Transforming Growth Factor ␤ Regulates P-Body Formation through Induction of the mRNA Decay Factor Tristetraprolin Fernando F. Blanco,a Sandhya Sanduja,b Natasha G. Deane,c Perry J. Blackshear,d Dan A. Dixona ‹Department of Cancer Biology, University of Kansas Medical Center, Kansas City, Kansas, USAa; Whitehead Institute for Biomedical Sciences, Cambridge, Massachusetts, USAb; Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USAc; Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USAd

ransforming growth factor ␤ (TGF-␤) is a multifunctional cytokine that regulates the cellular proliferation, migration, differentiation, and apoptosis necessary for intestinal epithelium homeostasis (1). TGF-␤ primarily mediates these effects through serine-threonine kinase type I and II receptors (T␤RI and T␤RII, respectively), where ligand binding to T␤RII leads to recruitment and phosphorylation of T␤RI. This allows activation and phosphorylation of receptor-regulated Smads 2 and 3, which then combine with Smad4, and the combination translocates to the nucleus to drive the transcription of several target genes (2). In this context, TGF-␤ serves as a potent growth inhibitor in normal intestinal epithelial cells (1). However, through selective genetic alterations in the TGF-␤ signaling pathway during colorectal tumorigenesis, TGF-␤ serves as an oncogene by promoting the survival, invasion, and metastasis of colorectal cancer cells (2). Normal cellular growth is associated with the rapid decay of growth- and inflammation-associated mRNAs (3). A key feature present in these transcripts’ 3= untranslated region (UTR) is the adenylate- and uridylate (AU)-rich element (ARE) (4), and bioinformatic prediction models indicate that ⬃8 to 16% of the human transcriptome contains an ARE (5, 6). AREs mediate their regulatory function through association with multiple RNA-binding proteins that can promote mRNA decay, mRNA stabilization, and translational silencing (7). Among the best-characterized AREbinding proteins involved in promoting the decay of AREmRNAs is tristetraprolin (TTP; ZFP36 and TIS11). TTP is a member of a small family of tandem Cys3His zinc finger RNA-binding proteins, and various studies have established TTP as a central regulator of various inflammation and cancer-associated mRNAs (e.g., tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, COX-2, and vascular endothelial growth factor [VEGF]) by targeting these mRNAs for degradation (8, 9). Rapid decay of ARE-mRNAs is initiated by poly(A) tail shortening (deadenylation), followed by exosome-mediated 3=-to-5= degradation, or through decapping and subsequent 5=-to-3= exo-

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nucleolytic degradation (4, 10). In many cases, ARE-mRNAs targeted for rapid decay require the components necessary for 5=to-3= degradation and are localized to processing (P) bodies (11– 13). These small cytoplasmic foci contain the components of the 5=-to-3= decay machinery along with other factors involved in microRNA (miRNA)- and small interfering RNA (siRNA)-mediated silencing, nonsense-mediated decay, and translational silencing (14). P-body assembly is a dynamic process that is tightly linked with the availability of free cytoplasmic mRNAs that drives the aggregation of mRNA-protein complexes in the cytoplasm into microscopically visible P bodies (15, 16). While information identifying and characterizing P-body components in various species has demonstrated the role of P bodies in posttranscriptional regulation (17, 18), further insight is needed to understand how these dynamic structures regulate ARE-mRNA expression in response to relevant physiological signaling. Through activation of Smad-dependent and -independent pathways, TGF-␤ has been shown to promote expression of a large number of genes, many of which contain ARE motifs (19–21). While many of these ARE-containing genes can be considered growth promoting when overexpressed (3), it would appear that limiting their expression should be a necessary component for Smad-mediated growth inhibition. However, the mechanism by which TGF-␤ influences the levels of ARE-mRNAs is not understood. In this article, we describe the ability of TGF-␤/Smad signaling to promote enhanced P-body formation in intestinal epi-

Received 8 August 2013 Returned for modification 23 August 2013 Accepted 28 October 2013 Published ahead of print 4 November 2013 Address correspondence to Dan A. Dixon, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/MCB.01020-13

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Transforming growth factor ␤ (TGF-␤) is a potent growth regulator and tumor suppressor in normal intestinal epithelium. Likewise, epithelial cell growth is controlled by rapid decay of growth-related mRNAs mediated through 3= untranslated region (UTR) AU-rich element (ARE) motifs. We demonstrate that treatment of nontransformed intestinal epithelial cells with TGF-␤ inhibited ARE-mRNA expression. This effect of TGF-␤ was promoted through increased assembly of cytoplasmic RNA processing (P) bodies where ARE-mRNA localization was observed. P-body formation was dependent on TGF-␤/Smad signaling, as Smad3 deletion abrogated P-body formation. In concert with increased P-body formation, TGF-␤ induced expression of the ARE-binding protein tristetraprolin (TTP), which colocalized to P bodies. TTP expression was necessary for TGF-␤-dependent P-body formation and promoted growth inhibition by TGF-␤. The significance of this was observed in vivo, where colonic epithelium deficient in TGF-␤/Smad signaling or TTP expression showed attenuated P-body levels. These results provide new insight into TGF-␤’s antiproliferative properties and identify TGF-␤ as a novel mRNA stability regulator in intestinal epithelium through its ability to promote TTP expression and subsequent P-body formation.

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thelial cells and colonic tissue. This novel feature of TGF-␤ signaling occurs via induction of the RNA decay factor TTP that allows TTP-driven delivery and subsequent decay of ARE-mRNAs at P bodies. We attribute this role of TGF-␤ in the regulation of ARE-mediated mRNA decay to be a necessary component to its tumor-suppressive activity within the intestinal epithelium. MATERIALS AND METHODS

