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Carcinogenesis Advance Access originally published online on September 3, 2007
Carcinogenesis 2007 28(12):2491-2500; doi:10.1093/carcin/bgm195
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The type III TGF-β receptor signals through both Smad3 and the p38 MAP kinase pathways to contribute to inhibition of cell proliferation

Hye Jin You1, Monique W. Bruinsma1, Tam How1, Julie H. Ostrander1 and Gerard C. Blobe1,2,*

1 Department of Medicine
2 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA

* To whom correspondence should be addressed. Tel: +1 919 668 1352; Fax: +1 919 668 2458; Email: blobe001{at}mc.duke.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Transforming growth factor β (TGFβ) has an important role as a negative regulator of cellular proliferation. The type III transforming growth factor β receptor (TβRIII) has an emerging role as both a TGFβ superfamily co-receptor and in mediating signaling through its cytoplasmic domain. In L6 myoblasts, TβRIII expression enhanced TGFβ1-mediated growth inhibition, with this effect mediated, in part, by the TβRIII cytoplasmic domain. The effects of TβRIII were not due to altered ligand presentation or to differences in Smad2 phosphorylation. Instead, TβRIII specifically increased Smad3 phosphorylation, both basal and TGFβ-stimulated Smad3 nuclear localization and Smad3-dependent activation of reporter genes independent of its cytoplasmic domain. Conversely, SB431542, a type I transforming growth factor β receptor (TβRI) inhibitor, as well as dominant-negative Smad3 specifically and significantly abrogated the effects of TβRIII on TGFβ1-mediated inhibition of proliferation. TβRIII also specifically increased p38 phosphorylation, and SB203580, a p38 kinase inhibitor, specifically and significantly abrogated the effects of TβRIII/TGFβ1-mediated inhibition of proliferation in L6 myoblasts and in primary human epithelial cells. Importantly, treatment with the TβRI and p38 inhibitors together had additive effects on abrogating TβRIII/TGFβ1-mediated inhibition of proliferation. In a reciprocal manner, short hairpin RNA-mediated knockdown of endogenous TβRIII in various human epithelial cells attenuated TGFβ1-mediated inhibition of proliferation. Taken together, these data demonstrate that TβRIII contributes to and enhances TGFβ-mediated growth inhibition through both TβRI/Smad3-dependent and p38 mitogen-activated protein kinase pathways.

Abbreviations: GIPC, GAIP-interacting protein, C-terminus; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HMEC, human mammary epithelial cell; MAPK, mitogen-activated protein kinase; NOSE, normal ovarian surface epithelial; PI3K, phosphatidylinositol 3-kinase; shRNA, short hairpin RNA; TGFβ, transforming growth factor β; TβRI, type I transforming growth factor β receptor; TβRII, type II transforming growth factor β receptor; TβRIII, type III transforming growth factor β receptor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
The transforming growth factor β (TGFβ) superfamily is composed of homodimeric polypeptide growth factors, including TGFβ isoforms, bone morphogenetic proteins, growth and differentiation factors, activins and inhibins. The TGFβ signaling pathway has essential roles in regulating many cellular responses including proliferation, differentiation, migration and apoptosis (1–5). There are three TGFβ isoforms, TGFβ1, TGFβ2 and TGFβ3, which are encoded by distinct genes and expressed in both a tissue-specific and a developmentally regulated manner. TGFβ exerts its biological function through binding to three high-affinity cell-surface receptors, the TGFβ type I, type II and type III receptors (TβRI or ALK-5, TβRII and TβRIII or betaglycan, respectively). TβRI and TβRII contain serine/threonine protein kinases in their cytoplasmic domains. Upon ligand binding, TβRII recruits and phosphorylates TβRI, activating its kinase activity to initiate intracellular signaling. TβRI signals by directly phosphorylating Smad2 and Smad3, which form a complex with the common mediator Smad4, translocate to nucleus and regulate gene transcription (6,7). While Smad-dependent pathways represent a major mechanism for TGFβ signaling, crosstalk and signaling through Smad-independent pathways, including the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways has been reported (8–14). However, the mechanism by which TGFβ activates Smad-independent pathways including MAPK pathways remains unclear.

TβRIII is the most abundant TGFβ receptor and is traditionally thought to function as a co-receptor, binding TGFβ and presenting it to TβRII (15). This is particularly important for the TGFβ2 isoform, which cannot bind TβRII independently. In addition, several studies support essential, non-redundant roles for TβRIII in mediating TGFβ sensitivity in intestinal goblet cells (16) in the mesenchymal transformation of chick embryonic heart development (17) and for mouse embryonic development (18). We recently reported the frequent loss of TβRIII expression in human breast (19), prostate (20) and ovarian cancers (21), with loss of expression correlating with disease progression and re-expression studies establishing a direct role for TβRIII in regulating cancer cell migration and invasion in vitro and angiogenesis and metastasis in vivo.

