20-fold by TGFh and 7-fold by TGFhRI(T204D) in RIE-1 cells, findings in close agreement with those reported in other cell lines [37]. In RIE-Ras cells, reporter gene activity was 4- to 5-fold reduced relative to RIE cells, irrespective of whether cells were treated with TGFh or transfection with TGFhRI(T204D), indicating that the block in TGFh signalling occurs downstream of the heteromeric, membraneassociated TGFhRI/TGFhRII. Because of differences in transfection efficiency between RIE and RIE-Ras cells, these experiments were performed in triplicate and were corrected for differences in efficiency by determination of h-galactosidase activity. In addition, an alternate TGFh reporter construct, SBE4-LUC, was used in similarly designed experiments, again in triplicate. These experiments also show that TGFhRI(T204D) did not restore TGFh-mediated SBE4-luciferase activation to control levels. In a second series of experiments, full-length TGFhRII was transiently overexpressed along with the 3TP-lux reporter in parental and RIE-Ras cells. Overexpression of TGFhRII in RIE-Ras cells was confirmed by Western analysis (not shown). Unlike observations previously reported in thyroid carcinoma cells [22,38], increased expression of TGFhRII in RIE-Ras cells did not restore sensitivity to TGFh (Fig. 2). The results shown in Fig. 2 indicate that overexpression of TGFhRII or constitutively active TGFhRI does not

Fig. 2. TGFh resistance in Ras-transformed RIE cells cannot be overcome by overexpression of mutant, activated TGFhRI or overexpression of wild-type TGFhRII. RIE cells (open bars) and RIE-Ras cells (filled bars) were transiently cotransfected with p3TP-lux or SBE4-LUC and a pCMV-h-gal control plasmid as described in Materials and methods. The data set on the left side of the abscissa represents results from cells transfected with 3TP-lux reporter construct along with TGFhRI(T204D) (designated TGFhRI*) or full length, wild-type TGFhRII. Cells were treated with TGFh1 or vehicle for 24 h as indicated, followed by determination of luciferase activity. Because of differences in transfection efficiency between the RIE and RIE-Ras cell lines, results were normalized for h-gal activity and expressed relative to treatment with vehicle alone. In the right panel, results are shown from repeated experiments using the SBE4-LUC reporter construct and TGFhRI(T204D).

B. Jiang et al. / Cellular Signalling 15 (2003) 699–708

‘‘rescue’’ TGFh signalling in RIE cells transformed by oncogenic Ras. These results question the significance of reduced receptor expression as an important factor in TGFh resistance in Ras-transformed cells. The integrity of TGFh signalling immediately downstream of TGFhRI serine/ threonine kinase activation was determined by measurement of Smad2 activation by TGFh in parental RIE cells and RIE-Ras cells (Fig. 3A). These results were complemented by analogous observations in HT-29, SW-620 and DLD-1 colon cancer cell lines (Fig. 3B). HT-29 is a microsatellitestable human colon adenocarcinoma cell line with wild-type Ras activity and reduced TGFhRII levels [17,33]. SW-620 is a microsatellite-stable, human metastatic colon adenocarcinoma cell line that contains oncogenic Ras activity, allelic loss of Smad4 and reduced TGFhRII levels [17,33,34,39]. DLD-1 is a microsatellite-instable human colon adenocarcinoma cell line that does not express cell surface TGFhRII [35], thereby serving as a negative control. Activation of Smad2 was assayed by Western analysis using an antiphosphorylated Smad2 antibody. Smad2 phosphorylation increased markedly 15 min after treatment with TGFh1 in all cell lines except DLD-1 (Fig. 3A,B). This observation permitted two conclusions. First, increased Smad2 phosphorylation occurred relatively equally in cells that are sensitive (RIE-1) and insensitive (RIE-Ras, HT-29 and SW620) to growth inhibition by TGFh. Second, a low level of TGFhRII expression in RIE-Ras, HT-29 and SW-620 cells did not attenuate phosphorylation in response to TGFh treatment. Collectively, these results suggest that the activation of proximal elements in the TGFh signalling cascade proceeds unabated in transformed intestinal epithelial cells resistant to growth inhibition to TGFh and expressing relatively low levels of TGFhRII. In HT-29 and SW-620 cells that contain wild-type and activated Ras, respectively,