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Cell culture. RIE-1 rat intestinal epithelial cells were cultured as described previously (22). YAMC conditionally immortalized murine colonocytes and Smad3-deficient YAMC (YAMC⌬Smad3) cells were maintained under permissive conditions as described previously (23) at 33°C in RPMI 1640 medium containing 25 mM HEPES and 2 mM L-glutamine (Invitrogen, Carlsbad, CA) and supplemented with 5% fetal bovine serum (HyClone, Logan, UT), insulin, transferrin, and selenium (ITS; Sigma-Aldrich, St. Louis, MO), and 5 U/ml of murine gamma interferon (IFN-␥; Roche, Indianapolis, IN). Experiments using YAMC and YAMC⌬Smad3 cells were performed after 48 h of culture under nonpermissive conditions of 37°C without IFN-␥. Primary mouse embryonic fibroblast (MEF) cultures were established from embryonic day 14.5 (E14.5) of wild-type (C57BL/6, Zfp36⫹/⫹) and TTP-knockout (C57BL/6, Zfp36⫺/⫺) mice and cultured as described previously (24). All animal procedures were performed under a protocol approved by the IACUC of University of Kansas Medical Center (KUMC). Immunofluorescence microscopy. Cells grown on coverslips were washed twice with phosphate-buffered saline (PBS) containing 10 mM glycine and fixed in 2% paraformaldehyde for 15 min at room temperature (RT). Following fixation, cells were permeabilized with 0.02% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in PBS. Cells were blocked with 5% normal goat serum (Jackson ImmunoResearch, West Grove, PA) in PBS containing 1% IgG-free bovine serum albumin (BSA; Jackson ImmunoResearch, West Grove, PA) and incubated overnight at 4°C with primary antibody diluted in blocking solution. Hedls was detected using anti-Hedls polyclonal antibody (25). Dcp1a was detected using mouse monoclonal anti-Dcp1a antibody (ab57654; Abcam, Cambridge, MA), and TTP was detected using a rabbit polyclonal antibody against amino acids 19 to 33 of human TTP (GenScript, Piscataway, NJ). Ki67 staining was accomplished by using a rabbit monoclonal antibody to Ki67 (ab16667; Abcam, Cambridge, MA). Secondary antibody was diluted in blocking buffer (PBS containing 3% IgG-free BSA) and incubated for 1 h at RT, after which the cells were counterstained with DAPI (4=,6-diamidino-2-phenylindole). Hedls detection was done using fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (catalog no. FI-1200; Vector Laboratories, Burlingame, CA) at a dilution of 1:100 or DyLight 649-conjugated goat anti-rabbit IgG (catalog no. 111-495-003; Jackson ImmunoResearch, West Grove, PA) at a 1:400 dilution. Dcp1a was detected with Cy3-labeled donkey anti-mouse IgG (catalog no. 715-165150; Jackson ImmunoResearch, West Grove, PA) at a 1:400 dilution. TTP was detected with Texas Red-labeled horse anti-rabbit IgG (catalog no. FI-2100; Vector Laboratories) at a dilution of 1:100. Ki67 was detected with FITC-labeled horse anti-rabbit IgG (catalog no. FI-2100; Vector Laboratories) at a dilution of 1:100. Visualization of P bodies in murine colonic tissue was accomplished using colons obtained from 16-week-old Smad3⫺/⫺ and 6-week-old TTP⫺/⫺ mice, along with respective wild-type littermates. For detection in Smad3⫺/⫺ mouse colonic tissue, the large intestine was extracted, flushed with saline, cut longitudinally, and then fixed in 10% buffered formalin for 24 h and paraffin embedded. Colon sections (4 ␮m) were deparaffinized in xylene for 5 min and rehydrated in ethanol. Slides were then subjected to antigen retrieval in citrate buffer in a steam bath for 30 min, blocked in 10% normal donkey serum in PBS-containing 1% IgGfree BSA, followed by a secondary blocking in 40 ␮g/ml AffiniPure Fab fragment goat anti-mouse antibody (catalog no. 115-007-003; Jackson ImmunoResearch, West Grove, PA), and incubated with anti-Dcp1a mouse monoclonal antibody (ab57654; Abcam, Cambridge, MA) as described

above. Fluorescence detection was accomplished by using a Cy3-labeled donkey anti-mouse IgG (catalog no. 715-165-150; Jackson ImmunoResearch, West Grove, PA) at a 1:400 dilution for 1 h at RT, and fluorescence was visualized by confocal microscopy. Immunohistochemical (IHC) analysis of Dcp1a and TTP expression was performed using mouse monoclonal anti-Dcp1a (ab57654; Abcam, Cambridge, MA) at a 1:1,000 dilution and TTP polyclonal antibody (ab33058; Abcam, Cambridge, MA) at a 1:200 dilution. Staining was performed as described previously (26). To visualize P bodies in TTP⫺/⫺ mouse colonic tissue, the large intestine was freshly harvested, flushed with saline, cut longitudinally, and snap-frozen in a dry ice-ethanol bath. The tissue was then sectioned in a cryostat at ⫺20°C at a 10-␮m thickness, after which the slides were incubated in 2% paraformaldehyde for 15 min. The sections were blocked in 10% normal donkey serum in PBS-containing 1% IgG-free BSA, followed by a secondary blocking in 40 ␮g/ml AffiniPure Fab fragment goat antimouse antibody. Sections were then incubated with anti-Dcp1a mouse monoclonal antibody as described above or anti-TTP polyclonal antibody (GenScript, Piscataway, NJ) at a dilution of 1:200 overnight at 4°C. Dcp1a was detected using Cy3-labeled donkey anti-mouse antibody at a dilution of 1:400. TTP was detected with DyLight 649-conjugated goat anti-rabbit IgG at a 1:400 dilution. DAPI was used to detect nuclei, and single-plane images were acquired by confocal microscopy. Epifluorescence microscopy was accomplished using a Zeiss Axiovert 200 microscope (Carl Zeiss, Inc.) equipped with a digital camera (Axiocam MRc5) at a ⫻63 oil objective (numerical aperture [NA], 1.4). Images were acquired using AxioVision (version 4.8.2) software (Carl Zeiss, Inc.) and imported into Adobe Photoshop (version 8.0) software. Confocal microscopy was done using a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Inc.) at a ⫻40 oil objective (NA, 1.3) at RT. The image input levels were adjusted with a histogram stretching to obtain the best visual reproduction, ensuring that linearity and the brightness scale were maintained. The images were exported into Adobe Illustrator software to compose the figures. To analyze changes in P-body number after TGF-␤ treatment, the number of transfected cells indicated below was blindly scored for P bodies by visual examination. P bodies with a pixel range for particle brightness of between 30 and 230 units, determined using ImageJ software (National Institutes of Health), were considered to have staining above the background level of staining and were selected for P-body analysis. Colocalization studies were done using the acquisition of single-plane images on a Zeiss LSM 510 Meta confocal microscope at RT, and the value of Pearson’s correlation coefficient was established using Zeiss LSM 510 Meta software. Senescence-associated ␤-galactosidase (SA-␤-Gal) detection and [3H]thymidine incorporation. Wild-type and TTP-knockout MEFs were cultured on the basis of a 3T3 protocol as described previously (27). Briefly, 3 ⫻ 105 cells at passage 2 were plated in 10-cm plates coated with 3% gelatin (Sigma-Aldrich, St. Louis, MO) in the presence or absence of 36 pg/ml TGF-␤ added twice per passage. Cells were trypsinized every 72 h and reseeded at the same density. At day 10, cells were fixed in 20% formaldehyde, 2% glutaraldehyde in PBS at RT and stained at pH 6.0 with X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside; Cell Signaling Technology, Danvers, MA). Staining was visualized using an Evos XL Core transmitted-light inverted microscope equipped with a high-sensitivity CMOS color camera. Images were captured at RT under ⫻20 magnification using a LPLAN PH2 objective lens (NA, 0.4). The image input levels were adjusted with a histogram stretching to obtain the best visual reproduction, ensuring that linearity and the brightness scale were maintained. The images were exported into Adobe Illustrator software to compose the figures. [3H]thymidine incorporation was performed in cultures of MEFs of the phenotypes indicated below, plated at a density of 20,000 cells per well in 6-well plates, and treated with 1.2 ng/ml TGF-␤ for 48 h. For the last 4 h of treatment, 5 ␮Ci of [3H]thymidine was added to the culture, and [3H]thymidine incorporation was assayed as previously described (28).