TβRIII has a short cytoplasmic domain without kinase activity. While this cytoplasmic domain is not essential for mediating the presentation role, it does contribute to TβRIII/TGFβ-mediated inhibition of proliferation (22). We also established specific functions of the cytoplasmic domain including mediating interaction with GAIP-interacting protein, C-terminus (GIPC), a PDZ domain-containing protein, to stabilize TβRIII on the cell surface and enhance TGFβ signaling (22), and phosphorylation by TβRII, which serves to recruit the scaffolding protein β-arrestin2, mediate the co-internalization of TβRII and TβRIII and down-regulate TGFβ signaling (23). Here, we explore the mechanism by which TβRIII enhances TGFβ-mediated inhibition of proliferation.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Materials and plasmids
Dulbecco's modified Eagle's medium was purchased from Invitrogen Co. (Carlsbad, CA), and defined fetal bovine serum was from Hyclone (Logan, UT). Fugene 6 was from Roche Molecular Biochemicals and SB203580, LY294002, SP600125 and PD98059 were purchased from Calbiochem (La Jolla, CA). SB431542 was from Sigma (St Louis, MO). TGFβ1 and TGFβ2 were from R&D systems (Minneapolis, MN). G418 was from Calbiochem. Mouse monoclonal antibody specific for Smad2, rabbit polyclonal antibodies specific for phospho-Smad2, phosphor-Smad3, p38, phosphor-p38 and horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from Cell Signaling Technology (Beverly, MA) and rabbit polyclonal antibody against Smad3 was from Calbiochem. Alexa488-conjugated anti-mouse or CyTM3-conjugated anti-rabbit secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). pcDNA3-Flag-p38{alpha} (AF) was generously provided by Dr Han at Scripps Institute (CA).

Cell culture and adenoviral infection
Rat L6 myoblasts (CRL-2256) and HaCaT cells were obtained from the American Type Culture Collection. L6 myoblasts and HaCaT cells were maintained as monolayers in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum. L6-Neo, L6-TβRIII and L6-TβRIII-Cyto stable cell lines were selected in 0.6 mg/ml G418 and maintained in growth media containing 0.3 mg/ml G418 as described previously (23,24). Normal human mammary epithelial cell (HMEC) strains 1001-15 and 1001-16 were purchased from Clonetics (San Diego, CA). HMECs were grown in mammary epithelial cell basal medium (Clonetics) supplemented with 4 µg/ml bovine pituitary extract, 5 µg/ml insulin (Sigma), 20 ng/ml epidermal growth factor (UBI, Lake Placid, NY) and 0.5 µg/ml hydrocortisone (Sigma) as described previously (25). Normal ovarian surface epithelial (NOSE) cells were generously provided by Dr Andrew Berchuck and grown in RPMI1640 supplemented with 10% fetal bovine serum. Adenoviral infection of short hairpin RNA (shRNA) of TβRIII was performed by plating cells, adding virus as indicated and incubating for 2 days for experiments. All cells were grown in 5% CO2 at 37°C in a humidified atmosphere.

Binding and cross-linking assay
Radioligand binding and cross-linking of 125I-TGFβ1 to TGFβ receptors in each cell lines were performed by incubating subconfluent cells with KRH buffer [50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, 130 mM NaCl, 5 mM MgSO4, 1 mM CaCl2 and 5 mM KCl] containing 0.5% bovine serum albumin for 30 min at 37°C, and then with 100 pM 125I-TGFβ1 for 3 h at 4°C. 125I-TGFβ1 was cross-linked with 0.5 mg/ml disuccinimidyl suberate and quenched with 20 mM glycine. Cells were then washed with KRH buffer, lysed in RIPA buffer, immunoprecipitated with the indicated antibodies and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and phosphorimaging analysis of dried gels.

Transcription reporter luciferase assay
Cells (3 x 104) were grown in a 24-well plate and transfected with a p3TP vector containing the luciferase gene under the regulation of a promoter based on the TGFβ-inducible promoter (a part of the PAI-1 promoter), pE2.1 vector containing the luciferase gene under 36 bp-pE2.1 element of PAI-1 gene (6,26,27) or pARE-FAST-1 (13) and the pRL-SV40 vector (Promega) expressing Renillar luciferase under the control of SV40 promoter to control for transfection efficiency using Fugene 6. Besides those reporter plasmids, some cells were transfected with pcDNA3, pcDNA3-Flag-p38{alpha} (WT), pcDNA3-Flag-p38{alpha} (AF) (28), pcDNA3-Flag-Smad3 or pcDNA3-Flag-Smad3DE (29). After 24 h, the cells were incubated with or without TGFβ1 (100 pM) or TGFβ2 (200 pM) for 20 h before harvest. Luciferase activities were measured in a multi-label counter (PerkinElmer) using the dual-luciferase reporter assay system (Promega).

[3H]thymidine incorporation assay
Cells (3 x 104 cells per ml) were grown in 96-well plates. After 24 h, cells were incubated with or without 0–1000 pM TGFβ1 for additional 24 h before harvest. Then, 10 µCi/ml of [3H]thymidine (Amersham Bioscience) was added during the last 4 h of incubation. The incorporation of acid-insoluble [3H]thymidine was measured by liquid scintillation counting. Growth inhibition was calculated as the ratio of radioactivity with TGFβ treatment to radioactivity in the absence of TGFβ treatment.

Immunofluorescence
To localize Smads, cells were plated on coverslips and grown for 48 h. Then, cells were treated with TGFβ1 for 40 min. The reaction was stopped with cold phosphate-buffered saline. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 5% bovine serum albumin/phosphate-buffered saline, followed by incubation with anti-Smad2 or anti-Smad3 antibodies (1:200) at room temperature for 2 h. Thereafter, they were washed, incubated with Alexa488-conjugated anti-mouse or CyTM3-conjugated anti-rabbit antibodies (1:200), washed, mounted on glass slides and sealed. Images were obtained with a Nikon eclipse Te2000-U fluorescence microscope and analyzed.