703

phosphorylation of Smad2 also occurred equally well, indicating that additional defects in growth inhibitory signalling must occur in these cell lines despite the marked differences in Ras activity (Fig. 3C). To confirm the presence or absence of hyperactive Ras activity in the cell lines used in our study, Ras activity was measured using an immunoaffinity assay based on the binding of activated RasGTP, but not Ras-GDP, to Raf1 RDB agarose [40]. As shown in Fig. 3C, RIE-Ras, SW-620 and A431 (a positive control) cells showed increased levels of Ras activity when compared with RIE cells transfected with Ras40C, a Ras effector domain mutant [41], HT-29 cells which have a wildtype Ras allele and the parental RIE line (not shown). Thus, the level of Ras activation in these cell lines coincides with that predicted on the basis of prior work and is not constitutively activated in HT-29 cells as previously hypothesized [42]. Prior studies have shown that phosphorylation and complex formation with phosphorylated Smad2 is required for nuclear distribution of Smad4 [43]. Having shown intact phosphorylation of Smad2 in TGFh-resistant intestinal epithelial cell, the subcellular localization of Smad4 in response to TGFh was next examined to determine if hyperactive Ras activity interferes with movement of Smad4 from the cytoplasm to the nucleus, as previously reported in transformed mammary epithelial cells [24]. Cytosolic and nuclear protein lysates were prepared from the RIE and RIE-Ras cell lines 30 min after treatment with TGFh and subjected to SDS/ polyacrylamide electrophoresis. Western blotting for Smad4 expression demonstrated that translocation into the nucleus in RIE-Ras cells occurred at a level equivalent to the parental RIE line (Fig. 4). Interestingly, the amount of total cellular Smad4 that translocated to the nucleus in response to TGFh was relatively small, as a large fraction remained in the

Fig. 3. Smad2 is phosphorylated by TGFh1 in TGFh growth-resistant transformed lines with low levels of TGFhRII expression as well as TGFh-sensitive parental RIE cells. (A) RIE and RIE-Ras cells were treated with 5 ng/ml TGFh1 or vehicle for 15 min. Whole cell lysates were prepared and subjected to immunoblot analysis for phosphorylated Smad2 as described in Materials and methods. (B) Lysates were also prepared from HT-29, SW-620 and DLD-1 human colon cancer cell lines and analysed for phosphorylated Smad2 protein to confirm that Smad2 activation occurs in TGFh-resistant cells with low levels of TGFhRII expression. The DLD-1 cell line is a negative control by virtue of microsatellite instability and loss of TGFhRII expression. (C) Ras-activity was determined in selected cell lines to confirm hyperactivity. Lysates from subconfluent cells were isolated and Ras activity determined in a pull-down assay based on binding of activated Ras to immobilized Raf-1 as described in Materials and methods.

704

B. Jiang et al. / Cellular Signalling 15 (2003) 699–708

Fig. 4. Smad4 translocates into the nucleus in TGFh-resistant RIE-Ras cells. RIE and RIE-Ras cells were treated with the indicated concentration of 5 ng/ml TGFh for 30 min. Nuclear cytosolic fractions were prepared as described in Materials and methods and subjected to SDS gel electrophoresis and Western blotting for Smad4 expression (top panel). The purity of the cytosolic and nuclear preparations was determined by detection of TGFhRII, a marker for the cytosolic fraction and histone HI, a marker for the nuclear fraction. Finally, equal protein loading was assessed by expression of h-actin. The immunoblot shown is representative of three separate experiments.