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ng/ml TGF-␤ for 24 h. Actinomycin D (ActD; 5 ␮g/ml; Fisher Scientific, Fair Lawn, NJ) was added to the growth medium at specified times, and total RNA was prepared at the indicated time points and analyzed by qPCR. IP. Immunoprecipitation (IP) of TTP-bound mRNAs was done as described previously (26). Briefly, RIE-1 cells were grown in the presence or absence of 5 ng/ml TGF-␤ for 24 h. Cells were lysed in polysome lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 7.0, 0.5% NP-40, 1 mM DTT) containing 100 U/ml RNase inhibitor (Ambion, Austin, TX) and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Five hundred micrograms of cytoplasmic lysate was incubated with 30 ␮g of antiTTP polyclonal antibody (catalog no. H-120; Santa Cruz Biotechnology, Santa Cruz, CA) or control IgG (catalog no. 2729; Cell Signaling Technology, Danvers, MA) precoated to protein A/G PLUS agarose (Santa Cruz Biotechnology) overnight at 4°C. Immunoprecipitates were collected by centrifugation and washed 5 times with lysis buffer. Total RNA was isolated from immunoprecipitates using 1 ml TRIzol reagent and then used for cDNA synthesis. Analysis of mRNA by qPCR in IP samples was done as described above. Statistical analysis. The data are expressed as the mean ⫾ standard error of the mean (SEM) and were compared by Student’s t test using GraphPad Prism software (GraphPad Software). P values of ⱕ0.05 were considered statistically significant.

RESULTS

TGF-␤ promotes P-body formation. Considerable evidence exists demonstrating that TGF-␤ is a potent regulator of epithelial cell growth and loss of TGF-␤ signaling contributes to intestinal tumorigenesis (1). Consistent with this, TGF-␤ has been shown to influence ARE-containing gene expression through modulation of mRNA decay (22, 34, 35). Based on these observations, we hypothesized that an antiproliferative property of TGF-␤ signaling occurs through enhanced ARE-mRNA decay and P-body formation. To test this, small intestine epithelial cells (RIE-1 cells) and colonocytes (YAMC cells) were utilized as nontransformed cell models of intestinal epithelium. RIE-1 cells were derived from normal small intestinal crypts from rats (36), and YAMC cells were derived from murine colon crypts conditionally immortalized with a temperature-sensitive simian virus 40 large T antigen (37). Both cell types display properties of normal intestinal epithelial cells (e.g., polarized growth, formation of tight adherens junctions, contact-mediated growth inhibition, and TGF-␤-mediated growth inhibition) and rapid ARE-mRNA turnover (21, 22, 38). To determine the effects of TGF-␤ on ARE-mRNA decay, we examined P bodies in TGF-␤-stimulated and nonstimulated cells by immunofluorescence microscopy. Hedls (EDC4), a well-characterized component of the decapping complex, was used as an endogenous P-body marker (25). As shown in Fig. 1A, RIE-1 cells treated with TGF-␤ for 24 h exhibited an ⬃2-fold increase in the average number of P bodies per cell. This effect of TGF-␤ appeared to be specific to P-body formation, since the formation of stress granules was not apparent in RIE-1 cells after treatment with TGF-␤ (data not shown). This TGF-␤-dependent induction of P bodies was transient, and removal of TGF-␤ for 24 and 48 h resulted in a return to baseline P-body levels (Fig. 1B). Using another well-characterized component of the decapping complex and P-body marker, Dcp1a (25), a similar increase in P bodies was observed with TGF-␤, with colocalization between Dcp1a and Hedls occurring (Fig. 1C). TGF-␤ treatment did not significantly increase the levels of Hedls or Dcp1a (Fig. 1D), indicating that this increase in P bodies with TGF-␤ was dependent on another factor.