Subcellular fractionation
To assess the localization of Smads, protein extract was prepared as followed (30). Cells (5 x 105) were grown in a 60 mm dish for 48 h and treated with TGFβ (100 pM) for 40 min. Then, the cells were washed with phosphate-buffered saline and re-suspended in eight packed cell volumes of hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM phenylmethylsulfonylfluoride and 0.5 mM dithiothreitol). Cells were allowed to swell for 10 min on and transferred to a Dounce homogenizer for processing with a type B pestle. Nuclei were collected from ruptured cells by centrifugation for 15 min at 4000 r.p.m. and suspended in one-half packed pellet volume of cold, low-salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 0.02 M KCl, 0.2 mM ethylenediaminetetraacetic acid, 0.2 mM phenylmethylsulfonylfluoride and 0.5 mM dithiothreitol). High-salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 1.2 M KCl, 0.2 mM phenylmethylsulfonylfluoride and 0.5 mM dithiothreirol) equal to one-half packed pellet volume was added, and the nuclei were further extracted with gentle rocking for 30 min at 4°C. Nuclei were collected by centrifugation for 30 min at 14 500 r.p.m., dialyzed and applied to biochemical assays.

Western blotting
Protein samples were heated to 95°C for 5 min and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 8 or 10% acrylamide gels, followed by transfer to polyvinylidene difluoride membranes for 1 h at 350 mA with a NOVEX semi transfer unit. Membranes were then blocked for 1 h in Tris-buffered saline with 0.01% Tween 20 with 5% non-fat dried milk, after which they were incubated for 2 h with primary antibody in Tris-buffered saline with 0.01% Tween 20 with 2% bovine serum albumin, followed by 1 h with horseradish peroxidase-conjugated anti-mouse or rabbit antibody. The blots were developed with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech), and quantification of band intensity on XAR-5 film (Eastman Kodak Co.) was measured by Quantity One software (Bio-Rad, Chicago, IL).

shRNA-mediated silencing of human TβRIII in human epithelial cells
TβRIII knockdown was achieved by shRNA-mediated silencing of TβRIII via adenoviral transduction. Briefly, human TβRIII small interfering RNAs were designed and purchased from Dharmacon (Lafayette, CO). A 66 bp oligonucleotide linker containing human TβRIII-specific sense and corresponding anti-sense sequences, flanking a 6 base hairpin, was generated and subcloned into pSiren-DNR-DsRed-Express, which was then utilized to prepare an adenoviral vector which expresses shRNA-targeting TβRIII (shRIII) (Clontech, Mountain View, CA). Adeno X-LP-shRIII or GFP adenovirus was produced in the Phoenix cell packaging line. For shRNA knockdown, HaCaT cells, HMECs and NOSE007 cells were infected with Adeno-X-shRIII or control GFP adenoviruses, the selective knockdown of TβRIII verified by 125I-TGFβ1 binding and cross-linking and reverse transcription–polymerase chain reaction.

Statistical analysis
All data are expressed as percentages of control and shown as means ± SDs. Statistical comparisons between groups were made using Student's t-tests. Values of P < 0.01 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
TβRIII increases TGFβ1-induced growth inhibition
We previously demonstrated that TβRIII-enhanced growth inhibition in TGFβ2-stimulated L6 myoblasts and that the cytoplasmic domain of TβRIII contributed to this response (22,24). As TGFβ2 does not bind TβRII independently and thus is more dependent on TβRIII for signaling (31,32), to establish a broader role for TβRIII and to identify the signaling mechanism by which TβRIII mediates TGFβ-induced growth inhibition, we examined TGFβ1-mediated growth inhibition in the L6 myoblast model system. L6 myoblasts express no TβRIII, while expressing TβRII and TβRI, and are minimally responsive to TGFβ1-mediated growth inhibition. While empty vector-transfected and selected L6-Neo cells expressed no TβRIII, TβRIII and TβRIII-Cyto (TβRIII lacking a cytoplasmic domain) expression vector-transfected and selected L6-TβRIII and L6-TβRIII-Cyto cells expressed TβRIII and TβRIII-Cyto, respectively, at comparable levels (Figure 1A). These stable cell lines consist of pools of individual clones, minimizing clonal variability. TGFβ1 decreased [3H]thymidine incorporation in L6-TβRIII cells with up to 60% inhibition at 32 h relative to untreated L6-TβRIII cells (Figure 1B). In contrast, TGFβ1 either modestly stimulated or inhibited thymidine incorporation in L6-Neo cells by <20% (Figure 1B). To investigate the role of cytoplasmic domain of TβRIII in TGFβ1-mediated growth regulation, we examined TGFβ1-mediated growth inhibition in L6-Neo, L6-TβRIII and L6-TβRIII-Cyto cells over a dose range of TGFβ1 from 0 to 1000 pM (Figure 1C and D). In L6-TβRIII cells, TGFβ1 induced growth inhibition beginning at 10 pM and reaching a maximum of 50% inhibition at 100 pM TGFβ1. In contrast, in L6-TβRIII-Cyto cells, the effects of TGFβ1 were markedly abrogated, with <20% inhibition at 100 pM TGFβ1. The significant difference in TGFβ1-mediated growth inhibition between L6-TβRIII and L6-TβRIII-Cyto cells was maintained from 50 to 1000 pM of TGFβ1. In addition, TβRIII restored TGFβ2-induced growth inhibition, in a cytoplasmic domain-dependent manner (Figure 1E), consistent with our previous studies (24). These results demonstrate that TβRIII has an important role in TGFβ1 as well as TGFβ2-mediated growth regulation, mediated, in part, through its cytoplasmic domain.