cytosol after treatment. The distribution of a known nonnuclear protein (TGFhRII) and a known nuclear protein (histone H1) was examined to verify the purity of cytosolic and nuclear lysates. The expected protein distribution was seen, including down-regulation of TGFhRII in Ras-transformed cells, as previously described [17]. In response to TGFh, phosphorylated Smad2 translocates into the nucleus in a complex with Smad3 and Smad 4. The data in Fig. 4 suggests that a small fraction of total cellular Smad4 translocates into the nucleus independent of the sensitivity of Ras-transformed cells to growth inhibition by TGFh. To assess further the extent to which the Smad complex translocates into the nucleus, in separate experiments, translocation of phosphorylated Smad2 was also examined. Fig. 5 confirms the observation that Smad proteins enter the nucleus in response to the TGFh in both TGFh-sensitive RIE-1 cells as well as TGFh-resistant RIERas cells, as phosphorylated Smad2 enters the nucleus

equally well in these two cell lines. Using an alternate approach, Smad complex formation was examined in RIE and RIE-Ras cells treated with TGFh1 for 30 min. Nuclei were purified and nuclear lysates were incubated with antiphosphorylated Smad2. Immunoprecipitated phosphorylated Smad2 and associated proteins were separated on SDS/polyacrylamide gels and Western blots were analysed for Smad4 protein. Again, nuclear translocation of Smad4 was observed, this time in conjunction with activated Smad2 (Fig. 6, top panel). Effective separation of cytoplasm from the starting nuclear preparation was confirmed by immunoblot analysis of TGFhRII expression in cytoplasmic and nuclear extracts. No TGFhRII was detected in the nuclear extracts, as expected (Fig. 6, bottom panel). Immunofluorescence staining of Smad4 in logarithmically growing RIE and RIE-Ras cells was used to determine further the distribution of Smad4 after treatment with TGFh. Prior to treatment with TGFh, Smad4 immunostaining is

Fig. 5. Phosphorylated Smad2 also translocates into the nucleus in TGFh-resistant RIE-Ras cells. RIE and RIE-Ras cells treated with 5 ng/ml TGFh or vehicle for 30 min. Cytosolic and nuclear lysates were prepared as described in Materials and methods. Immunoblot were prepared and probed for phosphorylated Smad2. Immunoblotting for h-actin was used to confirm equivalent loading. The immunoblot shown is representative of three separate experiments.

B. Jiang et al. / Cellular Signalling 15 (2003) 699–708

705

Fig. 6. A complex between phosphorylated Smad2 and Smad4 translocates into the nucleus in TGFh-resistant RIE-Ras cells. Nuclear lysates were prepared from RIE and RIE-Ras cells treated with 5 ng/ml TGFh for 30 min. Smad2/Smad4 complex was immunoprecipitated with anti-phosphorylated Smad2 antibody and probed with anti-Smad4 antibody to detect complex formation. To confirm the purity of starting material, immunoblots from the initial nuclear and cytosolic fractions were prepared using antibody to TGFhRII as a marker for cytosolic protein.

primarily cytoplasmic in both cell lines (Fig. 7). Nuclear accumulation of Smad4 occurred following TGFh treatment of each line, although, consistent with observation in Fig. 4, significant residual cytoplasmic Smad4 immunofluorescence remains. Collectively, these results signify that the blockade to TGFh growth inhibitory signalling in Ras-transformed cells occurs downstream of Smad4 redistribution into the nucleus.