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DNA and siRNA transfections. The pcDNA-MS2 and pcDNA-MS2YFP plasmids used for fluorescent visualization of RNA have been previously described (29). The COX-2 3= UTR (30) was inserted into pcDNAMS2 at NotI and XhoI sites. mCherry-tagged Dcp1a (pmCherry-Dcp1a) was created by cloning the human Dcp1a-coding region into the XhoI and EcoRI sites of the pmCherry-C1 vector (Clontech Laboratories, Mountain View, CA). The MS2 system was transiently cotransfected with pmCherry-Dcp1a into RIE-1 cells using Lipofectamine Plus (Invitrogen, Carlsbad, CA) for 24 h, and the cells were allowed to grow in the presence or absence of 5 ng/ml TGF-␤1 (R&D Systems, Minneapolis, MN), as indicated below. ARE-mediated mRNA decay was accomplished using a luciferase (Luc) reporter construct bearing the COX-2 3= UTR (Luc⫹3=UTR) as previously described (30). Promoter activity was assessed using a rat TTP promoter reporter construct (⫺5315/⫹58LUC) (31) and Smad-responsive reporter p6SBE-Luc (32). RIE-1, YAMC, and YAMC⌬Smad3 cells were transfected using Lipofectamine Plus (Invitrogen, Carlsbad, CA) for 48 h and then treated with 5 ng/ml TGF-␤ for 48 h (RIE-1 cells) or 12 h (YAMC cells). Cells were lysed in reporter lysis buffer and assayed using a luciferase assay system (Promega, Madison, WI). Reporter gene activities were normalized to total protein activity; all results represent the averages of triplicate experiments. RIE-1 cells were transfected with a predesigned siRNA against TTP (catalog no. AM16708A; Ambion, Austin, TX) or a negative control no. 1 siRNA (Ambion) using the siQUEST reagent (Mirus, Madison, WI) according to the manufacturer’s protocol. Cells were transfected for 48 h with 50 nM siRNA and then treated with 5 ng/ml TGF-␤1 for 48 h. Western blotting. Western blot analyses were performed as described previously (33) using antibodies against TTP (catalog no. H-120; Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:200 for 16 h at 4°C. Detection of Dcp1a and Hedls was accomplished using mouse monoclonal anti-Dcp1a at 1:1,000 (catalog no. ab57654; Abcam, Cambridge, MA) and rabbit polyclonal anti-Hedls at 1:1,000 (catalog no. 2548; Cell Signaling Technology, Danvers, MA). Membranes were stripped and reprobed using ␤-actin antibody (clone C4; MP Biomedicals, Solon, OH). Detection and quantitation of blots were carried out as described previously (30). RNA analysis. Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA synthesis was performed using 1 ␮g of total RNA in combination with oligo(dT) and Improm-II reverse transcriptase (Promega, Madison, WI). qPCR analysis was performed as described previously (33) using a 7300 PCR assay system (Applied Biosystems, Foster City, CA) and SYBR green PCR master mix (Applied Biosystems) containing primers for TTP (sense, 5=-ATGGATCTCTCTG CCATCTACG-3=; antisense, 5=-GCGAAGTAGGTGAGGGTGAC-3=), luciferase (sense, 5=-ACGGATTACCAGGGATTTCAGTC-3=; antisense, 5=-AGGCTCCTCAGAAACAGCTCTTC-3=), COX-2 (sense, 5=-GGAGC TCCATTCTCCTTGAA-3=; antisense, 5=-CTGCTTGTACAGCGATTGG A-3=), c-myc (sense, 5=-CAACGTCTTGGAACGTCAGA-3=; antisense, 5=-TCGTCTGCTTGAATGGACAG-3=), VEGF-A (sense, 5=-ATGAACTT TCTGCTCTCTTGG-3=; antisense, 5=-TATGTGCTGGCTTTGGTGAG3=), and ␤2-microglobulin (sense, 5=-ACCGTGATCTTTCTGGTGCTT G-3=; antisense, 5=-TAGCAGTTGAGGAAGTTGGGCT-3=). GAPDH (sense, 5=-CCACTCACGGCAAATTCAACGGCA-3=; antisense, 5=-TCT CCAGGCGGCACGTCAGATCC-3=) was used as a control for normalization. The fold change in mRNA expression levels was normalized to the cycle threshold (CT) using nontreated cells. Detection of COX-2, c-fos, c-myc, and VEGF-A mRNAs in siRNA-transfected RIE-1 cells was accomplished using a rat TGF-␤ signaling targets PCR array (catalog no. PARN235-Z; SA Biosciences, Qiagen Inc., Valencia, CA), and quantitative PCR (qPCR) was performed according to the manufacturer’s protocol. The luciferase mRNA half-life (t1/2) was determined in RIE-1 cells transfected with the Luc⫹3=UTR plasmid and treated with 5 ng/ml TGF-␤ for 48 h. Endogenous mRNA turnover was evaluated in RIE-1 cells transfected with control siRNA or siRNA for TTP for 48 h and then treated with 5

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FIG 1 TGF-␤ signaling promotes P-body formation in nontransformed intestinal epithelial cells. (A) RIE-1 cells treated with 5 ng/ml TGF-␤ for 24 h were immunostained with anti-Hedls antibody to visualize P bodies (green signal). DAPI was used to visualize nuclei. (B) RIE-1 cells were treated with TGF-␤ for the indicated times up to 24 h, after which cells were cultured in the absence of TGF-␤ for an additional 24 and 48 h. The graph presents the average number of P bodies per cell ⫾ SEM (n ⫽ 50 cells per group). (C) RIE-1 cells treated with 5 ng/ml TGF-␤ for 24 h were immunostained with anti-Dcp1a (red signal) and anti-Hedls (green signal) antibodies. Colocalization between Dcp1a and Hedls is shown in yellow in the merged images. DAPI was used to visualize nuclei. (D) RIE-1 cells treated with 5 ng/ml TGF-␤ for 24 h were assayed for Hedls and Dcp1a protein expression by Western blotting. Actin was used as a loading control. Bars ⫽ 10 ␮m. *, P ⱕ 0.05; **, P ⱕ 0.01.

In nontransformed cells, TGF-␤ signals through the canonical Smad pathway (1), and previous studies have demonstrated the importance of Smad3 in signaling the growth-inhibitory effects of TGF-␤ (28, 39). The role of TGF-␤/Smad signaling in induction of P-body assembly was examined using YAMC colonocytes and an isogenic variant derived from Smad3⫺/⫺ (YAMC⌬Smad3) mice (23). As shown in Fig. 2A, YAMC cells treated with TGFexhibited a similar induction in P bodies as RIE-1 cells. However, TGF-␤ treatment did not promote induction of P bodies in

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YAMC⌬Smad3 cells (Fig. 2B), indicating that the TGF-␤/Smad pathway is a physiological driver of P-body formation in intestinal epithelial cells. TGF-␤ promotes recruitment of ARE-mRNA to P bodies. AREs serve as cis-acting 3= UTR elements that target various mRNAs for rapid decay (40). Based on the observation that AREmRNAs are observed in P bodies (12), we determined if TGF-␤ could promote localization of ARE-mRNAs to P bodies as a means to control their expression. To examine the effects of TGF-␤ upon

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FIG 2 Smad3 is required for TGF-␤ induction of P bodies. YAMC (A) and YAMC⌬Smad3 (B) cells were treated with 5 ng/ml TGF-␤ for 8 h and immunostained using anti-Hedls antibody to visualize P bodies (red signal). DAPI was used to visualize nuclei. The bar graphs present the average number of P bodies per cell ⫾ SEM (n ⫽ 50 cells per group). Bars ⫽ 10 ␮m. ***, P ⱕ 0.0001; N.S., not significant.

the subcytoplasmic localization of an ARE-containing mRNA, the reporter construct pMS2-COX-2 3= UTR, which expressed a chimeric RNA consisting of 12 tandem MS2 RNA hairpins and the ARE-containing 3= UTR of COX-2 (30) (Fig. 3A), was prepared. Cotransfection with the vector pMS2-YFP, which expressed the fluorescent yellow fluorescent protein (YFP)-tagged MS2-binding protein that contained a nuclear localization signal (NLS), allowed detection of chimeric MS2-COX-2 3= UTR RNA subcellular localization (as the complex MS2-YFP/MS2-COX-2 3= UTR) by fluorescence microscopy. To detect P bodies, mCherry-tagged Dcp1a (pmCherry-Dcp1a) was also cotransfected with pMS2-COX-2 3= UTR and pMS2-YFP to mark P bodies. As shown in Fig. 3B, the majority of the MS2-YFP signal appeared to be nuclear in RIE-1 cells due to the presence of the NLS in the MS2-YFP protein. In unstimulated RIE-1 cells, MS2-COX-2 3= UTR RNA was observed in the cytoplasm with limited colocalization with Dcp1a (Fig. 3B, left). In contrast, treatment of cells with TGF-␤ resulted in increased P-body formation and enhanced colocalization of MS2COX-2 3= UTR RNA to the Dcp1a signal (Fig. 3B, right), indicating that TGF-␤ promoted the association of ARE-mRNA with P bodies. Control experiments using MS2 RNA lacking an ARE (MS2-⌬3= UTR) showed no reporter RNA colocalization with Dcp1a in the presence or absence of TGF-␤ (Fig. 3C).