Figure 1
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  		Fig. 1. TβRIII increases TGFβ1-mediated growth inhibition in L6 myoblasts. (A) The indicated cells were plated on a six-well plate, grown for 48 h in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and then subjected to 125I-TGFβ1-binding and cross-linking. Both total cell lysate and immunoprecipitates with HA antibody were analyzed. (B–E) Cells were plated on a 96-well plate and grown for 24 h in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, after which cells were treated with 100 pM TGFβ1 for the indicated times (B) or 24 h with 0–1000 pM of TGFβ1 (C). The effect over the 0–200 pM dose range of TGFβ1 is shown in (D). (E) Cells on a 96-well plate were treated with 100 pM of TGFβ1 or 200 pM of TGFβ2 for 24 h. [3H]thymidine was added 4 h prior to harvest, and the incorporation of acid-insoluble [3H]thymidine was assessed. Growth inhibition was calculated as the ratio of radioactivity with TGFβ1 treatment to radioactivity in the absence of TGFβ treatment. Data are expressed as means ± SDs of at least three independent experiments. Statistical significance was assessed using unpaired Student’s t-tests (*P < 0.005). All results shown are representative of at least three independent experiments.

 
TβRIII enhances Smad3-dependent signaling
TβRIII is traditionally thought to increase TGFβ signaling through enhancing ligand binding to TβRII, i.e. ligand presentation (15). However, we had previously reported and confirmed here (Figure 1A) that TβRIII-Cyto was indistinguishable from TβRIII in terms of TGFβ1 ligand presentation to TβRII (24). In addition, TβRIII did not shift the TGFβ1 dose response relative to TβRIII-Cyto, with maximal inhibition at 100 pM TGFβ1 in both cell lines (Figure 1C and D), suggesting that the effects of TβRIII on TGFβ1-mediated inhibition of proliferation were independent of ligand presentation effects.

TGFβ1 activates TβRI by forming ternary complex of TβRII with TβRI followed by transphosphorylation. Activated TβRI then phosphorylates Smad2 and Smad3, which accumulate in the nucleus with Smad4 to activate gene expression. To define the mechanism by which TβRIII enhances TGFβ1-mediated growth inhibition in L6 myoblasts, we examined the activation of Smad2 and Smad3 in TGFβ1-stimulated L6-Neo, L6-TβRIII and L6-TβRIII-Cyto cell lines. While TGFβ1-induced Smad2 phosphorylation was modestly decreased in the L6-TβRIII and L6-TβRIII-Cyto cell lines relative to the L6-Neo cell line, TGFβ1-induced Smad3 phosphorylation was consistently increased in the L6-TβRIII and L6-TβRIII-Cyto cell relative to the L6-Neo cell line (Figure 2A).


Figure 2
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  		Fig. 2. TβRIII increases Smad3 activation in TGFβ1-stimulated L6 myoblasts. (A) Subconfluent cells were treated with TGFβ1 (100 pM) for indicated times and lysed. Phosphorylation of Smad2 and Smad3 was analyzed by western blotting with phospho-specific Smad2 and Smad3 antibodies and total Smad2 and Smad3 levels determined by Smad2 and Smad3 antibodies. (B and C) Subconfluent cells were plated on coverslips and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. After 24 h, cells were treated with TGFβ1 (100 pM) for 40 min, fixed with 4% paraformaldehyde and labeled with anti-Smad2 (B, green) or anti-Smad3 (C, red) antibodies. Fluorescence-labeled proteins were visualized by fluorescence microscopy. (D) Subconfluent cells were exposed to TGFβ1 (100 pM) for 40 min and lysed. Nuclear fraction (Nu) and cytosolic fraction (Cy) were prepared as described in Materials and Methods and localization of Smads was analyzed by western blotting. Quantification of band intensity in each fraction was carried out using Quantity One software (Bio-Rad) and expressed as the relative ratio (%) in each set (E). The results shown are representative of at least three independent experiments. Statistical significance of differences was assessed using unpaired Student’s t-tests (*P < 0.05).

 
To assess whether these differences in Smad phosphorylation were reflected by differential nuclear accumulation of Smad2 and Smad3, we examined nuclear accumulation by both immunofluorescence microscopy and cell fractionation studies. While TGFβ1 induced Smad2 (Figure 2B) and Smad3 (Figure 2C) nuclear accumulation in both L6-Neo and L6-TβRIII cell lines, TβRIII expression increased both basal and TGFβ1-induced nuclear localization of Smad3 (Figure 2C). To quantify these effects, we performed cell fractionation studies. In unstimulated L6-Neo cells, most Smad3 was located in cytoplasm and TGFβ1 induced nuclear accumulation of ~13% of cellular Smad3. In unstimulated L6-TβRIII and L6-TβRIII-Cyto cell lines, 8.7 and 23.7% of cellular Smad3 was localized in the nucleus under basal conditions, respectively, and this was further increased by TGFβ1 treatment to 33.2 and 41.0%, respectively (Figure 2D and E). Taken together, these results support a role for TβRIII in specifically increasing Smad3 phosphorylation and nuclear accumulation. Interestingly, as enhanced phosphorylation and nuclear accumulation of Smad3 was also observed in L6-TβRIII-Cyto cells, these studies suggest that cytoplasmic domain of TβRIII is not essential for these effects.