The TGFh-induced transcriptional apparatus consists of Smad proteins associated with a complex array of potential coactivating and corepressing proteins [44]. Thus, a large number of potentially intersecting loci between signalling by activated Ras and the TGFh transcriptional apparatus exists. Recent data suggest that the TIEG subfamily of Sp1-like transcriptional repressors is one potential locus of interaction between Ras and TGFh signalling. Both TIEG1 and 2 are

Fig. 7. Immunofluorescent staining of Smad4 translocation into the nucleus in TGFh-treated parental RIE and RIE-Ras cells. Cells were treated with 10 ng/ml TGFh or vehicle for 1 h and prepared for Smad4 immunofluorescence staining using an affinity-purified mouse monoclonal antibody to Smad4 as described in Materials and methods. The secondary antibody was a FITC-conjugated goat anti-mouse antibody. Nuclei were stained with propidium iodide and photographed using a Zeiss LSM 410 confocal microscope.

706

B. Jiang et al. / Cellular Signalling 15 (2003) 699–708

TGFh-inducible early response genes that inhibit epithelial cell proliferation and induce apoptosis [45,46]. We have previously found that TIEG proteins are transcriptional repressors that function by recruitment of mSin3A/HDAC complexes via the conserved alpha helical repression motif [31]. More recently, TIEG2 has been identified as a target of EGF/Ras/MEK1/ERK2-mediated signalling. Phosphorylation of TIEG2 by ERK2 strongly antagonizes its repression activity by virtue of disrupting its binding to the corepressor mSin3A. Signalling disrupts mSin3A binding to the Mad1like Sin3-interacting domain of TIEG2, an Sp1-like repressor [30]. These observations led us to hypothesize that TIEG2 repression activity may be down-regulated in RIE-Ras cells. To test this hypothesis, we linked the TIEG2 N-terminal transcriptional regulatory domain (TIEG2-Nterm) to the Gal4 DNA binding domain (Gal4DBD) and performed transcriptional reporter assays in both parental RIE and RIE-Ras cells. Gal4 DBD alone was used as a control. Fig. 8 shows that Gal4 TIEG2-Nterm represses Gal4 TK reporter activity in RIE cells in a dose-responsive manner, a finding consistent with our previous reports that TIEG2 functions as a transcriptional repressor. Basal activity of the Gal4 TK reporter is not significantly different in RIE and RIE-Ras cells (1.17 F 0.027 vs. 1.14 F 0.091), respectively. Gal4 TIEG2-Nterm (50 ng) represses the reporter activity in RIE

Fig. 8. TIEG2-mediated transcriptional repression is blocked in TGFhresistant RIE-Ras cells. Gal4 reporter assays were performed to determine the effect of Ras hyperactivity on TIEG2 mediated transcriptional repression. RIE and RIE-Ras cells were cotransfected with an expression plasmid containing the N-terminus of TIEG2 linked to the Gal4 DNA binding domain and a reporter plasmid containing luciferase downstream of five tandem Gal4 DNA binding sites. Because of differences in transfection efficiency using the RSV-Renilla luciferase control plasmid, data are normalized for transfection efficiency. Results shown are the mean plus standard deviation of three determinations for each data point. Similar results were found in three independent assays. A similar blockade of transcriptional repression was also seen with the closely related family member TIEG1 (not shown).

cells about 3-fold as compared to control vector (0.37 F 0.012 vs. 1.17 F 0.027). The repression activity is attenuated in RIE-Ras cells as compared to RIE cells (0.83 F 0.012 vs. 0.45 F 0.069 at 25 ng and 0.74 F 0.026 vs. 0.37 F 0.012 at 50 ng) at similar expression levels (data not shown). These results suggest that transcriptional repression by TIEG2 is inhibited in RIE-Ras cells in a dose-responsive manner. Similar inhibition of transcriptional repressor activity by Ras was also observed for TIEG1 (data not shown).