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Induction of TTP is necessary for TGF-␤-mediated P-body formation. Several cellular factors involved in posttranscriptional regulation comprise P bodies (14); however, how such a factor(s) promotes controlled P-body assembly in response to TGF-␤ signaling is not known. Based on the ability of TTP to promote AREmediated decay and localize to P bodies (41, 42), we sought to determine if a causal link exists between TTP and TGF-␤-mediated P-body formation. Characteristic of most immediate early response genes, TTP expression is low in a variety of cell types and induced in response to growth factor signaling (8). In response to TGF-␤, RIE-1 cells showed a 7-fold induction of TTP mRNA after 16 h (Fig. 4A). A time-dependent induction of TTP protein was also observed, with significant induction seen as early as 6 h of TGF-␤ treatment (Fig. 4B). Similar induction of TTP mRNA and protein was observed in YAMC colonocytes treated with TGF-␤ (Fig. 4C and D). However, Smad3 deletion abrogated TGF-␤-mediated induction of TTP (Fig. 4CD). Examination of the TTP promoter sequence from human, mouse, and rat shows several putative Smad binding elements, which confer responsiveness to TGF-␤ (31, 43, 44). A luciferase expression construct driven by the rat TTP promoter or a control construct bearing 6 Smad-responsive elements (p6SBE-Luc) was

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FIG 3 TGF-␤ promotes ARE-containing mRNA recruitment to P bodies. (A) Schematic of the MS2 system for fluorescence-based mRNA visualization. The mRNA containing MS2 sites present upstream of the ARE-containing COX-2 3= UTR is bound by YFP-tagged MS2 binding protein, allowing fluorescent visualization of the mRNA. (B and C) RIE-1 cells were transfected with the MS2 dual plasmid system using the MS2-COX-2 3= UTR (B) or control MS2-⌬3=UTR (C) mRNA expression constructs, along with MS2-YFP and mCherry-tagged Dcp1a to visualize mRNA and P bodies, respectively. Cells were treated with 5 ng/ml TGF-␤ for 16 h and visualized for MS2-YFP-bound mRNA (pseudocolored green) and P bodies (red). Arrowheads, colocalization between MS2-COX-2 3= UTR mRNA and P bodies (shown in yellow). DAPI was used to visualize nuclei. Results shown are representative of those from three experiments. Bars ⫽ 10 ␮m.

transfected into RIE-1, YAMC, and YAMC⌬Smad3 cells and treated with TGF-␤. As shown in Fig. 4E, treatment of RIE-1 and YAMC cells with TGF-␤ resulted in a significant increase in TTP promoter activity, whereas Smad3-deficient YAMC cells did not show TGF-␤ induction. These results demonstrate the ability of TGF-␤ to promote TTP expression on the transcriptional level and are consistent with prior observations showing TGF-␤ induction of the human TTP promoter in inflammatory cells (43). To determine if TGF-␤ induction of TTP and P bodies impacted ARE-mRNA expression, RIE-1 cells were transfected with a luciferase reporter containing the COX-2 3= UTR (Luc⫹3=UTR) and treated with TGF-␤. As shown in Fig. 5A, TGF-␤ inhibited luciferase expression ⬎2-fold in the presence of the ARE-containing 3= UTR and did not significantly influence expression of a control reporter (Luc⌬3=UTR). However, this effect was not observed under conditions of TTP silencing (with siRNA for TTP

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[siTTP]), indicating the role of TTP in mediating ARE-mRNA expression levels. Since P bodies can regulate gene expression by promoting translational repression and mRNA degradation (11), changes in mRNA half-life were evaluated. RIE-1 cells transfected with the Luc⫹3=UTR reporter and stimulated with TGF-␤ were treated with actinomycin D (ActD) to halt transcription and determine the mRNA half-life. Figure 5B shows that TGF-␤ promoted the accelerated decay of Luc⫹3=UTR mRNA (t1/2 ⫽ 49 min) compared to that for nontreated cells (t1/2 ⫽ 87 min). This change in mRNA turnover was dependent on the presence of the 3= UTR ARE, since TGF-␤ did not significantly influence the halflife of control Luc⌬3=UTR mRNA (Fig. 5B). When transfected into cells, elevated levels of TTP have been shown to promote increased P-body numbers and colocalize with human Dcp1a (hDcp1a) (12). To determine if induction of endogenous TTP by TGF-␤ led to a similar localization of TTP to P

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were assayed by qPCR using GAPDH as a loading control and normalized to those for nontreated cells. Each value represents an average of triplicates ⫾ SEM. (B) RIE-1 cells treated with 5 ng/ml TGF-␤ were assayed for TTP protein expression by Western blotting. Actin was used as a loading control. (C and D) YAMC and YAMC⌬Smad3 cells were treated with 5 ng/ml TGF-␤ for the indicated times. TTP mRNA (C) and protein (D) were assayed as described above. (E) RIE-1, YAMC, and YAMC⌬Smad3 cells were transfected with luciferase reporters containing 5.3 kb of the rat TTP promoter or 6 Smad-responsive elements (p6SBELuc) and treated with 5 ng/ml TGF-␤, as indicated in Materials and Methods. Luciferase activity was normalized to that of total protein and is the average of 3 experiments ⫾ SEM. *, P ⱕ 0.05; **, P ⱕ 0.01; N.S., not significant.