To further investigate the role of TβRIII in Smad3-dependent transcriptional responses, we investigated the activation of the Smad3-dependent reporter, pE2.1-luciferase (26) and a Smad3- and AP1-dependent reporter, 3TP-luciferase (7,27,33–35) in these stable cell lines. Consistent with differences in Smad3 phosphorylation and nuclear accumulation, TβRIII expression in the L6-TβRIII cells increased Smad3-dependent transcriptional responses of both the pE2.1 (Figure 3A) and 3TP-luciferase (Figure 3B) reporters, with a slightly diminished effect in the L6-TβRIII-Cyto cell line. To confirm the role of Smad3 and its activity in TβRIII-mediated enhanced transcriptional activation in response to TGFβ, a dominant-negative mutant of Smad3 (Smad3DE) which cannot be phosphorylated by TβRI (29) was introduced L6-Neo, L6-TβRIII and L6-TβRIII-Cyto cells. Although expression of Smad3DE reduced 3TP induction in response to TGFβ1 and TGFβ2, this diminished activation was most pronounced in the L6-TβRIII-Cyto cells (Figure 3D), further suggesting that the cytoplasmic domain was not involved in TβRIII-mediated Smad3 signaling. To determine whether these effects were specific to Smad3, we also examined activation of the Smad2-dependent reporter, pARE with its co-activator, FAST-1 (13,36,37). In L6-Neo cells, TGFβ only modestly induced transcriptional activation of pARE with a maximum of 2-fold induction, and expression of TβRIII did not significantly alter TGFβ1-mediated pARE transcriptional activation (Figure 3C). Taken together, the ability of TβRIII to specifically increase Smad3 (and not Smad2) phosphorylation, nuclear accumulation and Smad3-dependent transcriptional responses suggest that TβRIII contributes to TGFβ-mediated signaling by enhancing Smad3 activation. The ability of TβRIII-Cyto to mediate these effects and for Smad3DE to potently block TβRIII-Cyto/TGFβ-mediated gene induction also suggests that the effects of TβRIII on Smad3 signaling are largely independent of its cytoplasmic domain.


Figure 3
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  		Fig. 3. TβRIII enhances Smad-dependent promoter activation. Cells were plated on 24-well plate and grown for 24 h in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, after which cells were transfected with pE2.1 (A), p3TP (B), ARE-FAST-1 (C), p3TP with pcDNA3 or pcDNA3-Flag-Smad3DE (D) and pRL-SV40 using Fugene 6. After 24 h, cells were exposed to 100 pM TGFβ1 for additional 20 h and subjected to dual-luciferase activity measurement. Data are expressed as means ± SDs of at least three independent experiments. Statistical significance of differences was assessed using unpaired Student's t-tests (*P < 0.01).

 
p38 kinase and TβRI inhibitors reverse TβRIII-mediated increases in TGFβ1-induced growth inhibition
Although TβRIII enhanced Smad3 activation and Smad3-dependent transcriptional responses, these differences were not enough to explain the enhanced TGFβ1-induced inhibition in L6-TβRIII. Furthermore, TβRIII-Cyto was also effective in Smad3 activation and Smad3-dependent transcriptional responses, while not able to mediate the effects of TβRIII on inhibition of proliferation. These observations suggested that Smad3-dependent signaling may not be wholly responsible for the effects of TβRIII on inhibition of proliferation. Accordingly, we investigated the role of both Smad-dependent and Smad-independent signaling pathways in TGFβ1–TβRIII-induced growth inhibition. TGFβ1 has been demonstrated to signal through several Smad-independent signaling pathways, including the MAPK and PI3K/Akt pathways (8,9,11,13,14). We therefore examined the effect of PD98059, a mitogen-activated protein kinase kinase inhibitor; SB203580, a p38 kinase inhibitor; SP600125, a c-Jun N-terminal kinase inhibitor; LY294002, a PI3K inhibitor as well as SB431542 (38,39), a TβRI inhibitor on TGFβ1–TβRIII-mediated inhibition of proliferation (Figure 4). While PD98059 and LY294002 slightly enhanced TGFβ1-mediated inhibition of thymidine incorporation to ~80% (Figure 4A and B) and SP600125 had no significant effect (Figure 4C), SB20358 significantly abrogated the effect of TβRIII such that only 30% inhibition of proliferation was observed in the L6-TβRIII cell line (Figure 4D). In addition, SB431542 also abrogated the effect of TβRIII, but to a lesser extent, with ~40% inhibition of proliferation observed in the L6-TβRIII cell line (Figure 4E). We also examined the effects of these inhibitors on TβRIII/TGFβ2-mediated growth inhibition. While PD98059 and SP600125 had no significant effect, SB203580 and SB431542 also abrogated TβRIII/TGFβ2-mediated growth inhibition (Figure 4F), suggesting a role for both the p38 and TβRI pathways in TβRIII-mediated growth inhibition in response to TGFβ1 and TGFβ2.


Figure 4
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  		Fig. 4. SB203580, a p38 kinase inhibitor, and SB431542, an ALK-5 inhibitor, attenuate TGFβ1–TβRIII-mediated growth inhibition in L6 myoblasts. (A–E) Cells were plated on a 96-well plate and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. After 24 h, cells were treated with 100 pM of TGFβ1 in the presence of the indicated amounts of dimethyl sulfoxide (DMSO), PD98059, a mitogen-activated protein kinase kinase inhibitor (A), LY294002, a PI3K inhibitor (B), SP600125, a c-Jun N-terminal kinase inhibitor (C), SB203580, a p38 kinase inhibitor (D) or SB431542, an ALK-5 inhibitor (E) and incubated for additional 24 h. Inhibitors were added 30 min prior to TGFβ1 treatment. Some cells were treated with 200 pM of TGFβ2 (F) in the presence of DMSO, SB431542 (0.5 µM), SB203580 (5 µM), PD98059 (10 µM) or SP600125 (10 µM) for additional 24 h. [3H]thymidine was added 4 h prior to harvest and the incorporation of acid-insoluble [3H]thymidine was assessed. Growth inhibition was calculated as the ratio of radioactivity with TGFβ1 treatment to radioactivity in the absence of TGFβ treatment. Data are expressed as means ± SDs of at least three independent experiments. Statistical significance was assessed using unpaired Student’s t-tests (*P < 0.005).