4. Discussion Escape from growth inhibitory TGFh signalling is a common event in neoplastic diseases, including colorectal neoplasia. The precise loci for defective TGFh signalling can be identified in approximately 50% of colorectal cancers, leaving a sizeable portion with unrecognized genetic or epigenetic defects in TGFh signalling. A potential epigenetic phenomenon is hyperactive Ras activity. Activating Ras mutations occur in about 50% of colorectal cancers [15] and Ras-transformed cells in culture are commonly resistant to growth inhibition by TGFh [16,18,21 –24,47,48]. Several laboratories have focused attention on the mechanism of TGFh-resistance in Ras-transformed intestinal epithelial cells [5,23,24,28]. Proposed mechanisms include decreased expression of TGFhRII [16,23], ineffective Smad complex assembly and nuclear translocation [24], and degradation of Smad4 prior to nuclear translocation [28]. In the present report, we examined more intensively the importance of decreased TGFhRII expression in Ras-transformed intestinal epithelial cells as a mechanism for reduced TGFh sensitivity. Observations in the RIE-1 model of Ras transformation were supplemented with observations in selected human colon cancer cell lines. The 5- to 10-fold decreased levels of TGFhRII protein expression in Rastransformed intestinal epithelial cells previously noted by several laboratories, including our own, [23] does not attenuate Smad activation. Smad2 phosphorylation also occurs in two TGFh-resistant, human colon carcinoma cell lines (HT-29 and SW620), both of which have markedly decreased expression of TGFhRII relative to nontransformed cells [17]. Thus, proximal Smad signalling in intestinal epithelial cells with reduced levels of TGFhRII proceeds unaltered, and suggests that regulation of receptor levels in most circumstances is likely to be insufficient to have biological consequences, at least with respect to Smad signalling. It is feasible that signalling by other proposed TGFh-dependent pathways, such as Ras/Erk1/2 [49] or Jun pathways [50], is modified by reduced receptor expression. Our study also demonstrates that Smad proteins form complexes and translocate into the nucleus in Ras-transformed TGFh-resistant cell lines. These observations contrast, in part at least, with those reported by Kretzschmar et al. [24] who found that Smad2 and Smad3 do not accumulate in the nucleus of mammary and lung epithelial cells

B. Jiang et al. / Cellular Signalling 15 (2003) 699–708

overexpressing oncogenic Ras. This was explained by phosphorylation of Erk1/2 consensus sites in the linker region of Smad2 and Smad3, a site distinct from the TGFh phosphorylation site in the SSxS domain in the carboxyterminus of Smad2 and Smad3. The result is inhibition of Smad accumulation in nucleus. An analogous Ras-mediated phosphorylation event interferes with translocation of Smad1 in BMP-treated cells [51]. It should be noted that inhibition of Smad translocation in these studies was not absolute. Interestingly, in TGFh-resistant, Ras/ErbB2-transformed mammary epithelial cells [52], TGFh induces functionally active Smad complexes that do translocate into the nucleus and activate transcription from the Smad7 promoter as well as an artificial reporter based on the Smad-binding element (SBE), findings that are congruous with our own. In aggregate, these studies suggest that if Ras interferes with TGFh signalling by phosphorylation of the linker region of Smad2 and Smad3, the effect may be dependent on the cell line being studied. In our study, Smad activation was assessed by examination of endogenous Smad proteins, as opposed to transfected, tagged exogenous proteins, suggesting that methodological differences in reported studies may also be operative. Finally, our results also suggest that the kinetics, stoichiomety and consequences of Ras accumulation in an inducible expression system may differ substantially from long-term overexpression characteristic of colorectal cancers cells and our own experimental model. Thus, methodological differences may explain the apparent absence in our studies of Ras-mediated Smad4 degradation as recently reported by Saha et al. [28]. In summary, the decreased expression of TGFhRII previously reported in Ras-transformed intestinal epithelial cells and TGFh-resistant human colon cancer cell lines is insufficient to attenuate TGFh signalling. In fact, in our experiments, Smad signalling proceeds beyond the point of Smad complex translocation into the nucleus. By demonstration of reduced TIEG2 activity in functional transcriptional assays, we propose a potential point of interference between hyperactive Ras signalling and TGFh-mediated transcriptional events. Additionally, the Ras pathway may impinge on TGFh-mediated transcription by interference with additional proteins, such as TGIF, as proposed by others [29]. It has been emphasized that extensive negative regulation of TGFh signalling is important in the maintenance of cellular growth homeostasis [53]. It is therefore not surprising that an increasingly accumulating body of data indicates extensive cross-talk and multiple loci of interference between proliferative Ras signalling and inhibitory TGFh signalling, depending in part on the cellular context or the system under study. Additional study is required to ascertain whether inhibition of TIEG proteins is fully responsible for the altered response to TGFh in Ras-RIE cells. Interference between Ras and TGFh signalling may occur at multiple points along the TGFh signalling pathway, including transcription from TIEG subfamily of Sp-1 like proteins.