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bodies, both RIE-1 and YAMC cells were transfected with GFPtagged hDcp1a and immunostained for TTP. As shown in Fig. 6A, both cell types showed an average number of P bodies per cell consistent with the results obtained when staining for an endogenous P-body marker (Fig. 1), and cells treated with TGF-␤ exhibited an ⬃2-fold increase in the average number of P bodies per cell. Importantly, TTP colocalized with GFP-hDcp1a both in the presence and in the absence of TGF-␤, and cells treated with TGF-␤ exhibited heightened colocalization, as indicated by a higher Pearson correlation coefficient for colocalization (data not shown). To determine if induction of TTP by TGF-␤ was necessary for enhanced P-body formation, RIE-1 cells were transfected with an siRNA against TTP or a scrambled siRNA control and treated with TGF-␤. Knockdown of TTP expression silenced endogenous and TGF-␤-induced TTP mRNA and protein expression (Fig. 6B). As a consequence of TTP knockdown, a marked decrease in P-body levels, detected by endogenous Dcp1a staining, was observed in

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both the presence and the absence of TGF-␤ (Fig. 6C). These results were further validated by an independent approach using mouse embryonic fibroblasts (MEFs) derived from wild-type (TTP⫹/⫹) and TTP-knockout (TTP⫺/⫺) mice. P bodies were detected in these cells using endogenous Dcp1a as a marker. Wildtype MEFs treated with TGF-␤ exhibited a 2-fold increase in Pbody numbers compared to the numbers for the nontreated controls. Conversely, TTP⫺/⫺ MEFs showed decreased levels of P bodies and were refractory to TGF-␤ induction of P bodies (Fig. 6D and E). The observed loss of P bodies under conditions of TTP downregulation resulted in RIE-1 cells being unable to attenuate ARE reporter gene expression in response to TGF-␤ (Fig. 5). To determine if this was also observed for endogenous expression of the TTP target genes, those for COX-2, c-fos, c-myc, and VEGF-A (7, 8), RIE-1 cells were transfected with a control siRNA or siTTP and then treated with TGF-␤ for 48 h (Fig. 7A). In control siRNAtreated cells, TGF-␤ promoted an ⬃1.5-fold increase in steady-

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FIG 6 TGF-␤ promotes P-body formation through induction of TTP. (A) RIE-1 and YAMC cells were transfected with GFP-hDcp1a and treated with 5 ng/ml TGF-␤ for 24 h (RIE-1 cells) or 8 h (YAMC cells). Immunofluorescent staining was done using anti-TTP antibody (red signal). Colocalization of TTP at P bodies is shown in merged panels as yellow. DAPI was used to visualize nuclei. Inset values indicate the average number of P bodies per cell ⫾ SEM (n ⫽ 30 cells per group). (B) RIE-1 cells were transfected with siTTP or control siRNA (siControl) and treated with 5 ng/ml TGF-␤ for 24 h. TTP mRNA levels were assayed by qPCR using GAPDH as a loading control and normalized to those for nontreated cells transfected with control siRNA. The data shown represent the average of triplicates ⫾ SEM. RIE-1 cells treated similarly were assayed for TTP protein expression by Western blotting. Actin was used as a loading control. (C) RIE-1 cells transfected with siTTP or control siRNA were treated with 5 ng/ml TGF-␤ for 24 h and subjected to immunofluorescence for endogenous Dcp1a (green signal). DAPI was used to visualize nuclei. The average number of P bodies per cell ⫾ SEM (n ⫽ 50 cells per group) is shown. (D) Wild-type (TTP⫹/⫹) and TTP-knockout (TTP⫺/⫺) MEFs were treated with 1.2 ng/ml TGF-␤ for 48 h and immunostained for endogenous Dcp1a to visualize P bodies (green signal). DAPI was used to visualize nuclei. (E) P bodies in MEFs were quantified by counting a total of 50 individual cells per group, and the amounts are presented as the average number of P bodies per cell ⫾ SEM. Bars ⫽ 10 ␮m. ***, P ⱕ 0.0001; N.S., not significant.

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FIG 7 TGF-␤ regulates ARE-mRNA expression in a TTP-dependent manner. (A) RIE-1 cells were transfected with a control siRNA (siControl) or siTTP and then treated with 5 ng/ml TGF-␤ for 48 h. qPCR analysis was performed as described in Materials and Methods for expression of the TTP target mRNAs: COX-2, c-fos, c-myc, and VEGF-A. Relative mRNA levels were normalized to those for the respective nontreated controls and are the average of 3 experiments ⫾ SEM. (B) RIE-1 cells were transfected with a control siRNA or siTTP and then treated with 5 ng/ml TGF-␤ for 24 h, after which ActD was added at the indicated times. COX-2, c-myc, VEGF-A, and ␤2-microglobulin mRNA half-lives were analyzed by qPCR using GAPDH as a loading control. Data shown are the average of 3 experiments ⫾ SEM. (C) RIE-1 cells were treated with 5 ng/ml TGF-␤ for 24 h, and equal amounts of cytoplasmic lysates were subjected to IP using control IgG or anti-TTP antibodies. RNA purified from immunoprecipitates was subjected to qPCR to detect COX-2, c-myc, VEGF-A, and GAPDH mRNAs. Fold induction was calculated relative to that for the nontreated controls and is the average of 3 experiments ⫾ SEM. *, P ⱕ 0.05; **, P ⱕ 0.001; N.S., not significant.

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⬃2-fold compared to that for wild-type MEFs in the presence or absence of TGF-␤. These findings indicate a role for TTP in facilitating TGF-␤-mediated inhibition of cell proliferation, whereas the ability of TGF-␤ to promote cellular senescence appeared to be independent of TTP. TTP is a physiological regulator of P-body assembly in colonic epithelium. TGF-␤’s role in normal gastrointestinal epithelial homeostasis includes control of cell proliferation, and genetic evidence has shown that proliferative pathways are activated if TGF-␤ receptors or Smads are mutated or downregulated (1). Based on our results demonstrating that deletion of Smad3 compromised TGF-␤ dependent P-body assembly (Fig. 2B), we examined endogenous P bodies in colonic epithelium from Smad3⫺/⫺ mice (Fig. 9A). In wild-type mice, the immunofluorescent staining pattern of Dcp1a revealed abundant visible, discrete foci characteristic of P bodies (Fig. 9A, top). However, fewer P bodies were observed in colonic crypts of Smad3⫺/⫺ mice. IHC analysis showed that similar levels of overall Dcp1a staining were seen between wild-type and Smad3⫺/⫺ mouse colon tissue, indicating that Dcp1a expression was not a limiting factor (Fig. 9A, bottom). Our observations also indicate TTP to be a link between TGF␤/Smad signaling and P-body formation. In support of this, decreased TTP expression was observed in the colonic epithelium of Smad3-deficient mice (Fig. 9B). To directly test if TTP loss in vivo impacted P-body formation, colonic sections from TTP-knockout mice were stained with Dcp1a to detect P bodies. Similar to the results presented above, colonic crypts from wild-type mice displayed pronounced levels of P bodies (Fig. 9C). We attribute the differences in the Dcp1a immunofluorescent staining pattern to be due to tissue preparation (formalin fixed, paraffin embedded versus fresh frozen), whereas in TTP-deficient (TTP⫺/⫺) mice, P-body levels were significantly reduced in colon sections, indicating a critical role for TTP with regard to P-body formation in colonic epithelium. These results demonstrate the presence of abundant P bodies in normal gastrointestinal epithelium and are consistent with previous findings showing abundant P bodies to be present in mouse bronchial epithelium (46). Furthermore, control of P-body levels was dependent upon TGF-␤/Smad signaling of TTP expression both in vitro and in vivo. DISCUSSION