 
The p38 and TβRI inhibitors used have the potential to inhibit other kinases, including the potential for the p38 inhibitor to inhibit TβRI and vice versa (38,39). In addition, p38 kinase has been reported to mediate TGFβ1-induced apoptosis independently of Smads (11) and to phosphorylate Smads2/3 in their linker region (10). Therefore, to assess the specificity of the inhibitors used and whether there is any crosstalk between p38 kinase and Smad-dependent signaling pathways in TGFβ1–TβRIII-mediated inhibition of proliferation, we examined the effect of SB431542 in TGF-β1-induced p38 kinase activation and the effect of SB203580 on TGFβ1-induced Smad phosphorylation. SB431542 did not affect TGFβ1-mediated p38 kinase phosphorylation in L6-TβRIII cells (Figure 5A). In addition, SB203580 did not inhibit Smad2 or Smad3 phosphorylation in the L6-Neo, L6-TβRIII or L6-TβRIII-Cyto cell lines, whereas SB431542 effectively inhibited but did not abrogate Smad2 and Smad3 phosphorylation in these cell lines (Figure 5B). These results demonstrate that, at the doses used, the SB203580 and SB431542 were specific for their respective pathways. Whether there was any crosstalk between p38 kinase and Smad-dependent pathways in L6 myoblasts was further confirmed by investigating the effect of wild-type (p38{alpha}-WT) or dominant-negative mutant (p38{alpha}-AF) of p38{alpha} on TGFβ1- or TGFβ2–TβRIII-mediated gene induction in L6-TβRIII cells. Expression of either p38{alpha}-WT or p38{alpha}-AF did not alter TGFβ-induced 3TP induction (Figure 5C), suggesting that p38 kinase does not significantly crosstalk with TGFβ-induced Smad-dependent transcriptional activation in this system.


Figure 5
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  		Fig. 5. The p38 kinase and TβRI pathways do not significantly crosstalk in L6 myoblasts. (A) Subconfluent cells were treated with TGFβ1 (100 pM) for 30 min in the presence of dimethyl sulfoxide (DMSO), SB431542 (0.5 µM) or SB203580 (5 µM) and lysed. Phosphorylation of Smad2 and Smad3 was analyzed by western blotting. The results shown are representative of at least three independent experiments. (B) Subconfluent cells were treated with TGFβ1 (10 pM) for 2 h in the presence of DMSO or SB431542 (0.5 µM). Inhibitors were added 30 min prior to TGFβ1 treatment. Phosphorylation of p38 kinase was analyzed by western blotting with a p38 phospho-specific antibody. The results shown are representative of at least three independent experiments. (C) L6-TβRIII cells on 24-well plate were transfected with p3TP and pRL-SV40 with pcDNA3, pcDNA3-Flag-p38{alpha} (WT) or pcDNA3-Flag-p38{alpha} (AF). After 24 h, cells were treated with TGFβ1 (100 pM) or TGFβ2 (200 pM) for additional 20 h and subjected to dual-luciferase activity (DLA) measurement. Data are expressed as means ± SDs of at least three independent experiments. Statistical significance was assessed using unpaired Student’s t-tests (*P < 0.01).

 
TβRIII increases p38 kinase phosphorylation
As the inhibitor studies suggested that the p38 kinase pathway may have a significant role in TGFβ1–TβRIII-mediated inhibition of proliferation, we investigated whether TβRIII expression altered p38 kinase phosphorylation and whether the cytoplasmic domain of TβRIII contributed to p38 kinase activation. In L6-Neo cells, TGFβ1 phosphorylated p38 kinase in a time-dependent fashion from 0.5 to 2 h (Figure 6A), and this activation was increased in L6-TβRIII cells. TGFβ1 also induced p38 kinase phosphorylation in L6-TβRIII-Cyto cells; however, this activation was always less robust than in L6-TβRIII cells, suggesting a role for the TβRIII cytoplasmic domain in TGFβ1-mediated p38 kinase activation. Taken together, these data provide support for a role for p38 kinase in mediating at least a portion of TβRIII/TGFβ1-mediated inhibition of proliferation.


Figure 6
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  		Fig. 6. TGFβ1–TβRIII-mediated growth inhibition is mediated both by p38 kinase and TβRI pathways. (A) Subconfluent L6 myoblasts were treated with TGFβ1 (10 pM) for indicated times and lysed. Phosphorylation of p38 kinase was analyzed by western blotting with a p38 phos-phospecific antibody. The results shown are representative of at least three independent experiments. (B) The effects of SB203580 (2 µM) and SB431542 (0.2 µM for L6 myoblasts and 0.4 µM for HMECs) were assessed in TGFβ1 (100 pM for L6 myoblasts and 20 pM for HMECs)-mediated growth inhibition by measuring incorporation of [3H]thymidine in L6-Neo, L6-TβRIII (left panel) and HMECs (right panel). Data are expressed as means ± SDs of at least three independent experiments. Statistical significance was assessed using unpaired Student's t-tests (*P < 0.005). The results shown are representative of at least three independent experiments. (C) HaCaT (left top), HMEC (left bottom) or NOSE007 cells (right) were plated on a 96-well plate and grown in growth media. After 24 h, cells were infected with shRIII or GFP adenovirus. After 48 h, cells were treated with TGFβ1 (20 pM for HaCaT, HMEC; 100 pM for NOSE007) for 24 h. [3H]thymidine was added 4 h prior to harvest and the incorporation of acid-insoluble [3H]thymidine was assessed. Growth inhibition was calculated as the ratio of radioactivity with TGFβ1 treatment to radioactivity in the absence of TGFβ treatment. Data are expressed as means ± SDs of at least three independent experiments. Statistical significance was assessed using unpaired Student's t-tests (*P < 0.005). Cell-surface expression of TβRIII was demonstrated by binding and cross-linking assay (each panel, right). The results shown are representative of at least three independent experiments.