707

Acknowledgements This work was supported by R01 DK57128 (JB). References [1] Massague J, Wotton D. EMBO J 2000;19:1745 – 54. [2] Barnard JA, Beauchamp RD, Coffey RJ, Moses HL. Proc Natl Acad Sci U S A 1989;86:1578 – 82. [3] Koyama S, Podolsky DK. J Clin Invest 1989;83:1768 – 73. [4] Grady WM, Myeroff LL, Swinler SE, Rajput A, Thiagalingham S, Lutterbaugh J, et al. Cancer Res 1999;59:320 – 4. [5] Filmus J, Kerbel RS. Curr Opin Oncol 1993;5:123 – 9. [6] Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J, et al. Science 1995;268:1336 – 8. [7] Wang J, Sun L, Myeroff L, Wang X-H, Gentry LE, Yang J, et al. J Biol Chem 1995;270:22044 – 9. [8] Eppert K, Scherer SW, Ozcelik H, Pirone R, Hoodless PA, Kim HJ, et al. Cell 1996;86:543 – 52. [9] Takenoshita S, Tani M, Moghi A, Nagashima M, Nagamachi Y, Bennett WP, et al. Carcinogenesis 1998;19:803 – 7. [10] Thiagalingham S, Lengauer C, Leach FS, Schutte M, Hahn SA, Overhauser J, et al. Nat Genet 1996;13:343 – 6. [11] Takagi Y, Kohmura H, Futamura M, Kida H, Tanemura H, Simokawa K, et al. Gastroenterology 1996;111:1369 – 72. [12] Hogue ATMS, Hahn SA, Schutte M, Kern SE. Gut 1997;40:120 – 2. [13] Howe J, Roth S, Ringold JC, Summers RW, Jarvinen HJ. Science 1998;280:1086 – 8. [14] Khosravi-Far R, Campbell S, Rossman KL, Der CJ. Adv Cancer Res 1998;72:57 – 107. [15] Bos JL, Fearon ER, Hamilton SR, Verlaan-de V, van Boom JH, van der Eb AJ, et al. Nature 1987;327:293 – 7. [16] Filmus J, Zhao J, Buick RN. Oncogene 1992;7:521 – 6. [17] Winesett MP, Ramsey GW, Barnard JA. Carcinogenesis 1996;17: 989 – 95. [18] Houck KA, Michalopoulos GK, Strom SC. Oncogene 1989;4: 19 – 25. [19] Zhao J, Buick RN. Cancer Res 1995;55:6181 – 8. [20] Zhao J, Buick RN. Exp Cell Res 1993;204:82 – 7. [21] Wakefield LM, Smith DM, Masui T, Harris CC, Sporn MB. J Cell Biol 1987;105:965 – 75. [22] Turco A, Coppa A, Aloe S, Baccheschi G, Morrone S, Zupi G, et al. Int J Cancer 1999;80:85 – 91. [23] Bulus N, Peterson MS, Peeler MO, Sheng H-M, Sizemore N, Oldham SM, et al. Neoplasia 2000;2:357 – 64. [24] Kretzschmar M, Doody J, Timokhina I, Massague J. Genes Dev 1999;14:804 – 16. [25] Hu PP, Shen X, Huang D, Liu Y, Counter C, Wang XF. J Biol Chem 1999;274. [26] Blanchette F, Rivard N, Rudd P, Grondin F, Attisano A, DuBois CM. J Biol Chem 2001;276:33986 – 94. [27] de Caestecker MP, Parks WT, Frank CJ, Castagnino P, Bottaro DP, Roberts AB, et al. Genes Dev 1998;12:1587 – 92. [28] Saha D, Datta PK, Beauchamp RD. J Biol Chem 2001;276:29531 – 7. [29] Lo RS, Wotton D, Massague J. EMBO J 2001;20:128 – 36. [30] Ellenrieder V, Zhang JS, Kaczynski J, Urrutia R. EMBO J 2002;21: 2451 – 60. [31] Zhang J-S, Moncrieffe MC, Kaczynski J, Ellenrieder V, Prendergast FG, Urrutia R. Mol Cell Biol 2001;21:5041 – 9. [32] Barnard JA, Warwick GJ, Gold LI. Gastroenterology 1993;105:67 – 73. [33] Sepp-Lorenzino L, Ma Z, Rands E, Kohl NE, Gibbs JB, Oiliff A, et al. Cancer Res 1995;55:5302 – 9. [34] Calonge MJ, Massague J. J Biol Chem 1999;274:33637 – 43. [35] Malkhosyan S, McCarty A, Sawai H, Perucho M. Mutat Res 1996; 316:249 – 59.