TGF-␤ plays a central role in gastrointestinal homeostasis by controlling the proliferative activity and differentiation of intestinal epithelial cells, whereas escape from TGF-␤ responsiveness is associated with malignant transformation of epithelial cells (1, 2). It is well established that TGF-␤ can activate a cytostatic response through Smad-mediated transcriptional repression of growthpromoting transcription factors c-myc, ID1, ID2, and ID3 (where ID represents inhibitor of DNA binding), along with induction of cyclin-dependent kinase (CDK) cell cycle inhibitors (2, 47). The results presented here have uncovered an additional component of the TGF-␤ cytostatic response by promoting ARE-mRNA decay through enhanced P-body formation. This increase in P-body formation occurred through Smad-mediated transcriptional induction of the ARE-binding protein TTP. On the basis of TTP’s ability to deliver and facilitate P-body formation on ARE-mRNAs (12), our findings indicate TTP to be a direct link connecting TGF-␤ and the observed assembly of P bodies (Fig. 10). Our results indicate that signaling through the TGF-␤/Smad pathway promotes P-body formation as a means to control ARE-

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state ARE-mRNA levels, whereas in the absence of TTP, these mRNAs were upregulated approximately 4- to 11-fold in cells stimulated with TGF-␤, indicating the ability of TTP to control their respective levels. To determine if this ARE-mRNA upregulation was due to increased mRNA stability, the half-lives of COX-2, c-myc, and VEGF-A mRNAs were assessed. In RIE-1 cells treated with TGF-␤, rapid decay of COX-2, c-myc, and VEGF-A mRNAs was observed, yielding half-lives of ⬃32, 22, and 79 min, respectively (Fig. 7B). In contrast, COX-2, c-myc, and VEGF-A mRNAs were stabilized approximately 2-fold or greater in TTPknockdown cells, with observed half-lives of ⬃59, 38, and ⬎120 min, respectively. Similar trends were observed in the absence of TGF-␤ (data not shown). Decay of the housekeeping ␤2-microglobulin mRNA was not changed under TTP-knockdown conditions. To establish whether the observed effects on ARE-mRNA turnover were mediated through a direct TTP–ARE-mRNA association, immunoprecipitations (IPs) were done to determine whether endogenous ARE-mRNAs would associate with TTP in a TGF-␤-dependent manner. RIE-1 cells were treated with TGF-␤ for 24 h, and IP of cytoplasmic lysates was done using an anti-TTP antibody or control IgG. The association of COX-2, c-myc, and VEFG-A mRNAs with TTP was assayed by qPCR of the mRNA present in immunoprecipitates. As shown in Fig. 7C, all AREmRNAs were significantly enriched in the TTP IP samples from cells stimulated with TGF-␤, whereas capture of the negative-control GAPDH mRNA was unchanged. Taken together, these results demonstrate the essential role of TTP in TGF-␤-mediated induction of P-body formation and subsequent regulation of AREmRNA expression. TTP promotes TGF-␤-mediated growth inhibition. Inhibition of cell proliferation and induction of senescence are characteristic cytostatic outcomes of TGF-␤ signaling in nontransformed cells (1, 37, 45). Based on the results presented here, we sought to determine if these growth-suppressive effects induced by TGF-␤ occur through its induction of TTP and P-body formation. To test this, wild-type and TTP⫺/⫺ MEFs were grown in the presence or absence of TGF-␤ for 48 h, and cell proliferation was assayed by staining for the proliferation marker Ki67. As shown in Fig. 8A and B, TGF-␤ attenuated proliferation of wild-type MEFs 2.5-fold. However, TTP⫺/⫺ MEFs were less responsive to the antiproliferative effect of TGF-␤, exhibiting a 1.4-fold change in the percentage of proliferating cells. As an independent measure of proliferation, [3H]thymidine incorporation was performed in MEF cultures. Consistent with the results obtained with Ki67 staining, TGF-␤ was unable to inhibit [3H]thymidine incorporation in TTP⫺/⫺ MEFs to the extent observed in wild-type MEFs (Fig. 8C). Similar results in RIE-1 cells were observed for Ki67 staining after siRNA knockdown of TTP expression, making them less sensitive to TGF-␤-mediated growth inhibition than control transfected cells (data not shown). To evaluate whether the presence of TTP can influence TGF␤-mediated induction of senescence, wild-type and TTP⫺/⫺ MEFs were cultured in the presence of TGF-␤ over a 10-day period. As shown in Fig. 8D and E, TGF-␤ promoted a senescent phenotype in wild-type MEFs, with a 1.8-fold increase in the percentage of senescence-associated ␤-galactosidase (SA-␤-Gal)positive cells being detected. While TGF-␤ was also able to induce a similar 1.7-fold increase in SA-␤-Gal-positive cells in TTP⫺/⫺ MEFs, the overall extent of senescent TTP⫺/⫺ MEFs was decreased

TGF-␤ Regulates P-Body Formation

Downloaded from http://mcb.asm.org/ on April 17, 2015 by UCSF Library & CKM FIG 8 Loss of TTP expression abrogates TGF-␤-mediated growth inhibition in nontransformed cells. (A) Wild-type (TTP⫹/⫹) and TTP-knockout (TTP⫺/⫺) MEFs were cultured in the presence or absence of 1.2 ng/ml TGF-␤ for 48 h and immunostained with anti-Ki67 antibody to visualize proliferative cells (green signal). DAPI was used to visualize nuclei. Bar ⫽ 75 ␮m. (B) Cells positive for the proliferation marker Ki67 were quantified by determining the average number of Ki67-positive cells per ⫻20 field of view for 10 individual fields. Results are represented as an average of the percentage of Ki67-positive cells per ⫻20 field of view (F.O.V.) ⫾ SEM. (C) Wild-type and TTP⫺/⫺ MEFs were assayed for TGF-␤-mediated growth inhibition after 48 h of TGF-␤ treatment by [3H]thymidine incorporation. Results represent the average [3H]thymidine incorporation for triplicate wells ⫾ SEM. (D) MEFs were cultured in the presence or absence of TGF-␤ for 10 days and stained for SA-␤-Gal activity (blue signal). Bar ⫽ 30 ␮m. (E) MEFs positive for SA-␤-Gal were quantified by determining the average number of positive cells per ⫻20 field of view for 10 individual fields, and the amounts are represented as an average of SA-␤-Gal-positive cells per ⫻20 field of view ⫾ SEM. **, P ⱕ 0.001; ***, P ⱕ 0.0001; N.S., not significant.