 
p38 kinase and TβRI-mediated signaling mediate TGFβ1-induced growth inhibition
The present results suggested a role for both a p38/Smad-independent pathway and the TβRI/Smad3-dependent pathway in TGFβ1–TβRIII-mediated enhanced growth inhibition. To determine whether both pathways were involved, we investigated the effect of p38 kinase and TβRI inhibition on TGFβ1–TβRIII-mediated growth inhibition. When cells were exposed to either SB203580 or SB431542 at submaximal concentrations (2 and 0.2 µM, respectively), TGFβ1-mediated growth inhibition was only partially inhibited in L6-TβRIII cells (Figure 6B, left). In contrast, exposure to SB203580 and SB431542 together completely abrogated TGFβ1-mediated growth inhibition in L6-TβRIII cells (Figure 6B, left). These data suggest that the p38 kinase and TβRI-mediated Smad3-dependent signaling pathways work in concert in TβRIII-mediated growth inhibition.

TβRIII/TGFβ1-induced inhibition of proliferation and growth arrest in human keratinocytes, mammary epithelial cells and ovarian surface epithelial cells
To investigate the relevance and broader significance of TβRIII in TGFβ1-induced growth inhibition, we initially tested several normal epithelial cell lines and demonstrated that human keratinocytes (HaCaT cell line) and HMECs were quite sensitive to TGFβ1-mediated growth inhibition (up to 90%). In addition, ovarian surface epithelial cells (NOSE007 cell line) were sensitive to TGFβ1-mediated growth inhibition (up to 50%). The endogenous expression of TβRIII in those cells was verified by reverse transcription–polymerase chain reaction (data not shown) and binding and cross-linking assays (Figure 6C).

To provide direct support for TβRIII in contributing to inhibition of proliferation in these cell lines, we used shRNA to TβRIII to knock down TβRIII expression in these cell lines. In HaCaT cells, shRNA to TβRIII decreased TβRIII expression by ~80% (Figure 6C, left top). This decrease in TβRIII expression was accompanied by partial but significant attenuation of TGFβ-mediated inhibition of proliferation, suggesting that TβRIII was responsible, at least in part, for mediating the effects of TGFβ1 on inhibition of proliferation in HaCaT cells (Figure 6C, left top). Similarly, TGFβ-induced growth inhibition was partially but consistently attenuated when TβRIII expression was decreased in HMECs (Figure 6C, left bottom) and in NOSE007 cells (Figure 6C, right).

As these results suggested a contribution of TβRIII to TGFβ-mediated inhibition of proliferation in a broader population of epithelial cell types, we investigated whether the pathways operative in L6 myoblasts were being utilized in these cell types. In HMECs and HaCaT cells, the ALK-5 inhibitor, SB431542 partially inhibited TGFβ-mediated inhibition of proliferation (Figure 6B, right and data not shown). While the p38 kinase inhibitor SB203580 had less potent effects in HMECs and in HaCaT cells, this inhibitor also partially inhibited TGFβ-mediated inhibition of proliferation (Figure 6B, right, data not shown). In addition, when HMECs were treated with both inhibitors at low doses (0.4 µM of SB431542 and 2 µM of SB203580, respectively), TGFβ-mediated growth inhibition was nearly abrogated, suggesting that both pathways were utilized in HMECs to mediate inhibition of proliferation. Taken together, these results support a role for TβRIII in TGFβ-mediated growth inhibition, through both p38 kinase and TβRI/Smad3-dependent signaling pathways.


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The TGFβ superfamily co-receptor, TβRIII/betaglycan, was the first TGFβ receptor cloned and characterized (40,41). However, the molecular domain structure of TβRIII including a short cytoplasmic domain with no clear signaling motifs (40,41) and reports of TβRIII-independent TGFβ signaling (42,43) suggested minor roles for TβRIII including ligand presentation (15). Although recent studies have suggested essential, non-redundant roles for TβRIII in embryonic development (17,18), TGFβ signaling (24) and carcinogenesis (19,20), how TβRIII mediates its effects remains unclear. We had previously reported an essential role for the cytoplasmic domain of TβRIII in TGFβ2-mediated inhibition of proliferation (24). Here, we establish a role for TβRIII in contributing to TGFβ1- and TGFβ2-mediated inhibition of proliferation, broadening the significance of TβRIII's role and raising important questions about TβRIII's function apart from ligand presentation. A presentation-independent role for TβRIII in TGFβ-mediated inhibition of proliferation is supported by (i) a contribution by the TβRIII cytoplasmic domain, which is dispensable for ligand presentation [(24), Figure 1A], (ii) the ability of TβRIII to enhance both TGFβ1- and TGFβ2-mediated inhibition of proliferation to a similar extent, even though TGFβ1 is not as dependent on TβRIII for presentation [(24), Figure 1B] and (iii) the ability of TβRIII to increase the maximum inhibition of proliferation without shifting the dose–response curve for TGFβ1 (Figure 1C and D). Instead, through examination of both Smad-dependent and Smad-independent signaling pathways, we present evidence supporting a role for Smad3, TβRI and the p38 MAPK pathway in mediating TβRIII's effects.