708

B. Jiang et al. / Cellular Signalling 15 (2003) 699–708

[36] Gangarosa LM, Sizemore N, Graves-Deal R, Oldham SM, Der CJ, Coffey RJ. J Biol Chem 1997;272:18926 – 31. [37] Wieser R, Wrana JL, Massague J. EMBO J 1995;14:2199 – 208. [38] Coppa A, Mincione G, Lazzereschi D, Ranieri A, Turco A, Lucignano B, et al. J Cell Physiol 1987;172:200 – 8. [39] Geiser AG, Burmester JK, Webbink R, Roberts AB, Sporn MB. J Biol Chem 1992;267:2588 – 93. [40] de Rooij J, Bos JL. Oncogene 1997;14:623 – 5. [41] Khosravi-Far CR, White M, Westwick J, Solski P, ChrzanowskaWodnicka M, Van Aelst L, et al. Mol Cell Biol 1996;16:3923 – 33. [42] Bissonnette M, Khare S, von Lintig F, Wali R, Nguyen L, Zhang Y, et al. Cancer Res 2000;60:4602 – 9. [43] Souchelnytskyi S, Tamaki K, Engstrom U, Wernstedt C, Ten Dijke P, Heldin C-H. J Biol Chem 1997;272:28107 – 15. [44] Attisano A, Wrana JL. Curr Opin Cell Biol 2000;12:235 – 43.

[45] Tachibana I, Imoto M, Adjei PN, Gores GJ, Subramaniam M, Spelsberg TC, et al. J Clin Invest 1997;99:2365 – 74. [46] Cook T, Genelein B, Mesa K, Mladek A, Urrutia R. J Biol Chem 1998;273:25929 – 36. [47] Longstreet M, Miller B, Howe PH. Oncogene 1992;9:1549 – 56. [48] Chakrabarty S, Fan D, Varani J. Int J Cancer 1990;46:493 – 9. [49] Hartsough MT, Mulder KM. J Biol Chem 1995;270:7117 – 24. [50] Engel ME, McDonnell MA, Law B, Moses HL. J Biol Chem 1999; 274:37413 – 20. [51] Kretzschmar M, Doody J, Massague J. Nature 1997;389:618 – 22. [52] Chen C-R, Kang Y, Massague J. Proc Natl Acad Sci U S A 2001;98: 992 – 9. [53] Engel ME, Datta PK, Moses HL. J Cell Biochem, Suppl 1998;30/31: 111 – 22.