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through P-body assembly. TGF-␤ signaling promotes TTP expression through Smad-mediated transcription. Increased TTP expression allows binding of ARE-mRNAs and trafficking to P bodies for subsequent decay.

FIG 9 TTP is a physiological driver of P-body formation in the normal intestinal epithelium. (A) (Top) Formalin-fixed, paraffin-embedded colonic sections derived from wild-type (Smad3⫹/⫹) or Smad3-knockout (Smad3⫺/⫺) mice were stained for Dcp1a to visualize P bodies (green signal). DAPI was used to visualize nuclei. (Bottom) IHC detection of Dcp1a in wild-type and Smad3⫺/⫺ mouse colon tissue. Representative tissue sections were examined for Dcp1a expression and counterstained with hematoxylin. Bars ⫽ 100 ␮m. (B) IHC analysis of TTP expression in wild-type and Smad3⫺/⫺ mouse colon tissue. Bar ⫽ 50 ␮m. (C) Fresh-frozen colonic sections from wild-type (TTP⫹/⫹) or TTP-knockout (TTP⫺/⫺) mice were stained with anti-Dcp1a or anti-TTP antibodies to visualize P bodies (green signal) or TTP expression (red signal). DAPI was used to visualize nuclei. Bar ⫽ 100 ␮m.

mRNA levels and impact cell proliferation. This physiological signaling is in contrast to other established inducers of P-body formation involving stresses, such as glucose starvation, osmotic stress, UV irradiation, inhibition of translation initiation, and microtubule disruption, which can lead to inhibition of translation and mRNA decay (11, 15, 17, 48–51). Other findings more

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consistent with those presented here have shown enhanced Pbody formation to occur in cells in response to serum and insulin stimulation and through activation of the AKT and Jun N-terminal protein kinase (JNK) pathways (52–55). Interestingly, activation of these pathways promoted P-body formation through independent posttranslational events involving phosphorylation of P-body components EDC3, Dcp1a, and the translational initiation factor 4E-transporter (4E-T), leading to increased P-body formation and localization of these factors to P bodies (53–55). Cyclic AMP-dependent protein kinase A (PKA) signaling has also been shown to be involved in P-body assembly regulation in the yeast Saccharomyces cerevisiae (56), and TGF-␤ signaling has been shown to activate PKA (57). Although the role of TTP was not investigated in these studies, it is intriguing to speculate the contribution of TTP in P-body induction in these studies on the basis of these signaling pathways’ ability to facilitate TTP expression (8, 58–62). Previous studies have demonstrated the ability of TGF-␤ to mediate posttranscriptional regulation of oncogenic and chemokine ARE-mRNAs (34, 63). Although the mechanism by which TGF-␤ modulates mRNA turnover is not known, our results indicate that it occurs through TGF-␤ induction of TTP and P bodies. An alternative explanation suggests miRNA involvement. Recent findings have shown that TGF-␤ induces expression of specific miRNAs through a mechanism involving receptor-regulated Smad (R-Smad) binding of primary miRNAs (pri-miRNAs) and recruitment of the Drosha microprocessor complex, leading to pre-miRNA maturation (64, 65). The observed increase in P bodies with TGF-␤ treatment shown here would provide a nucleation site for subsequent mRNA decay, in that components of miRNA-mediated suppression are concentrated in P bodies (11).

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FIG 10 TGF-␤-mediated induction of TTP regulates ARE-mRNA expression

TGF-␤ Regulates P-Body Formation

ACKNOWLEDGMENTS We thank J. Lykke-Andersen for anti-Hedls antibody and GFP-hDcp1a, J.-Y. Wang for MS2 plasmids, and N. Kaneda for rat TTP promoter. This work was supported by the National Institutes of Health

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(R01CA134609) and the American Cancer Society (RSG-06-122-01CNE).

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Further work examining the TGF-␤-induced epithelial-to-mesenchymal transition (EMT) has identified a posttranscriptional component of this process. Various transcripts involved in EMT contain 3= UTR elements bound by a ribonucleoprotein complex containing heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) and eukaryotic elongation factor-1 A1 (eEF1A1), which mediates translational inhibition. Interestingly, TGF-␤ signaling promotes phosphorylation of hnRNP E1 and its release from the mRNA, allowing eEF1A1-mediated translational elongation to proceed (66, 67). While this conserved 3= UTR element appears to be distinct to AU-rich elements (68), these findings further highlight the functional contribution of TGF-␤ in posttranscriptional regulation. The TGF-␤/Smad pathway exerts an essential growth-inhibitory and tumor suppressor role in the gastrointestinal epithelium. Similar to this, expression of TTP was shown to inhibit cell growth and tumorigenesis in various cell and tumor models through mechanisms promoting decreased proliferation, loss of viability, senescence, and apoptosis (26, 69–73). In colon cancer cells, TTP can inhibit cell growth and tumorigenesis through downregulation of COX-2 and VEGF (33, 74). These similarities in growth suppression suggest that the cytostatic effects induced by TGF-␤ are in part through its induction of TTP and indicate that TTP can serve in a tumor suppressor capacity downstream of TGF-␤/Smad signaling. During colorectal tumorigenesis, inactivating mutations and downregulation of TGF-␤ signaling components are observed, and recent findings indicate a tumor-suppressive function of Smad3, as 4.3% of primary tumors display mutations in SMAD3 (1, 75). In support of this, Smad3-deficient mice spontaneously form invasive colorectal adenocarcinomas (76). The results presented here indicate that loss of Smad3 abrogates the TGF-␤ cytostatic response partially through loss of Smad-mediated induction of TTP expression. As a consequence, ARE-mRNA decay is compromised due to deficiencies in P-body formation, and the observed lack of P bodies in the colonic epithelium of Smad3-deficient mice provides insight into the hyperproliferative and tumorigenic phenotype observed in these animals. TTP is expressed at modest levels in human and murine colonic epithelium (33, 77), indicating a posttranscriptional function of TGF-␤/Smad signaling in the regulation of growth and inflammatory mediators by maintaining cellular TTP and P-body levels. However, loss of TTP expression is observed in ⬎75% of colorectal adenomas and adenocarcinomas, allowing enhanced tumor-promoting ARE-mRNA expression (9, 33, 70). Similarly, TTP-deficient mice develop multiple inflammatory syndromes resulting from increased levels of tumor necrosis factor alpha, COX-2, and inflammatory factors due to defects in ARE-mRNA turnover (78, 79). While the mechanisms promoting TTP loss in colorectal cancer are largely undefined, our findings suggest that during tumorigenesis inactivating mutations and downregulation of TGF-␤ signaling components directly alter TTP and P-body levels, leading to pathogenic ARE-mRNA stabilization. Taken together, these results provide an additional insight into the growthpromoting effects of TGF-␤ observed in colorectal tumors.

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