A role for Smad-dependent signaling in mediating some of TβRIII's effects is supported by (i) TβRIII-enhanced Smad3 (but not Smad2) phosphorylation and nuclear accumulation (Figure 2) and (ii) TβRIII-enhanced Smad3-dependent reporter gene transcription (Figure 3) and the ability of dominant-negative Smad3 and the TβRI inhibitor SB431542 to markedly abrogate the effects of TβRIII (Figures 3C and 4E). This current report adds to a growing body of literature distinguishing the functions and role of TGFβ signaling through Smad2 and Smad3. For example, Piek et al. (44) demonstrated different contributions of Smad2 and Smad3 in TGFβ1-mediated signaling, with TGFβ1-mediated induction of matrix metalloproteinase-2 selectively dependent on Smad2 and TGFβ1-mediated induction of c-fos, Smad7 and TGFβ1 selectively dependent on Smad3. More recently, Kim et al. (45) reported a specific role for Smad3 in mediating the cytostatic effects of TGFβ as RNAi-mediated knockdown of Smad3 abrogated TGFβ-mediated inhibition of proliferation, whereas RNAi-mediated knockdown of Smad2 enhanced TGFβ-mediated inhibition of proliferation. As TβRIII is ubiquitously expressed, the current studies suggest that TβRIII may selectively activate Smad3, with Smad3 having specific functions in regulating TGFβ-mediated inhibition of proliferation. Establishing the mechanism of Smad3-specific activation and the identification of TβRIII target genes are active areas of the current investigation.

A role for Smad-independent signaling in partly mediating TβRIII's effects is supported by TβRIII-enhanced p38 phosphorylation (Figure 6A), and the ability of the p38 inhibitor to markedly abrogate the effects of TβRIII (Figure 4D and F). How does TβRIII mediate signaling to the p38 MAPK pathway? Crosstalk between the Smad pathways and MAPK pathways does not appear to be involved, as the TβRI inhibitor had no effect on TGFβ1-induced p38 phosphorylation (Figure 5A), and expression of either p38{alpha} or p38{alpha}-AF did not affect TGFβ1- and TGFβ2-mediated 3TP induction (Figure 5C). Instead, the data support a role for the cytoplasmic domain of TβRIII. We have previously demonstrated that TβRIII is phosphorylated by active TβRII on its cytoplasmic domain (24). Although the full consequences of this phosphorylation remain to be elucidated, one consequence is the recruitment of β-arrestin2 and the subsequent down-regulation of TβRIII and TβRII expression and of TGFβ signaling (23). In addition, β-arrestin2 has been reported to scaffold interacting receptors to signaling pathways including MAPK pathways (46). Also, we have established that TβRIII interacts with the scaffolding protein GIPC (22), which stabilizes TβRIII on the cell surface and increases TGFβ signaling. The interaction of GIPC with TβRIII could potentially scaffold TβRIII to signaling pathways. Whether β-arrestin2 or GIPC has a role in TGFβ–TβRIII-mediated signaling to the p38 MAPK pathway is being investigated.

Crosstalk between signaling pathways is quite common and the TGFβ-signaling pathway is no exception. The p38 kinase has been reported to phosphorylate the linker region of Smad3, and this is important for the full transactivation potential of Smad3 (10). In the present studies, the p38 inhibitor, SB203580, had no effect on Smad2 or Smad3 phosphorylation at the C-terminus (Figure 5B), suggesting that although p38 kinase might be involved in TGFβ1-induced Smad3 activation, it does not crosstalk during TGFβ1–TβRIII-mediated growth inhibition. Smad3 can also be regulated by the PI3K/Akt pathway (8,9). However, in the present studies, the PI3K inhibitor, LY294002, as well as the mitogen-activated protein kinase kinase inhibitor, PD98059, did not abrograte the effects of TβRIII on TGFβ1-mediated growth inhibition (Figure 4A and B). Instead, each slightly enhanced TGFβ1-mediated growth inhibition, suggesting that TGFβ1–TβRIII-mediated activation of Smad3 and subsequent growth inhibition is not mediated through PI3K or ERK, but that these pathways may contribute in other ways to enhance TGFβ1-mediated growth inhibition. The kinase inhibitor studies suggest contributions of both TβRI and p38 in TGFβ1- and TGFβ2-mediated growth inhibition in L6 myoblasts and in HMECs. As TGFβ through TβRIII can stimulate Smad3 through both TβRI and p38, which appear to be necessary for the maximum effects of TβRIII on inhibition of proliferation, we are currently investigating how these two pathways crosstalk/integrate to mediate these effects on inhibition of proliferation.

Although the TGFβ-signaling pathway has defined roles in human carcinogenesis, initially as a tumor suppressor and then as a tumor promoter (4), only recently have studies demonstrated a role for TβRIII in the pathogenesis of human cancers. We and others have reported that loss of TβRIII expression through epigenetic silencing, loss of heterozygosity and transcriptional regulation occurs early in breast (19), kidney (47), ovarian (21) and prostate cancer (20), with loss correlating with disease progression. Functional studies in cancer cell lines and murine cancer models have defined a role for TβRIII in negatively regulating motility, invasion and tumor-associated angiogenesis (19,20). The current studies in non-transformed cells suggest another potential role for loss of TβRIII expression, namely release from TGFβ-mediated inhibition of proliferation early in cancer formation. Interestingly, although most cancers become resistant to TGFβ-mediated inhibition of proliferation, known defects in the pathway (i.e. in Smad2/3/4 and TβRII/TβRI) provide the mechanism for only a fraction of these cases. As TβRIII expression is frequently decreased or lost in human cancers (i.e. 65% of breast cancers and 60% of prostate cancers) and here we define decreased TβRIII expression as a mechanism for functional resistance to TGFβ-mediated inhibition of proliferation, decreased TβRIII expression could be one of the most common mechanisms for TGFβ resistance in human cancers. The role of TβRIII in regulating inhibition of proliferation in these and other cancers is being investigated.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Institute of Health/National Cancer Institute (R01-CA106307 [GenBank] ) to G.C.B.; Susan G. Komen Breast Cancer Foundation to H.J.Y.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received July 3, 2007; revised August 15, 2007; accepted August 17, 2007.


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